Multispecific antigen-binding protein with improved expression efficiency

Abstract

The present invention relates to a nucleic acid molecule encoding a multispecific antigen-binding protein and exhibiting significantly improved expression efficiency in vivo. In the multispecific antigen-binding protein of the present invention, specifically, a bispecific antibody that recognizes a tumor antigen or a viral antigen and a natural killer cell-specific activating receptor, the light chain variable region (VL) and the heavy chain variable region (VH) of a receptor-recognizing site of a natural killer cell-specific activating receptor are sequentially arranged in the N-terminus to the C-terminus direction, thereby increasing expression efficiency by up to 8-fold. Therefore, the present invention provides an optimal mRNA structure capable of most efficiently expressing bispecific antibodies in vitro and in vivo, thereby enabling effective use thereof for not only mass-production of recombinant bispecific antibodies but also as an excellent mRNA therapeutic agent for stable and continuous production of a therapeutically effective amount of bispecific antibodies in the body of a patient.

Description

Multispecific antigen-binding protein with improved expression efficiency

[0001] The present invention relates to a multispecific antigen-binding protein in which the translation efficiency from mRNA to protein is significantly improved by arranging variable regions within the antigen recognition site in a specific order. The present invention was made possible through the support of the National New Drug Development Project (RS-2023-00258675) of the National New Drug Development Agency, funded by the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, and the Ministry of Health and Welfare.

[0002]

[0003] Various structures have been proposed for multispecific antibodies that bind to multiple distinct antigens, depending on the composition of the antigen recognition sites for each target antigen and the binding patterns between them. These multispecific antibodies not only exhibit high specificity that cannot be achieved with conventional monoclonal antibodies but also provide superior efficacy by synergistically regulating signaling pathways through cross-linking of different receptors. For example, a bispecific antibody that simultaneously recognizes cancer cell surface antigens and activation receptors of immune cells, such as T cells or natural killer cells, can significantly enhance immunotherapeutic activity against tumors by simultaneously performing cancer cell targeting and tumor-specific activation of immune cells.

[0004] Natural Killer (NK) cells are a type of lymphocyte involved in atypical immunity and account for about 10% of blood cells. NK cells have the ability to kill tumor cells or cells infected by exogenous pathogens, and thus play a role in eliminating abnormal cells that could become pathological. Most NK cells in the body exist in an inactivated state under normal conditions, but since activated NK cells are required for therapeutic use, research on activating NK cells from normal blood or patient blood is actively underway.

[0005] Three activating receptors found in NK cells—NKp30, NKp44, and NKp46—act as natural cytotoxic receptors (NCRs) and play a crucial role in the antitumor and antiviral defense of NK cells. Since NKp46 is expressed exclusively by NK cells, it serves not only as an important activating receptor but also as a key marker for identifying, isolating, or detecting NK cells. Activation of NK cells by NKp46-specific antibodies leads to an increase in intracellular calcium ion concentration, induction of cytotoxicity, and release of lymphokines. In particular, Fcγ receptors expressed in tumor cells, such as B-cell malignancies, interact with the Fc domain of anti-NKp46 antibodies bound to NK cells, thereby enhancing the efficiency of apoptosis.

[0006] Meanwhile, although DNA is known to be relatively more stable and easier to handle than RNA in applications such as gene therapy, it has disadvantages. When delivered into a target genome, DNA can be inserted at unintended locations, potentially damaging the host's genes. Furthermore, it can be damaged by anti-DNA antibodies generated by the host's immune response, and the expression level of the target active protein is limited by various variables affecting transcription. In contrast, mRNA synthesizes proteins directly within the cytoplasm without the need for transcription in the nucleus. It poses no risk of damaging the host cell's genetic structure and has a short half-life, which prevents long-term genetic modification; thus, compared to DNA, it is more stable and easier to mass-produce.

[0007] Accordingly, although therapeutic methods involving the administration of mRNA encoding multiple antibodies to subjects instead of protein-based multiple antibodies offer the advantage of continuously producing multiple antibodies through the endogenous translation system present within cells, they often present difficulties, such as failure to express in specific tissues or aggregation after expression, leading to denaturation into inactive high molecular weight (HMW) proteins. Therefore, there is a need for the development of efficient multiple antibodies that can stably and continuously produce a therapeutically effective dose after being delivered into the patient's body in the form of mRNA.

[0008]

[0009] Throughout this specification, numerous papers and patent documents are referenced and cited. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the state of the art to which the present invention pertains and the content of the present invention.

[0010]

[0011] The inventors have made diligent research efforts to explore the structure of an efficient multispecific antigen-binding protein that can be stably and highly expressed in vivo after being delivered in the form of a nucleic acid molecule. As a result, the present invention was completed by discovering that the in vivo translation efficiency of the antigen-binding protein is significantly improved when the light chain variable region (VL) and the heavy chain variable region (VH) constituting the second antigen-binding site are arranged in a specific order, in a second antigen-binding site located at the C-terminus of the antigen-binding protein or located between the first antigen-binding site and the Fc domain within the antigen-binding protein.

[0012] Therefore, the objective of the present invention is to provide a nucleic acid molecule encoding a multispecific antigen-binding protein with improved expression efficiency.

[0013] Another objective of the present invention is to provide a multispecific antigen-binding protein expressed using the nucleic acid molecule.

[0014] Another objective of the present invention is to provide a composition for enhancing the production efficiency of a multispecific antigen-binding protein comprising the above-mentioned nucleic acid molecule as an active ingredient.

[0015] Another objective of the present invention is to provide a method for enhancing the production efficiency of a multispecific antigen-binding protein, comprising the step of introducing the nucleic acid molecule into a host cell.

[0016]

[0017] Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims, and drawings.

[0018]

[0019] According to one aspect of the present invention, the present invention provides a nucleic acid molecule encoding a multispecific antigen-binding protein comprising:

[0020] (1) A first antigen-binding site comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1);

[0021] (2) A second antigen-binding site comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2); and

[0022] (3) An Fc domain located between (1) and (2) above or coupled to the C-terminus of (2) above.

[0023] The inventors have made diligent research efforts to explore the optimal structure of a multispecific antigen-binding protein, specifically a bispecific antigen-binding protein, that can be stably and highly expressed in vivo after being delivered in the form of a nucleic acid molecule. As a result, they discovered that when the light chain variable region (VL) and the heavy chain variable region (VH), which constitute the C-terminus of the antigen-binding protein or the second antigen-binding site located between the first antigen-binding site and the Fc domain within the antigen-binding protein, are arranged in a specific order, they can be used as an excellent mRNA therapeutic agent that is stably and continuously highly expressed in vivo.

[0024] In this specification, the term “nucleic acid molecule” has a meaning that comprehensively includes DNA and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base sites are modified (Uhlman er al., Chemical Reviews (1990) 90:543-584). The sequence of the nucleic acid molecule encoding the entire antigen-binding protein molecule or the variable regions of the heavy chain and light chain of the present invention may be modified, and said modification includes the addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0025] The nucleic acid molecule of the present invention is interpreted to include a nucleotide sequence that exhibits substantial identity with respect to the nucleotide sequence mentioned in the present invention. The above-mentioned substantial identity refers to a nucleotide sequence that exhibits at least 80% homology, specifically at least 85% homology, more specifically at least 90% homology, and most specifically at least 95% homology when any other sequence is aligned to correspond as much as possible with the nucleotide sequence of the present invention and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0026] In this specification, the term “multispecific antigen binding protein” refers to an engineered molecule that specifically binds to two or more different antigens, and is also referred to as a multispecific antibody. In this specification, the term “antibody” refers to a peptide that recognizes a specific epitope of a target antigen and specifically binds to it, and includes not only the complete antibody form but also antigen-binding fragments (antibody fragments) of a full-length antibody molecule. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and has subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The invariant region of the light chain has kappa (κ) and lambda (λ) types.

[0027] In this specification, the term “antigen binding region” refers to a domain comprising a three-dimensional structure capable of immunospecifically binding to an epitope. Accordingly, the antigen binding region may include a hypervariable region of the antibody chain, specifically a VH and / or VL domain, at least a VH domain, or at least one complementarity determining region (CDR).

[0028] In this specification, the term “CDR (complementarity determining region)” refers to the amino acid sequence of a hypervariable region of an immunoglobulin heavy chain and a light chain. The heavy chains (HCDR1, HCDR2, and HCDR3) and light chains (LCDR1, LCDR2, and LCDR3) each contain three CDRs, and these CDRs provide major contact residues for the antibody to bind to an antigen or epitope.

[0029] In this specification, the term “antigen-binding fragment of an antibody” refers to a fragment having a significant antigen-antibody binding function within a full-length antibody molecule, and includes fragments containing the aforementioned antigen-binding site such as Fab, F(ab'), F(ab')2, Fv, and nanobodies (nanobody or sybody).

[0030] Fab, among the antibody fragments, has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain and a first constant region (CH1) of the heavy chain, and has one antigen binding site.

[0031] F(ab)' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0032] Fv is a minimal antibody fragment having only a heavy chain variable region and a light chain variable region. In two-chain Fv, the heavy chain variable region and the light chain variable region are connected by non-covalent bonds, and in single-chain Fv (ScFv), the heavy chain variable region and the single chain variable region are generally connected by covalent bonds through a peptide linker or directly at the C-terminus, so they can form a dimer-like structure similar to that of two-chain Fv.

[0033] In this specification, the term “heavy chain” means both the full-length heavy chain and fragments thereof, comprising an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen, a variable region domain VH, and three constant region domains CH1, CH2 and CH3.

[0034] In this specification, the term “light chain” means both the full-length light chain and fragments thereof, comprising a variable region domain VL and a constant region domain CL, which have an amino acid sequence having a sufficient variable region sequence to confer specificity to an antigen.

[0035]

[0036] According to a specific embodiment of the present invention, the multispecific antigen-binding protein encoded by the nucleic acid molecule of the present invention may be a bispecific antibody.

[0037] The bispecific antibody of the present invention has a first antigen-binding site located at the N-terminus of the entire bispecific antibody molecule, a second antigen-binding site located at the C-terminus relative to the first antigen-binding site, and an Fc domain may be located between the first and second antigen-binding sites or at the C-terminus of the entire bispecific antibody molecule.

[0038] According to one embodiment, an antigen-binding fragment that binds to a first antigen, specifically ScFv or Fab; and an antigen-binding fragment that binds to a second antigen, specifically ScFv, may be connected together with an Fc region in various sequences as shown in FIG. 1. For example, ScFv for the first antigen - Fc - ScFv for the second antigen may be connected sequentially from the N-terminus to the C-terminus (structure (A) of FIG. 1, ScFv-Fc-ScFv); ScFv for the first antigen - ScFv for the second antigen - Fc may be connected sequentially (structures (B) and (C) of FIG. 1, ScFv2-Fc); and Fab for the first antigen - Fc - ScFv for the second antigen may be connected sequentially (structures (D) and (E) of FIG. 1, Fab-Fc-ScFv).

[0039]

[0040] According to a specific embodiment of the present invention, the first antigen-binding site specifically recognizes a target antigen, and the second antigen-binding site specifically recognizes a natural killer cell-specific activation receptor.

[0041] In this specification, the term “target antigen” refers to any antigenic substance excluding effector antigens expressed by immune effector cells; specifically, it refers to an antigen that is specifically expressed in diseased lesion cells or pathogens, but is not expressed, is expressed at a low level, or is expressed in a state where access to the antigen recognition site is impossible in healthy normal tissues or normal cells. Accordingly, the target antigen can serve as a therapeutic or diagnostic target for the corresponding disease.

[0042] More specifically, the target antigen may be a viral antigen or a tumor antigen. More specifically, the target antigen may be a tumor antigen.

[0043] In this specification, the term “viral antigen” refers to an antigenic molecule expressed by a virus within an infected host, recognized by the host’s immune system, and inducing an immune response. The viral antigen may be a protein, glycoprotein, or lipid complex, and may include a surface protein of the virus (e.g., spike protein) or an internal component of the virus (e.g., capsid protein or nucleoprotein binding protein). The viral antigen recognized by the first antigen-binding site of the present invention may be, for example, PreS1, a human hepatitis B virus surface antigen, but is not limited thereto.

[0044] In this specification, the term “tumor antigen” refers to an antigen that exhibits a specific pattern of expression in tumor cells, which induces an immune response in the individual and serves as a therapeutic target for immunotherapy or as a diagnostic marker to determine the presence of a tumor.

[0045] According to one embodiment of the present invention, the bispecific antigen-binding protein of the present invention has a ScFv (structures (A), (B), and (C) of FIG. 1) or Fab (structures (D) and (E) of FIG. 1) located at the N-terminus as a first antigen-binding site that recognizes a tumor antigen or a viral antigen.

[0046] According to a specific embodiment of the present invention, the tumor antigen may be selected from the group consisting of CD19, CD20, CD22, BCMA, CD123, CD5, B7-H3, PD-L1, GPC-3, c-Met, AFP, TROP2, CLDN6, CLDN18.2, ROR1, EpCAM, Mesothelin, HER-2, EGFR, MUC1, and CEA, but is not limited thereto.

[0047] According to one embodiment of the present invention, the first antigen-binding site may specifically bind to CD20, in which case the first antigen-binding site includes a heavy chain variable region comprising the HCDR1 region of sequence 15, the HCDR2 region of sequence 16, and the HCDR3 region of sequence 17. More specifically, the first antigen-binding site additionally includes a light chain variable region comprising the LCDR1 region of sequence 18, the LCDR2 region of sequence 19, and the LCDR3 region of sequence 20.

[0048] According to a specific embodiment of the present invention, the first antigen-binding site may specifically bind to GPC-3, in which case the first antigen-binding site includes a heavy chain variable region comprising an HCDR1 region of sequence 21 or 27, an HCDR2 region of sequence 22 or 28, and an HCDR3 region of sequence 23 or 29. More specifically, the first antigen-binding site additionally includes a light chain variable region comprising an LCDR1 region of sequence 24 or 30, an LCDR2 region of sequence 25 or 31, and an LCDR3 region of sequence 26 or 32.

[0049] According to a specific embodiment of the present invention, the first antigen-binding site can specifically bind to Pre-S1, in which case the first antigen-binding site includes a heavy chain variable region comprising the HCDR1 region of sequence 33, the HCDR2 region of sequence 34, and the HCDR3 region of sequence 35. More specifically, the first antigen-binding site additionally includes a light chain variable region comprising the LCDR1 region of sequence 36, the LCDR2 region of sequence 37, and the LCDR3 region of sequence 38.

[0050] In this specification, the term “natural killer cell-specific activating receptor (NK cell activating receptor)” refers to a receptor protein that is specifically expressed on the surface of NK cells and plays an important role in recognizing and eliminating infected cells, cancer cells, or abnormal lesion cells under stress. It binds to specific ligands on the surface of target cells to induce signaling pathways that activate NK cells, thereby releasing cytotoxic substances and cytokines to eliminate lesion cells.

[0051] According to a specific embodiment of the present invention, the natural killer cell-specific activation receptor is selected from the group consisting of NKp46, NKp44, NKp30, CD16a, DNAM-1, 2B4, and NKG2D.

[0052] According to a specific embodiment of the present invention, the second antigen-binding site can specifically bind to NKp46, in which case the second antigen-binding site includes a heavy chain variable region comprising the HCDR1 region of sequence 39, the HCDR2 region of sequence 40, and the HCDR3 region of sequence 41. More specifically, the second antigen-binding site additionally includes a light chain variable region comprising the LCDR1 region of sequence 42, the LCDR2 region of sequence 43, and the LCDR3 region of sequence 44.

[0053] According to a specific embodiment of the present invention, the second antigen-binding site may specifically bind to CD16a, in which case the second antigen-binding site includes a heavy chain variable region comprising an HCDR1 region of sequence 45 or 51, an HCDR2 region of sequence 46 or 52, and an HCDR3 region of sequence 47 or 53. More specifically, the second antigen-binding site additionally includes a light chain variable region comprising an LCDR1 region of sequence 48 or 54, an LCDR2 region of sequence 49 or 55, and an LCDR3 region of sequence 50 or 56.

[0054] The range of antigen-binding proteins of the present invention includes variants having conservative amino acid substitutions in the CDR region. Additionally, the antibody or antigen-binding fragment of the present invention may include variants of the amino acid sequences described in the attached sequence list within a range capable of specifically recognizing the target antigen. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of amino acid sequence residues of the antibody and are made based on the relative similarity of amino acid side chain substituents, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0055] Furthermore, amino acid substitutions in proteins that do not alter the overall activity of the molecule are known in the art (H. Neurath et al., The Proteins, Academic Press, New York, 1979). The most common exchanges are those between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0056] Considering the variant having the aforementioned biologically equivalent activity, the amino acid sequence constituting the antibody of the present invention is interpreted to include a sequence that exhibits substantial identity with the sequence listed in the sequence list. The above substantial identity refers to a sequence that exhibits at least 61% homology, 70% homology according to one specific example, 80% homology according to another specific example, and 90% homology according to yet another specific example when the sequence of the present invention is aligned to correspond as much as possible with any other sequence and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods and algorithms for sequence comparison are disclosed in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31, etc.

[0057] According to a specific embodiment of the present invention, the first antigen-binding region is a Fab (fragment antigen-binding region) or a ScFv (single-chain variable fragment), and the second antigen-binding region is a ScFv.

[0058] According to a specific embodiment of the present invention, the second antigen-binding site has a second light chain variable region (VL2) and a second heavy chain variable region (VH2) positioned in sequence from the N-terminus to the C-terminus.

[0059] According to the present invention, the inventors confirmed that when the variable region within the ScFv constituting the second antigen-binding site is arranged in the order of VL-VH from the N-terminus to the C-terminus, it exhibits an increase in expression efficiency of up to 8 times compared to the opposite arrangement of VH-VL.

[0060] According to a specific embodiment of the present invention, the multispecific antigen-binding protein has a cytokine additionally bound to its C-terminus. More specifically, the cytokine is IL(interleukin)-15. More specifically, the IL-15 is an IL-15 substituted with one or more amino acid residues selected from the group consisting of residues 46 and 49 of sequence 65 of the Sequence List, more specifically an IL-15 including an E46G or V49R substitution, and most specifically an IL-15 including an E46G and V49R substitution.

[0061] According to a specific embodiment of the present invention, the cytokine is coupled to the C-terminus of the multispecific antigen-binding protein by a peptide linker represented by the following general formula 1:

[0062] General formula 1

[0063] X1(EAAAK) n X2

[0064] In the above general formula, X1 and X2 are each independently alanine (A) or not, and n is an integer from 1 to 3.

[0065] More specifically, X1 and X2 are alanine (A) and n is 2.

[0066] According to a specific embodiment of the present invention, the nucleic acid molecule of the present invention described above is an mRNA molecule. According to a specific embodiment of the present invention, the nucleic acid molecule used in the present invention may be an mRNA molecule, and more specifically, may be an IVT (in vitro transcribed) mRNA. When mRNA is used as the nucleic acid molecule of the present invention, various modifications may be applied to improve the expression (translation) efficiency of the antibody or antigen-binding fragment of the present invention, such as, for example, a change in the poly(A) tail length or a substitution of some adenine bases; a modification of the 5' cap; or the application of one or more modified nucleosides.

[0067] More specifically, all or part of the uracil (U) in the mRNA molecule is substituted with modified U represented by the following chemical formula 1:

[0068] Chemical formula 1

[0069]

[0070] In the above chemical formula, R1 and R2 are each independently hydrogen, C1-C3 alkyl, or C1-C3 alkoxy, and X and A are carbon or nitrogen and are different from each other. represents a single bond or a double bond.

[0071] In this specification, the term “alkyl” means a straight-chain or branched saturated hydrocarbon group, including, for example, methyl, ethyl, propyl, isopropyl, etc. C1-C3 alkyl means an alkyl group having alkyl units having 1 to 3 carbon atoms, and when C1-C3 alkyl is substituted, the number of carbon atoms of the substituent is not included.

[0072] In this specification, the term “alkoxy” refers to a radical formed by the removal of hydrogen from an alcohol, and in the case where a C1-C3 alkoxy is substituted, the number of carbon atoms of the substituent is not included.

[0073] According to the octet rule, if X or A is nitrogen, the bond in which X or A participates, respectively It is obvious that is a single bond.

[0074] According to a specific embodiment of the present invention, in the above formula, X is nitrogen, A is carbon, and R1 and R2 are hydrogen. The compound of Formula 1 in which X is nitrogen, A is carbon, and R1 and R2 are hydrogen is pseudouridine.

[0075] According to one embodiment of the present invention, in the above formula, X is nitrogen, A is carbon, R1 is C1 alkyl (methyl), and R2 is hydrogen. The compound of Formula 1, in which X is nitrogen, A is carbon, R1 is methyl, and R2 is hydrogen, is N1-methyl-pseudouridine.

[0076] According to one embodiment of the present invention, in the above formula, X is carbon, A is nitrogen, R1 is a C1 alkoxy (methoxy), and R2 is hydrogen. The compound of Formula 1, in which X is carbon, A is nitrogen, R1 is methoxy, and R2 is hydrogen, is 5-methoxyuridine.

[0077]

[0078] According to a specific embodiment of the present invention, the RNA molecule of the present invention may be an in vitro transcribed (IVT) mRNA molecule. In this specification, the term “IVT mRNA” refers to mRNA that is transcribed in vitro in a DNA-dependent manner. Template DNA may be linearized with a suitable restriction enzyme prior to in vitro transcription, or it may be synthesized in a linearized form without the reaction of a restriction enzyme. Reagents used for in vitro transcription of RNA may include, typically, bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); nucleoside triphosphates (NTPs) for four bases (adenosine triphosphate, guanosine triphosphate, cytidine triphosphate, and uridine triphosphate) and optionally cap analogs; modified nucleosides; RNase inhibitors, etc.

[0079] According to another aspect of the present invention, the present invention provides a multispecific antigen-binding protein expressed using the nucleic acid molecule of the present invention described above.

[0080] According to another aspect of the present invention, the present invention provides a gene delivery vehicle comprising a nucleic acid molecule of the present invention as described above.

[0081]

[0082] Since the multispecific antigen-binding protein-encoding nucleic acid molecules used in the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0083] In this specification, the term “gene carrier” refers to a medium for introducing and expressing a desired target gene in target cells. In this specification, the term “gene delivery” refers to the transport of a gene into a cell and has the same meaning as gene transduction. At the tissue and cellular level, since “gene delivery” has the same meaning as gene spread, the gene carrier may be described as a gene infiltration system and a gene spread system.

[0084] In this specification, the term “to express” means that a gene becomes replicable as an extrachromosomal factor within a subject’s cell by artificially introducing it using a gene carrier to cause the subject to express an exogenous gene or to increase the natural expression level of an endogenous gene. Accordingly, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.”

[0085] The gene delivery vehicle of the present invention may be included in the form of an expression cassette, which is a polynucleotide structure containing all elements necessary for the self-expression of the gene to be introduced. The expression cassette typically includes an expression regulatory sequence, a transcription termination signal, a ribosome binding site, and a translation termination signal operably linked to the gene. The expression cassette may be in the form of a self-replicating expression vector.

[0086] The gene delivery system used in the present invention may be any gene delivery system used for conventional gene insertion, and includes, for example, plasmids, adenoviruses, adeno-associated viruses (AAV), retroviruses, lentiviruses, herpes simplex virus, vaccinia virus, liposomes, and niosomes, but is not limited thereto.

[0087] According to another aspect of the present invention, the present invention provides a cell into which the gene delivery vehicle of the present invention described above has been introduced.

[0088] Since the nucleic acid molecules used in the present invention and the gene delivery vehicles containing them have already been described above, their description is omitted to avoid excessive duplication.

[0089] The nucleic acid molecule of the present invention maintains high structural stability and protein translation efficiency within various host cells, so it can be usefully utilized not only as a gene therapy agent delivering a bispecific antibody, which is a therapeutic protein, but also for the recombinant production of a bispecific antibody, which is a target protein. Accordingly, the cell into which the gene delivery vehicle of the present invention is introduced can be used without limitation as well as various prokaryotic cells (e.g., E. coli), plant cells (e.g., Nicotiana benthamiana cells), mammalian cells (e.g., CHO cells), and insect cells (e.g., sf-9 cells) that can be used for the recombinant production of a target protein, in addition to human cells.

[0090] According to another aspect of the present invention, the present invention provides a composition for enhancing the production efficiency of a multispecific antigen-binding protein comprising the nucleic acid molecule of the present invention described above as an active ingredient.

[0091] According to another aspect of the present invention, the present invention provides a method for enhancing the production efficiency of a multispecific antigen-binding protein, comprising the step of introducing a nucleic acid molecule of the present invention described above into a host cell.

[0092] Since the nucleic acid molecule of the present invention and the multispecific antigen-binding protein expressed therethrough have already been described above, a description thereof is omitted to avoid excessive duplication.

[0093] In this specification, the term “enhanced production efficiency of protein” means that the translation efficiency of the target protein, i.e., the multispecific antigen-binding protein encoded by the nucleic acid molecule of the present invention, is significantly increased in vivo or in vitro to a measurable degree, and may mean, for example, a case where the unique multispecific antigen-binding protein structure of the present invention described above is not present, for example, a case where the variable region within the ScFv constituting the second antigen-binding site is arranged in the order of VH-VL from the N-terminus to the C-terminus, and the efficiency is increased by at least 50%, specifically at least 100%.

[0094]

[0095] The features and advantages of the present invention are summarized as follows:

[0096] (a) The present invention provides a nucleic acid molecule encoding a multispecific antigen-binding protein, which has significantly improved expression efficiency in vivo.

[0097] (b) The multispecific antigen-binding protein of the present invention, specifically a bispecific antibody that recognizes a tumor antigen or a viral antigen and a natural killer cell-specific activating receptor, has an expression efficiency of up to 8 times by having the light chain variable region (VL) and the heavy chain variable region (VH) of the natural killer cell-specific activating receptor recognition portion arranged sequentially from the N-terminus to the C-terminus.

[0098] (c) Accordingly, the present invention provides an optimal mRNA structure capable of most efficiently expressing a bispecific antibody in vitro and in vivo, thereby being usefully utilized as an excellent mRNA therapeutic agent that not only enables the recombinant mass production of a target protein but also stably and continuously produces a therapeutically effective amount of the target protein in the body of a patient.

[0099]

[0100] Figure 1 is a schematic diagram showing the structure of a tumor antigen and NK cell antigen-recognizing dual antibody produced in the present invention, wherein the dark gray oval represents a variable region that recognizes the tumor antigen, the light gray oval represents a variable region that recognizes the NK cell antigen, and the dark gray circular represents an IL-15 variant (IL15v).

[0101] Figure 2 shows the structure of a linker that binds IL15v to the C-terminus of the Fc region in a scFv2-Fc-IL15v bispecific antibody that recognizes CD20 and NKp46 (Figure 2a), and the results of SDS-PAGE and Western blotting analysis to confirm whether aggregation of the bispecific antibody protein occurs accordingly (Figure 2b), respectively.

[0102] Figure 3 shows the results of a comparative evaluation of the NK cell-mediated Ramos cytotoxicity efficacy of IPH6501 antibody and scFv2-Fc and scFv2-Fc-L-IL15v, which are two forms of bispecific antibodies recognizing CD20 and NKp46.

[0103] Figure 4 is a figure showing the results of evaluating the NK cell-mediated cytotoxicity of scFv2-Fc-L-IL15v, a dual antibody recognizing CD20 and NKp46, on rituximab-resistant raji and daudi cells.

[0104]

[0105] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0106]

[0107] Examples

[0108] Example 1: Evaluation of protein expression efficiency according to the arrangement of variable regions in scFv

[0109] We intended to investigate the protein expression efficiency according to the VH and VL sequences within the scFv domain for NK cell antigens in various NK engager bispecific antibody structures containing antibody variable regions for tumor antigens human CD20 and human GPC-3, human hepatitis B virus surface antigen PreS1; and antigens expressed in natural killer cells human NKp46 and human CD16a. To this end, we synthesized tumor antigens or viral surface antigens having structures of ScFv-Fc-ScFv, ScFv2-Fc-IL15v, ScFv2-Fc, Fab-Fc-ScFv-IL15v, and Fab-Fc-ScFv; and bispecific antibodies targeting NK cell antigens (Fig. 1), and the amino acid sequences of each heavy chain and light chain variable region constituting the antigen recognition site of these bispecific antibodies are shown in Table 1 below.

[0110]

[0111] The coded optimized nucleic acid sequence was synthesized at Bionia Co., Ltd. (Daejeon). Using the synthesized gene as a template, the gene was amplified by PCR using pfu polymerase (Solgent, Cat# SPX16-R500). The obtained PCR product and the pCEP4 vector (Invitrogen, cat# V04450) were converted into a circular vector using an In-fusion cloning kit (Takara, cat# 639649) and then transformed into DH5alpha (RBC, cat# RH619). Colonies were cultured, and plasmid DNA was extracted using the miniprep method. Then, clones with the same nucleotide sequence as the bispecific antibody coding region were selected through DNA sequencing analysis.

[0112] Plasmid DNA was isolated and purified using the maxi-prep kit (Qiagen, cat# 12362) and then produced by transient expression in an Expi293 cell expression system. For expression, the cell culture supernatant obtained by centrifuging Expi293 cells after 6 days of culture was filtered using a 0.22 μm filter, and the antibody protein was purified from the filtrate using protein A (Repligen, cat# CA-PRI-0025).

[0113] As a result of measuring the production expression of bispecific antibodies of various structures, the tumor antigen recognition portion or virus surface antigen recognition portion (1) of the N-terminus of the bispecific antibody st Regardless of the structure and sequence of scFv or Fab), the NK cell antigen recognition site (2 nd When the variable region within scFv) is arranged in the order of VL-VH (“LH”) from the N-terminal to the C-terminal, production efficiency is increased by up to 8 times compared to the VH-VL (“HL”) arrangement.

[0114] Structure 1 st scFv or Fab2 nd scFv productivity antibody sequence antibody sequence mg / L multiple difference (2 ndscFv: LH / HL)scFv-Fc-scFv-CD20LH-CD16a-1HL4.24.7LHLH19.6scFv2-Fc-IL15v-CD20LH-NKp46HL72.2HLLH15.5scFv2-Fc-CD20LH-CD16a-2HL2.14.2HLLH9Fab-Fc-scFv-IL15v-CD20Fab-NKp46HL6.12.2LH13.3Fab-Fc-scFv-GPC3-1Fab-CD16a-1HL0.58LH4.3scFv2-Fc-IL15v-PreS1LH-NKp46HL1.43.3HLLH4.6

[0115] Example 2: Evaluation of protein aggregation based on the linker structure connecting the Fc region and IL-15

[0116] The inventors discovered that when IL-15 is bound to the bispecific antibody of the present invention, the linker structure connecting the IL-15 molecule and the bispecific antibody also affects whether the produced bispecific antibody aggregates, and sought to find an optimal linker structure. To this end, the variable region sequence of obinutuzumab, an anti-CD20 antibody, was used as an exemplary tumor antigen recognition site, and the variable region sequence of a humanized NKp46 antibody was used as an exemplary NK cell antigen recognition site. This was obtained by constructing hybridoma cells from mouse plasma cells obtained by immunizing mice with human NKp46 and then humanizing the antibody sequence obtained through screening. The code-optimized nucleic acid sequence encoding the bispecific antibody, in which the above antibody protein is linked with an IL-15 mutant (hereinafter IL15v) containing a human constant region and an E46G / V49R substitution, was synthesized at Bionia Co., Ltd. (Daejeon). Four types of bispecific antibody genes were synthesized using (G4S)3, G4S, PAPAP, and A(EAAAK)2A (hereinafter L, L5, L6, and L7, respectively) as linkers connecting the C-terminus of Fc and the N-terminus of IL15v (Fig. 2a), and their amino acid sequences are shown in Table 3 below. The construction of the expression vector and the production of the antibody protein were performed in the same manner as in Example 1.

[0117]

[0118] SDS-PAGE and Western blotting were performed to analyze the molecular weight of the purified proteins and confirm the formation of high molecular weight (HMW) proteins. First, the concentration of each antibody was determined by measuring the absorbance at a wavelength of 280 nm using the extinction coefficient of the antibody proteins. Each protein was mixed with 4 x sample buffer (Invitrogen, cat# B0007), reacted at 95°C for 5 minutes, and then subjected to electrophoresis on an SDS-PAGE gel. After the electrophoresis was complete, the gel was separated and transferred to a PVDF membrane. It was then treated with blocking buffer (TBS buffer supplemented with 0.05% w / v Tween20 and 5% v / v skim milk) and reacted for 1 hour, followed by treatment with an anti-human Fc antibody labeled with HRP (horseradish peroxidase). Chemiluminescence was measured using an iBright™ FL1500 Imaging System (Thermo, cat# A44115).

[0119] As a result of performing SDS-PAGE and Western blotting under non-reducing conditions, it was confirmed that all four types of bispecific antibodies were expressed as homodimers of approximately 180 kDa. In addition, it was confirmed that the HMW band observed in the three types of bispecific antibodies containing L, L5, and L6 linkers was reduced in the bispecific antibody containing the L7 linker (Fig. 2b).

[0120]

[0121] Example 3: Measurement of Antigen-Antibody Affinity

[0122] The binding affinity of each bispecific antibody produced in this invention to human CD20, human NKp46 (Acro biosystems, NC1-H52H4), human IL-15Rα (Acro biosystems, ILA-H5253), and human IL-2Rβ (Sino biologicals, 10696-H08B) was measured using an OCTET RH16 system (Sartorius). To measure the binding affinity to CD20, amino acid residues 163–187 containing the obinutuzumab binding epitope within the extracellular domain of human CD20 expressed on the surface of B cells were recombinantly expressed in animal cells and then purified.

[0123] The surface of the AHC (Sartorius, 18-5060) sensor was hydrated with 10 x pharmacokinetic buffer (Sartorius, 18-1105) or 1 x pharmacokinetic buffer diluted with 1 x PBS, and then immersed in the pharmacokinetic buffer for 60 seconds to establish a baseline. The sensor was captured by immersing it in a bispecific antibody protein diluted to a concentration of 10 nM in the said buffer for 5 minutes, followed by immersion in the pharmacokinetic buffer for 60 seconds, and then reacted with target proteins of different concentrations for 5 minutes. Subsequently, the sensor was reacted again in the pharmacokinetic buffer for 10 minutes to dissociate it.

[0124] The association constant (Kon), dissociation constant (Kdis), and equilibrium dissodication constant (KD) for the antibodies were determined using OCTET Data Analysis (SW version: 13.0.3.52) software. The results of the binding affinity analysis for human CD20, human NKp46, human IL-15Rα, and human IL-2Rβ are shown in Table 4 below.

[0125] CD20IL15Rα antibody KD (M)ka (1 / Ms)kdis (1 / s)FullR^2 antibody KD (M)scFv2-Fc-L-IL15v7.454E-108.E+056.096 E-040.9698scFv2-Fc-L-IL15v unconjugated scFv2-Fc-L5-IL15v3.016E-107.E+051.976 E-040.9857scFv2-Fc-L5-IL15v unconjugated scFv2-Fc-L6-IL15v3.984E-107.E+053.E-040.9771scFv2-Fc-L6-IL15v unconjugated scFv2-Fc-L7-IL15v6.339E-107.E+054.E-040.9708scFv2-Fc-L7-IL15v unconjugated NKp46IL15Rβ antibody KD (M)ka (1 / Ms)kdis (1 / s)FullR^2 antibody KD (M)Full R^2scFv2-Fc-L-IL15v4.539E-104.E+052.E-040.9969scFv2-Fc-L-IL15v3.795E-7*0.996 5scFv2-Fc-L5-IL15v6.662E-103.E+052.E-040.9924scFv2-Fc-L5-IL15v1.073E-6*0.996 9scFv2-Fc-L6-IL15v3.807E-103.E+051.E-040.9923scFv2-Fc-L6-IL15v5.898E-7*0.982 7scFv2-Fc-L7-IL15v3.95E-103.E+051.E-040.9987scFv2-Fc-L7-IL15v1.956E-7*0.9976

[0126] Steady-state affinity analysis is used due to fast kinetics.

[0127]

[0128] Example 4: NK cell-mediated Ramos cytotoxicity

[0129] To confirm the NK cell-mediated cancer cell death effect by an NK cell engager bispecific antibody that binds to CD20 and NKp46, ADCC efficacy was evaluated using the PBMC ADCC Bioassay Kit 5 x (Ramos) kit (Promega, CS3055A40). The kit provides Ramos cells engineered to express a luciferase domain, PBMCs, and a substrate along with the luciferase domain. When NK cells kill cancer cells by the bispecific antibody of the present invention, the luciferase domain inside the cancer cell is secreted, binds to another luciferase domain to become an activated form, and reacts with the substrate to emit light.

[0130] Ramos cells and PBMCs were cultured in a CO2 incubator for one day according to the manufacturer's instructions, then washed 1-2 times each using assay buffer (prepared from the composition in the kit). Afterward, 5,000 Ramos cells were placed in a 96-well multi-plate (Greiner, 650207) at a volume of 25 μL per well, followed by the sequential addition of 25 μL of sequentially diluted bispecific antibody and 25 μL of PBMC. Additionally, digitonin (Sigma, D141) was added as a positive control and assay buffer only as a negative control, and the mixture was incubated in a CO2 incubator for 48 hours. 70 μL of a solution containing LgBit protein and HiBiT Extracellular substrate mixed in HiBiT Extracellular buffer was added to the wells where the reaction had ended, and after incubation for 10 minutes, luminescence was measured using a Varioskan LUX multimode microplate reader (Thermo, VLBLATD0).

[0131] As a result, as shown in Figure 3, the CD20 x NKp46 dual antibody (ScFv2-Fc-L-IL15v) of the present invention exhibited high apoptotic activity compared to rituximab and IPH6501.

[0132]

[0133] Example 5: Evaluation of apoptotic effect on rituximab-resistant cell lines

[0134] Cell culture and production of rituximab-resistant cell lines

[0135] Raji and daudi cells were cultured in RPMI 1640 medium (Welgene, LM011-51) supplemented with 10% FBS (fetal bovine serum, Gibco, 10082-147) and 1% penicillin-streptomycin (Gibco, 15140122) under conditions of 5% CO2 and 37°C. For rituximab-resistant raji and daudi cells, rituximab (Roche, Mabthera 100 mg) was treated in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at concentrations increasing twofold from 0.125 μg / mL to 128 μg / mL. After treatment with rituximab, the culture medium was replaced and rituximab was administered again after recovery for 4-5 days. After all treatments up to 128 μg / mL were completed, rituximab 128 μg / mL was administered one more time to establish rituximab-resistant cells.

[0136]

[0137] NK cell isolation and culture

[0138] Blood from the LRS (leukoreduction system) chamber, derived from the donor platelet collection process, was centrifuged at 2,500 rpm for 30 minutes using a Ficoll Paque Plus (Cytiva, 17-1440-02) to separate the buffy coat, and this process was repeated once to isolate peripheral blood monocytes. The isolated peripheral blood monocytes were purified into NK cells using a negative selection method with a human NK cell isolation kit (Miltenyi Biotec, 130-092-657) and an LS isolation column (Miltenyi Biotec, 130-042-401). For this purpose, the cell count of peripheral blood monocytes was measured using a hematocytometer, and 1 x 10⁶ cells were found per 400 μl of PBS (phosphate-buffered saline) buffer containing 2% FBS. 8 After diluting to ensure the presence of cells, 100 μL of the NK cell biotin-antibody cocktail (Human NK cell Isolation Kit) was added and incubated at 4°C for 30 minutes. Subsequently, 300 μL of PBS buffer containing 2% FBS was added, followed by 200 μL of the NK cell microbead cocktail (Human NK cell Isolation Kit), and incubated at 4°C for 30 minutes. After the reaction was complete, the peripheral blood monocytes were placed in an LS column along with PBS buffer containing 2% FBS to isolate the NK cells, and the purified NK cells were cultured overnight at 37°C under 5% CO2 in RPMI 1640 medium containing 10% FBS and 1% penicillin-streptomycin.

[0139]

[0140] NK cell activation

[0141] Raji, Daudi, and rituximab-resistant Raji and Daudi cells were prepared as target cells and treated with the CD20 x NKp46 dual antibody of the present invention, rituximab, and IPH6501 antibody at a concentration of 0.02 or 0.004 nM. Then, NK cells and target cells were mixed in a 5:1 ratio in a 24-well plate and cultured at 37°C for 6 days.

[0142]

[0143] Flow cytometry

[0144] After culturing NK and target cells for 6 days, antibodies including APC anti-human CD56 (NCAM) (Biolegend, 362504), FITC anti-human CD3 (Biolegend, 317306), Alexa Fluor® 700 anti-human CD8 (Biolegend, 344724), and PerCP / Cyanine 5.5 anti-human CD19 (Biolegend, 302230) were added, and the cells were incubated at 4°C for 20 minutes. After the reaction was complete, the cells were washed with PBS buffer containing 2% FBS and analyzed using a BD FACS Celesta flow cytometer (BD Biosciences, 660344).

[0145] As a result of the analysis, the CD20 x NKp46 dual antibody (ScFv2-Fc-L-IL15v) of the present invention significantly reduced the survival rate of rituximab-resistant raji and daudi cells compared to rituximab as well as IPH6501 antibody (Fig. 4).

[0146]

[0147] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Nucleic acid molecules encoding multiple specific antigen-binding proteins, including the following: (1) A first antigen-binding site comprising a first heavy chain variable region (VH1) and a first light chain variable region (VL1); (2) A second antigen-binding site comprising a second heavy chain variable region (VH2) and a second light chain variable region (VL2); and (3) An Fc domain located between (1) and (2) above or coupled to the C-terminus of (2) above.

2. A nucleic acid molecule according to claim 1, characterized in that the first antigen-binding site specifically recognizes a target antigen, and the second antigen-binding site specifically recognizes a natural killer cell-specific activation receptor.

3. A nucleic acid molecule according to claim 2, characterized in that the target antigen is a tumor antigen.

4. A nucleic acid molecule according to claim 3, characterized in that the tumor antigen is selected from the group consisting of CD19, CD20, GPC-3, HER-2, CD5, BCMA, and CD123.

5. A nucleic acid molecule according to claim 2, characterized in that the natural killer cell-specific activation receptor is selected from the group consisting of NKp46, NKp44, NKp30, CD16a, DNAM-1, 2B4, and NKG2D.

6. A nucleic acid molecule according to claim 2, characterized in that the first antigen-binding region is a Fab (fragment antigen-binding region) or ScFv (single-chain variable fragment), and the second antigen-binding region is ScFv.

7. A nucleic acid molecule according to claim 6, wherein the second antigen-binding site is characterized in that the second light chain variable region (VL2) and the second heavy chain variable region (VH2) are positioned in order from the N-terminus to the C-terminus.

8. The nucleic acid molecule according to claim 1, wherein the multispecific antigen-binding protein is characterized by having a cytokine additionally bound to the C-terminus.

9. A nucleic acid molecule according to claim 8, wherein the cytokine is IL (interleukin)-15 substituted with one or more amino acid residues selected from the group consisting of residues 46 and 49 of sequence 65 of the sequence list.

10. The nucleic acid molecule according to claim 8, wherein the cytokine is coupled to the C-terminus of the multispecific antigen-binding protein by a peptide linker represented by the following general formula 1: General formula 1 X1(EAAAK) n X2 In the above general formula, X1 and X2 are each independently alanine (A) or not, and n is an integer from 1 to 3.

11. A nucleic acid molecule according to claim 1, characterized in that the nucleic acid molecule is an mRNA molecule.

12. A nucleic acid molecule according to claim 11, characterized in that all or part of the uracil (U) in the mRNA molecule is substituted with a modified U represented by the following chemical formula 1: Chemical formula 1 In the above chemical formula, R1 and R2 are each independently hydrogen, C1-C3 alkyl, or C1-C3 alkoxy, and X and A are carbon or nitrogen and are different from each other. represents a single bond or a double bond.

13. A multispecific antigen-binding protein expressed using a nucleic acid molecule of any one of claims 1 to 12.

14. A gene delivery vehicle comprising a nucleic acid molecule according to any one of claims 1 to 12.

15. A cell into which the gene carrier of claim 14 has been introduced.

16. A composition for enhancing the production efficiency of a multispecific antigen-binding protein comprising a nucleic acid molecule of any one of claims 1 to 12 as an active ingredient.

17. A method for enhancing the production efficiency of a multispecific antigen-binding protein, comprising the step of introducing a nucleic acid molecule of any one of claims 1 to 12 into a cell.

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