Immunoglobulin fc-fusion protein

Abstract

The present invention relates to an immunoglobulin Fc-fusion protein. Specifically, the fusion protein according to the present invention relates to an immunoglobulin Fc-fusion protein which has heterodimers obtained by reciprocal substitution (swapping mutations) from Ig A and Ig G as the basic configuration, and in which additional mutations are carried out to increase stability and maintain or enhance the characteristics of Ig G and Ig A such that the immunoglobulin Fc-fusion protein has an anti-inflammatory effect. Specifically, the immunoglobulin Fc-fusion protein comprises a first Fc domain and a second Fc domain, wherein at least one selected from the group consisting of the first Fc domain and the second Fc domain includes at least one amino acid modification, and the first Fc domain includes a reciprocal mutation of a portion of the Fc amino acid sequence of IgA with the corresponding amino acid of IgG, and the second Fc domain includes a mutual substitution of a portion of the Fc amino acid sequence of IgG with the corresponding amino acid of IgA.

Description

Immunoglobulin FC-fusion protein

[0001] The present invention relates to an immunoglobulin Fc-fusion protein.

[0002] In modern medicine, the regulation of immune responses plays a crucial role in the treatment of various diseases. In particular, antibody-based therapies are widely used to treat various inflammatory and immune-related diseases, and active research is underway to maximize the efficacy and efficiency of these antibody treatments.

[0003] IgA (Immunoglobulin A, abbreviated as Immunoglobulin A or Immunoglobulin A, as used throughout the description of the present invention) is an important component of humoral immunity, playing a particularly important role in mucosal immunity. For instance, IgA can regulate immune responses in the body by binding to Fcα receptors (FcαRI). As such signaling, when IgA binds to FcαRI, there are ITAM (Immunoreceptor Tyrosine-based Activation Motif) and ITAMi (Immunoreceptor Tyrosine-based Inhibitory Motif). Among these, the present invention inhibits ITAM and induces only ITAMi to provide an anti-inflammatory function.

[0004] In other words, when IgA binds to FcαRI, if two IgA molecules forming an immune complex simultaneously bind to two Fcα receptors of the FcαRI receptor, cross-linking of the receptor occurs. This cross-linking activates ITAM linked to FcαRI, transmitting signals that induce an inflammatory response, which in turn induces inflammatory responses such as the release of inflammatory cytokines. Therefore, by modifying IgA to allow monomeric IgA to bind to FcαRI without forming an immune complex, the cross-linking of the receptor is blocked. Consequently, the activation of ITAM by the FcαRI receptor is suppressed, and instead, an inhibitory signal is induced through ITAMi, thereby suppressing the release of inflammatory cytokines and preventing excessive activation of immune cells, which can provide an anti-inflammatory effect.

[0005] However, IgA has a relatively short half-life because it is rapidly eliminated from the bloodstream—for example, the half-life of IgA is about 5 to 6 days, which is very short compared to other immunoglobulins (e.g., the half-life of IgG is about 21 days) (IgG is an abbreviation for Immunoglobulin G, Immunoglobulin G, or Immunoglobulin G, and is referred to as such throughout the description of the invention)—and this short half-life means that when used as a therapeutic agent, it does not remain in the body for a long time, so its effect is not sustained and it must be administered continuously and frequently. This leads to problems that place a burden on the patient and increase treatment costs.

[0006] Furthermore, due to its structural characteristics, IgA has the problem of being difficult to purify efficiently using conventional antibody purification methods (e.g., chromatography using Protein A or Protein G). That is, Protein A or Protein G has a high affinity for the Fc domain of IgG but does not bind to the Fc domain of IgA, so they are not suitable for purifying IgA. Consequently, to purify IgA in large quantities, expensive resins must be used or complex additional processes are required, which lowers purification efficiency and increases production costs.

[0007] As such, IgA had limitations in being utilized as a therapeutic agent due to its relatively short half-life and the difficulty of purification. To address these issues, the inventors arrived at the present invention, an immunoglobulin Fc fusion protein fused with IgG.

[0008] In other words, by introducing the binding characteristics of IgG's FcRn to IgA, the short half-life of IgA is compensated for, and due to its high affinity with Protein A / Protein G, it can be utilized in the purification process, thereby improving purification efficiency.

[0009] In other words, the aim is to improve the productivity of Ig A-based therapeutics by introducing IgG characteristics while maintaining the FcαRI binding properties of the Ig A Fc domain, thereby extending the half-life and facilitating purification while preserving the functional advantages of Ig A.

[0010] To explain more specifically, first, since the Fc domain of IgA binds to FcαRI to perform specific immune regulatory functions, this hybrid Fc domain must maintain this binding ability to enable the regulation of inflammation and immune responses, which are the original functions of IgA.

[0011] Second, the Fc domain of IgG binds to FcRn to extend the antibody's half-life; the hybrid Fc domain introduces the FcRn binding characteristics of IgG to extend the antibody's retention period in the body and provide a longer therapeutic effect.

[0012] Third, the Fc domain of IgG has a high affinity for Protein A, so it can be used efficiently in the purification process. Therefore, if Protein A binding characteristics are introduced through the hybrid Fc domain, cost reduction and efficiency improvement can be expected in the antibody production and purification processes.

[0013] Finally, since the Fc domain of IgG can bind to FcγR to induce ADCC (antibody-dependent cytotoxicity) and this may be inappropriate when aiming for the inhibition of inflammatory responses and anti-inflammatory effects, the hybrid Fc domain is designed not to bind to FcγR to maximize the anti-inflammatory function.

[0014] Hereinafter, the present invention will be described in detail against the background described above.

[0015] As described above, one objective is to provide an immunoglobulin Fc fusion protein for the treatment or prevention of inflammation-related diseases.

[0016] In addition, one objective is to enable immunoglobulin Fc fusion proteins to possess enhanced anti-inflammatory effects compared to the IgG wild type, and to demonstrate the potential for developing various anti-inflammatory protein drugs using this as a platform.

[0017] In addition, one task is to make the immunoglobulin Fc fusion protein exhibit an anti-inflammatory effect by binding to FcαRI, which binds to immunoglobulin A.

[0018] In addition, one objective is to demonstrate that the disadvantage of the reduced half-life of the immunoglobulin Fc fusion protein, which cannot bind to immunoglobulin A, is compensated for by improving the binding affinity of FcRn.

[0019] Furthermore, it demonstrates that the immunoglobulin Fc fusion protein does not bind to FcγRI. This sets forth a goal to significantly reduce cytotoxicity caused by the induction of antibody-dependent cytotoxicity (ADCC) resulting from the binding of IgG to FcγRI.

[0020] In addition, immunoglobulin Fc fusion proteins provide excellent productivity. In particular, one goal is to increase the binding affinity of Protein A, making purification easier and reducing costs.

[0021] In addition, one objective is to provide a novel protein drug for anti-inflammatory or metabolism-related diseases (non-alcoholic fatty liver, rheumatoid arthritis, obesity, diabetes, etc.) and chronic inflammatory diseases, which is used in combination with a pharmaceutical composition using an immunoglobulin Fc fusion protein platform, or to provide a pharmaceutical composition containing the same as an active ingredient.

[0022] In addition, one objective is that the immunoglobulin Fc fusion protein platform can be used to manufacture novel protein drugs for anticancer-related diseases in combination with a pharmaceutical composition, or to be used as a pharmaceutical composition containing the same as an active ingredient.

[0023] To achieve the above objective, as one means of the present invention, an immunoglobulin Fc fusion protein with enhanced binding affinity is obtained by undergoing an additional mutation process following mutual substitution mutation (swapping mutation) from sequences derived from IgA and IgG, respectively.

[0024] In addition, based on this, the amino acid sequence of the fusion protein and the nucleic acid sequence coding therefor are provided as one means.

[0025] Furthermore, the present invention may provide a pharmaceutical composition for the prevention or treatment of immune diseases comprising, as an active ingredient, a fusion protein containing a modified immunoglobulin Fc region comprising the above-mentioned fusion protein, or provide a starting point for such a composition. Additionally, the present invention may provide a method for the prevention or treatment of immune diseases comprising the step of administering to an individual a fusion protein containing Fc in the form of an IgA and IgG heteromer and a pharmaceutically acceptable carrier, or provide a starting point therefor.

[0026] As a specific example, an immunoglobulin heteromer Fc-fusion protein is disclosed as one means according to the present invention, comprising a first Fc domain and a second Fc domain, wherein at least one selected from the group consisting of the first Fc domain and the second Fc domain comprises at least one amino acid modification, and wherein the first Fc domain is mutually substituted with a portion of the Fc amino acid sequence of IgA to a corresponding amino acid of IgG, and the second Fc domain is mutually substituted with a portion of the Fc amino acid sequence of IgG to a corresponding amino acid of IgA.

[0027] Additionally, an immunoglobulin heteromer Fc-fusion protein is disclosed, comprising a first Fc domain and a second Fc domain, wherein at least one selected from the group consisting of the first Fc domain and the second Fc domain comprises at least one amino acid modification, wherein the first Fc domain is a sequence derived from the Fc amino acid sequence of IgA and the second Fc domain is a sequence derived from the Fc amino acid sequence of IgG, wherein at least one variant among SW variant, CW variant, or PY variant is applied to at least one selected from the group consisting of the first Fc domain and the second Fc domain.

[0028] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, comprising an LS mutation processed in the second Fc domain.

[0029] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, comprising one that has been treated with N-bond glycosylation.

[0030] Herein, an immunoglobulin heteromer Fc-fusion protein comprising an IgA tailpiece domain is disclosed.

[0031] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, comprising a reduced binding affinity of FcγRI compared to the wild-type human antibody IgG4.

[0032] Herein, an immunoglobulin heteromer Fc-fusion protein comprising binding to Protein A is disclosed.

[0033] Here, the fusion protein has no binding affinity with FcγRI at pH 7.4, or 6.65 x 10⁻⁶. -6 An immunoglobulin heteromer Fc-fusion protein is disclosed, comprising having a dissociation constant (Kd) of less than

[0034] Here, the dissociation constant (Kd) value with enhanced bonding strength is 9.66 x 10⁻⁶ -7 including that which is less than

[0035] Introduces an immunoglobulin heteromer Fc-fusion protein.

[0036] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the N-linked glycosylated one has an enhanced binding affinity to FcαRI compared to the one not N-linked glycosylated.

[0037] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the first Fc domain comprises SEQ ID NO. 11 and the second Fc domain comprises SEQ ID NO. 12.

[0038] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the first Fc domain comprises SEQ ID NO. 13 and the second Fc domain comprises SEQ ID NO. 14. Nucleus

[0039] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the first Fc domain comprises SEQ ID NO. 15 and the second Fc domain comprises SEQ ID NO. 14.

[0040] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the first Fc domain comprises SEQ ID NO. 16 and the second Fc domain comprises SEQ ID NO. 17.

[0041] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the first Fc domain comprises SEQ ID NO. 18 and the second Fc domain comprises SEQ ID NO. 17.

[0042] Herein, an immunoglobulin heteromer Fc-fusion protein is disclosed, wherein the IgA tailpiece domain comprises either SEQ ID NO. 20 or SEQ ID NO. 21.

[0043] However, it is not limited thereto, and may be understood within the scope that a person skilled in the art can easily understand throughout the entire description of the invention.

[0044] One effect is to provide an example of an immunoglobulin heteromer-Fc fusion protein. That is, the immunoglobulin heteromer-Fc fusion protein is designed to target various disease-related proteins in the Fab portion.

[0045] In addition, the above immunoglobulin heteromer-Fc fusion protein has a high protein A binding affinity, showing high values ​​in terms of purity and yield.

[0046] In addition, the above immunoglobulin heteromer-Fc fusion protein has a longer half-life due to FcRn binding compared to wild IgA, and becomes able to bind to FcαRI, which IgG cannot bind to.

[0047] In addition, it does not bind to FcγRI, resulting in low cytotoxicity through the induction of antibody-dependent cytotoxicity (ADCC). Based on this novel antibody protein platform, the potential for its use as a novel protein drug for anti-inflammatory or metabolic diseases (non-alcoholic fatty liver disease, rheumatoid arthritis, obesity, diabetes, etc.) and chronic inflammatory diseases can be observed.

[0048] FIG. 1 is a representative schematic diagram of an immunoglobulin heteromer-Fc fusion protein according to the present invention, including a hinge and a CH2-CH3 domain portion.

[0049] FIG. 2 shows the bands of the PAGE test results for the first to seventh manufacturing examples. The first to seventh manufacturing examples are labeled as Example (1) ~ Example (7).

[0050] FIGS. 3a to 3g are schematic diagrams of the first to seventh manufacturing examples (the basic configuration and description are the same as FIG. 1a).

[0051] FIGS. 4a to 4g, FIGS. 5a to 5b, and FIGS. 6a to 6g are SPR graphs for each of the first to seventh manufacturing examples.

[0052] Figure 7 is a chromatography graph for the first to seventh preparation examples (Example (1) ~ Example (7)) according to pH.

[0053] The various embodiments or examples described in this document are illustrative for the purpose of clearly explaining the technical concept of the present invention and are not intended to limit it to specific embodiments. The technical concept of the present invention includes various modifications, equivalents, and alternatives of each embodiment or example described in this document, as well as embodiments or examples selectively combined from all or part of each embodiment or example.

[0054] All technical and scientific terms used in the present invention, unless otherwise defined, have the meaning generally understood by those skilled in the art to which the present invention pertains.

[0055] In general, the nomenclature and techniques used in connection with cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art. The methods and techniques of this disclosure are generally carried out in accordance with conventional methods well known in the art, and unless otherwise indicated, as described in the various general and more specific references cited and discussed throughout this specification.

[0056] Enzyme reaction and purification techniques are performed according to the manufacturer's specifications, as generally achieved in the art or as described herein. The nomenclature, laboratory procedures, and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical formulation, formulation, and delivery, and patient treatment.

[0057] Singular expressions used in this document may include the meaning of the plural form unless the context otherwise indicates, and this applies likewise to singular expressions described in the claims.

[0058] I. Definition

[0059] The present invention is not limited to specific methods and experimental conditions described as such methods, and conditions may vary. Furthermore, as the scope of the invention is defined by the claims, technical terms used herein should be understood merely for the purpose of describing specific embodiments and are not intended to be limiting.

[0060] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art of the present invention. Any methods and materials similar or identical to those described herein may be used in the practice or testing of the present invention, but specific methods and materials are described herein. All mentioned publications are incorporated herein by reference.

[0061] The “antibody” comprises an immunoglobulin molecule comprising four polypeptide chains linked together by disulfide bonds, two heavy (H) chains and two light (L) chains. Each heavy chain comprises a heavy chain variable region (abbreviated as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions may be further subdivided into hypervariable regions referred to as complementary determining regions (CDR) and may be arranged together with more conserved regions referred to as backbone regions (FR). Each VH and VL consists of three CDRs and four FRs aligned from the N-terminus to the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high-affinity" antibody is at least 10 at its target -9 M, at least 10 -10 M; at least 10 -11 M; or at least 10 as measured by surface plasmon resonance, e.g., BIACORE, or solubility-affinity ELISA -12M It refers to antibodies that have a binding affinity.

[0062] "Heavy chain" or "immunoglobulin heavy chain" comprises an immunoglobulin heavy chain constant region from any organism and, unless otherwise specified, a heavy chain variable domain. Unless otherwise specified, the heavy chain variable domain comprises three heavy chain CDRs and four FR regions. Fragments of the heavy chain comprise CDRs, CDRs and FRs, and combinations thereof. A typical "heavy chain" has the following variable domains (from N-terminus to C-terminus), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional fragments of the heavy chain can specifically recognize antigens (e.g., recognize antigens having KD in the micromolar, nanomolar, or picomolar ranges), that is, can be expressed and secreted from cells, and comprise fragments comprising at least one CDR.

[0063] "Fc protein" includes antibodies, bispecific antibodies, immunoadhesives, and other binding proteins comprising minimal functional portions of the immunoglobulin CH2 and CH3 regions. The "functional portion" relates to the CH2 and CH3 regions capable of binding to an Fc receptor (e.g., FcγR; or FcRn) and / or participating in the activity of the complement. If the CH2 and CH3 regions contain deletions, substitutions, and / or insertions or other modifications that prevent the CH2 and CH3 regions from binding to any Fc receptor and from activating the complement, the CH2 and CH3 regions are not functional.

[0064] "Fc protein" may include modifications of the immunoglobulin domain, the modifications including those affecting one or more effector functions of the binding protein (e.g., modifications affecting FcγR binding, FcRn binding and thus half-life, and / or ADCC or ADCC / CDC activity). These variations include, but are not limited to, the following variations and combinations thereof with respect to the EU numbering of the immunoglobulin constant region: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0065] For example, without restriction, the binding protein is an Fc protein, exhibiting an enhanced serum half-life (compared to the same Fc protein without the listed modification(s)), and having modifications at position 250 (e.g., E or Q); modifications at 250 and 428 (e.g., L or F); modifications at 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at 250 and / or 428; or modifications at 307 or 308 (e.g., 308F, V308F), and 434. In other examples, the variants may include 428L (e.g., M428L) and 434S (e.g., N434S) variants; 428L, 259I (e.g., V259I), and 308F (e.g., V308F) variants; 433K (e.g., H433K) and 434 (e.g., 434Y) variants; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) variants; 250Q and 428L variants (e.g., T250Q and M428L); and 307 and / or 308 variants (e.g., 308F or 308P).

[0066] Here, amino acid residue numbers may follow the Kabat numbering system used in the industry (such as EU index numbers as in Kabat et al., in *Proteins of Immunological Interest 5th Ed.*, US Department of Health and Human Services, NIH Publication No. 91-3242, 1991).

[0067] Here, "effector function" refers to biochemical events arising from the interaction between the antibody Fc domain and the Fc receptor or ligand. Effector function includes, but is not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC).

[0068] “Light chain” comprises an immunoglobulin light chain constant region from any organism, and unless otherwise specified, includes human kappa and lambda light chains. Unless otherwise specified, the light chain variable (VL) domain typically comprises three light chain CDRs and four backbone (FR) regions. Generally, the full-length light chain comprises a VL domain and a light chain constant region comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 from the N-terminus to the C-terminus. Light chains that may be used with the present invention include, for example, those that do not selectively bind to one of the first or second antigens selectively bound by the antigen-binding protein. Suitable light chains include those that can be identified by screening for the most commonly used light chains in an existing antibody library (wet library or in silico), and the light chains do not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Suitable light chains include those that can bind to one or both of the epitopes bound by the antigen-binding region of the antigen-binding protein.

[0069] The “variable domain” comprises an amino acid sequence of an immunoglobulin light chain or heavy chain (modified as desired) comprising the following amino acid regions in a sequence from the N-terminus to the C-terminus (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The “variable domain” comprises an amino acid sequence that can be folded into a standard domain (VH or VL) having a double beta sheet structure, wherein the beta sheets are connected by a disulfide bond between the first beta sheet and the second beta sheet.

[0070] "Amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be present at a specific and defined position. Amino acids include both naturally occurring and synthetic amino acids. In most cases, when recombinantly producing proteins, only naturally occurring amino acids are used.

[0071] "Protein" means at least two covalently bonded amino acids, including proteins, polypeptides, oligopeptides, and peptides. Proteins may consist of naturally occurring amino acids and peptide bonds, or synthetic peptide-mimicking structures, such as analogs like peptoids.

[0072] In the present invention, the term “about” may indicate a customary range of error for each value, as is widely known to those skilled in the art. This may mean ±20%, ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of the numerical value or range mentioned or claimed in one embodiment in the context of the numerical value or range described in the present invention. With respect to the length of a nucleotide or amino acid sequence, “about” may indicate that the nucleic acid or protein is not limited to the number of nucleotides or amino acids described, and may include some nucleotides or amino acids added or removed at both ends within a range that does not impede functional activity.

[0073] Expressions such as "comprising," "having," "having," etc. used in the present invention should be understood as open-ended terms that imply the possibility of including other embodiments in a manner similar to "comprising," unless otherwise stated in the phrase or sentence containing such expressions.

[0074] One embodiment of the present invention described in this invention is understood to include "comprising," "consisting of," and / or "essentially composed of" one embodiment.

[0075] The term "and / or" as used in the present invention may mean any one or more of the items, any combination of the items, or all of the items in the items associated with the term.

[0076] As used in the present invention, the term "amino acid" may refer to all naturally occurring L-α-amino acids. This definition may include norleucine, ornithine, and homocysteine.

[0077] As used in the present invention, the terms "variation" or "amino acid variation" may refer to a substitution, insertion, deletion, or combination thereof of an amino acid sequence compared to a reference (e.g., natural sequence) polypeptide or protein, and the term "variant" may refer to a molecule having some difference in an amino acid sequence compared to a reference polypeptide or protein due to such variation.

[0078] Additionally, the scope of variants of the present invention may include proteins or fragments or derivatives thereof that exhibit the same or similar biological activity, and derivatives that are separately modified during or after translation, for example, by glycosylation, cleavage by protein hydrolysis, linkage to antibody molecules or other cell ligands, etc.

[0079] Here, or throughout the entire invention, the meaning of terms or expressions must be understood regardless of the order of description.

[0080] The structure and effects of the present invention will be explained in more detail below with reference to the drawings and examples. However, these examples are provided for illustrative purposes only to aid in understanding the present invention, and the scope and range of the present invention are not limited by the following examples.

[0081] FIG. 1 is a representative schematic diagram of an immunoglobulin heteromer-Fc fusion protein according to the present invention, including a hinge and a CH2-CH3 domain portion.

[0082] FIG. 2 shows the bands of the PAGE test results for the first to seventh manufacturing examples. The first to seventh manufacturing examples are labeled as Example (1) ~ Example (7).

[0083] FIGS. 3a to 3g are schematic diagrams of the first to seventh manufacturing examples (the basic configuration and description are the same as FIG. 1a).

[0084] FIGS. 4a to 4g, FIGS. 5a to 5b, and FIGS. 6a to 6g are SPR graphs for each of the first to seventh manufacturing examples.

[0085] Figure 7 is a chromatography graph for the first to seventh preparation examples (Example (1) ~ Example (7)) according to pH.

[0086] Each component of Fig. 1 consists of N-glycan, IgA1, IgG4, and disulfide bond (no legend).

[0087] It is composed of a dimer, and the two thick horizontal lines in the middle represent disulfide bonds connecting the first Fc domain and the second Fc domain.

[0088] Based on the disulfide bond, a total of four blocks, two on the lower side, represent Fc domains (Fc regions), and in the Fc regions, the two blocks on the left are designated as the first Fc domains and the two blocks on the right as the second Fc domains. Additionally, the first Fc or second Fc domains represent CH2 domains and CH3 domains in order of proximity to the disulfide bond.

[0089] In addition, IgA1 and IgG4 refer to the amino acid sequences of human wild-type IgA1 and IgG4, or amino acid sequences derived therefrom.

[0090] Accordingly, as illustrated in Figure 1, the first Fc domain is based on the amino acid sequence of the Fc region (including the CH2-CH3 domain) derived from IgA1, but the CH3 domain is partially substituted with the amino acid sequence derived from IgG4, and it can be seen that N-glycans are attached (N-glycosylated) to parts of the CH2 and CH3 domains.

[0091] The second Fc domain is based on the amino acid sequence of the Fc region derived from IgG4, but parts of the CH2 and CH3 domains are partially substituted with sequences derived from IgA1, and there are no N-glycans attached.

[0092] Meanwhile, the part connecting the CH2 domain of the Fc region and PD-L1 is a hinge region composed of IgA2 or an amino acid sequence derived therefrom. Unless otherwise noted, the hinges on the left and right are identical sequences.

[0093] This explanation can be substituted with the description of each schematic diagram for the first to seventh preparation examples in FIG. 3a to 3g, and the Fab region, which is the upper part based on the disulfide bond, was represented as hPD-L1 in the first to seventh preparation examples, indicating that N-glycans can be attached (N-glycosylated) to the left and right blocks, respectively. However, here, hPD-L1 was utilized as a so-called "carrier protein."

[0094] Based on this, it is obvious that the contents described on the drawing can be fully understood by a person with ordinary knowledge.

[0095] II. Fusion protein and modified fusion protein components according to the present invention

[0096] 1. Overview

[0097] (1) Design purpose of the fusion protein according to the present invention

[0098] IgA exhibits anti-inflammatory effects when acting as a monomer, suggesting its potential as a protein drug platform for inflammation-related diseases. However, the Fc domain of IgA has limitations in functioning as a platform for protein drugs due to low productivity and purification rates, diverse glycosylation patterns, and a relatively short half-life.

[0099] To address this, the aim is to develop a fusion protein platform that overcomes the aforementioned limitations by performing fusion with IgG while maintaining the characteristics of IgA. Specifically, the developed novel fusion protein platform is intended to enhance production and purification efficiency by increasing binding affinity with Protein A, improve half-life by inducing recycling through increased binding affinity with FcRn, and possess the anti-inflammatory effects of IgA monomers by maintaining the binding with FcαRI, a characteristic of IgA.

[0100] (2) Setting up a candidate pool of immunoglobulin Fc fusion proteins

[0101] The fusion protein for the above purpose was designed as follows.

[0102] Heterodimer (IgG / IgA hybrid) technology for IgG and IgA is applied. Specifically, the aim is to enhance the stability of the fusion protein by improving binding affinity using a heterogeneous Fc domain. To achieve this, for example, a portion of the CH3 domain can be mutually substituted (swapped) between IgA and IgG.

[0103] In other words, it involves mixing certain characteristics of the two antibodies by substituting specific amino acid sequences of IgA with corresponding sequences of IgG, or vice versa, thereby optimizing binding characteristics, stability, and interactions as a heterodimer.

[0104] Furthermore, by applying Knob-into-hole (KIH) technology, it is possible to maximize its stability and increase the yield of the Fc domain.

[0105] In addition, to enhance the binding of the Fc domain within the CH3 domain, we intend to mutually substitute parts from IgA and IgG to improve the binding affinity between heterogeneous Fc domains while maintaining the functions of IgA and IgG.

[0106] In addition, SW / CW / PY mutations are applied to the CH2 domain. For instance, some amino acids in the CH2 domain of the IgA and IgG backbones were substituted with other amino acids.

[0107] In addition, the LS(M428L / N434S) mutation was applied to the CH3 domain, or an N-linked glycosylation and IgA tailpiece domain was included to improve binding affinity with FcRn.

[0108] Below, each item will be explained in detail.

[0109] 2. Heterodimers of IgG and IgA

[0110] 2.1. Intersubstitution Variants (Swapping Variants) for Enhancing Heteromer Stability

[0111] IgG and IgA are antibodies that play important roles in the immune system; IgG neutralizes pathogens in humoral immunity and activates the complement system, while IgA is found in mucous membranes and plays a role in fighting pathogens.

[0112] A heterodimer is formed by combining different protein subunits; in the present invention, it is formed by combining the respective Fc domains of IgG and IgA. Through this, a novel fusion protein having the functions and / or characteristics of IgG and IgA is obtained.

[0113] In particular, mutual substitution mutations or swapping mutations involve substituting specific amino acid sequences between antibodies as described above, thereby optimizing the binding characteristics, stability, and interactions between immune cells between antibodies.

[0114] For example, by combining the characteristics of IgA and IgG, new functions can be added or existing functions can be enhanced (maintenance and improvement of function), and by improving the binding affinity between antibodies, the stability of the heterologous Fc domain can be enhanced to improve persistence and in vivo responsiveness (enhancement of Fc binding), and additionally, the function of ITAMi can be enhanced in relation to FcαRI binding (enhancement of ITAMi function).

[0115] 2.2. Knob-Into-Hole (KIH)

[0116] Knob-into-hole (KIH) technology can be applied to improve the binding strength of the heterodimer Fc domain.

[0117] In other words, by introducing a Knob structure, which is a mutation that creates a protruding structure in the Fc domain of one antibody heavy chain, and a Hole structure, which is a mutation that creates a hole structure in the Fc domain of another antibody heavy chain, they can interlock to increase the binding strength between them.

[0118] At this time, the amino acid residues included in the hydrophobic core that contribute to the formation of homomers between the heavy chain CH3 domains of the immunoglobulin are Leu351, Thr366, Leu368, and Tyr407, and the amino acid numbers of the antibody chains are based on EU numbering (Cunningham, Pflumm et al. 1969).

[0119] This technique involves replacing large hydrophobic amino acid residues with small hydrophobic amino acids in one heavy chain CH3 domain with large hydrophobic amino acids to form a hole structure (Thr366Ser, Leu368Ala, Tyr407Val) and replacing small hydrophobic amino acid residues with large hydrophobic amino acids in the other heavy chain CH3 domain to form a knob structure (Thr366Trp), thereby making the formation of a heterodimeric heavy chain invariant site more favorable than a homodimeric heavy chain invariant site when two mutant pairs, namely CH3A (Thr366Ser, Leu368Ala, Tyr407Val) and CH3B (Thr366Trp), are co-expressed.

[0120] Furthermore, to further stabilize the formation of the heterodimeric heavy chain constant region, an additional mutation is performed with a hole structure in one heavy chain (Tyr394Cys), and an additional mutation is performed on the knob structure of the CH3 domain in another heavy chain (Ser354Cys). When two mutation pairs, namely CH3A (Tyr394Cys) and CH3B (Ser354Cys), are co-expressed, the formation of the heterodimeric heavy chain constant region can be more stable than that of the homodimeric heavy chain constant region.

[0121] 3. SW / CW / PY variants of the CH2 domain

[0122] The binding affinity between the Fc of the two antibodies can be enhanced by substituting specific amino acid positions in the CH2 domains of the IgA1 backbone and the IgG4 backbone, respectively.

[0123] The above specific amino acid positions are to substitute the amino acid sequences of IgG4 296, 297, 298, 299, 300, 301 (EU numbering for C-domain), CH2.84.3, CH2.84.4, CH2.85.4, CH2.85.3, CH2.85.2, CH2.85.1 (IMGT numbering for C-domain) with the amino acid sequences of IgA1.

[0124] In other words, the mutation sites in the CH2 domain and the pre- / post-mutation variants are L296C, C297W / L_CH2.84.3_C, C_CH2.84.4_W, or L296S, C297W / L_CH2.84.3_S, C_CH2.84.4_W variants, and L296P, C297Y / L_CH2.84.3_P, C_CH2.84.4_Y variants in the IgG sequence (EU numbering / IMGT numbering for C-domain).

[0125] 4. LS variant

[0126] Mutations M428L, N434S / M_CH3.107_L, and N_CH3.114_S (EU numbering / IMGT numbering for C-domain) occur, increasing the binding affinity with FcRn. As a result, the antibody's half-life is extended, the duration of antibody action in the body is prolonged, and anti-inflammatory effects can be sustained.

[0127] 5. N-linked Glycosylation and IgA tailpiece

[0128] 5.1. N-linked Glycosylation

[0129] N-bond glycosylation is the attachment of a carbohydrate, composed of several sugar molecules also known as oligosaccharides or glycans, to a nitrogen atom. The properties of N-bond glycans attached to glycoproteins are determined by the protein and the cell in which the protein is expressed.

[0130] Generally, this N-bond glycosylation helps polypeptides (proteins) acquire the appropriate structure to perform their functions and effects. For instance, it reduces or prevents errors, such as misfolding during the polypeptide structuring stage, and increases structural stability.

[0131] This N-bond glycosylation occurs in Asn-X-Ser or Asn-X-Thr and occurs in a specific pattern, and the positions of the N-glycans in the fusion protein according to the invention are 257, 452 (EU-numbering for C-domain).

[0132] 5.2. IgA tailpiece

[0133] The tailpiece can typically be viewed as an 18-amino acid region at the C-terminal end of the IgA heavy chain constant region or Fc region containing cysteine ​​residues essential for polymerization processes such as dimer formation, or an extension thereof.

[0134] In one embodiment, the structure of the present invention may be an IgA-derived tailpiece, or modified / mutated / inactive / cleaved / removed, or the cysteine ​​residue may be modified / mutated / inactive / cleaved / removed, or derived from any subunit of an IgA antibody, or modified / mutated / inactive / cleaved / removed.

[0135] In some embodiments, the Fc region of IgA may be engineered or modified to include enhanced or modified effector functions, such as the induction of antibody-dependent cell cytotoxicity (ADCC) or antibody-dependent cell phagocytosis (ADCP), or increased co-engaging or binding to an Fcα receptor, such as FcαRI. Suitable Fc mutants having (or conferring) these features are well known and described in the art, and any of these may be used.

[0136] As an example of the present invention, the IgA tailpiece sequence positions are 448 to 466 (EU numbering for C-doamin). More specifically, the sequence of the conventionally known IgA tailpiece is 'PTHVNVSVVMAEVDGTCY' (SEQ No. 19 or SEQ_19), but in the case of the present invention, it can be either 'PTHVNVSVVMAEVDGT' (SEQ No. 20 or SEQ_20) without the 'CY' at the end, or 'PTHVNVSVVMAEVDGTSY' (SEQ No. 21 or SEQ_21) in which the 'C' at the end is mutated to 'S'.

[0137] III. Sequence and structure of the fusion protein according to the present invention (schematic diagram)

[0138] The sequence and structure used in the present invention are illustrated in [Table 1] and Figures 2a to 2j below, respectively.

[0139] The number (SEQ No.) and schematic diagram for each sequence are summarized, and the specific amino acid sequence for each sequence is listed in [Table 1] below and the attached list of sequence numbers. Throughout the entire description of the present invention, including [Table 1] below, the notation method for amino acid sequence numbers is as sequence number OO or SEQ_OO. That is, SEQ_1 is the same as sequence number 1.

[0140] Here, for the convenience of explanation, among the two Fc domains of the antibody (referring to the domain containing CH2 and CH3 domains), the Fc domain on the left is referred to as the first Fc domain and the Fc domain on the right is referred to as the second Fc domain based on the schematic diagram. In addition, the sequences of the first Fc and second Fc domains are shown from top to bottom in each preparation example in [Table 1] below.

[0141] For example, in the case of the first manufacturing example, from the perspective of viewing the schematic diagram, the left side is the first Fc domain and the right side is the second Fc domain, and both the first Fc and second Fc domains are based on sequence number 1, and the second manufacturing example can be understood in the same way.

[0142] For example, in the case of the third manufacturing example, from the perspective of viewing the schematic diagram, the left side is the first Fc domain and the right side is the second Fc domain, where the first Fc is according to sequence number 3 and the second Fc is according to sequence number 4.

[0143] For example, if the sequences of the first Fc and second Fc domains are derived from homologous immunoglobulins or consist of the same sequence, as in the first or second preparation example, they may be homodimers; however, if they have different sequences, such as being formed from sequences derived from different subunits as in the third preparation example, they may be heterodimers.

[0144] Classification Sequence Number Specific Sequence 1 Preparation Example SEQ_1 (Sequence Number 1) FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTKHYTNSSQDVTVPSRVPP PPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVHLLPPPSEELALNELVTLTCLARGFSPKDVLVRWLQGSQELPREKYLTWASRQEPSQGTTTFAVTSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTHHHHHH 2nd Preparation Example SEQ_2 (Sequence Number 2)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKV NAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTGSAES KYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK3rd Preparation Example SEQ_3(Sequence No.3)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTKHYTNSSQDVTVPSRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSEQ_4(서열번호 4)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTKHYTNSSQDVTVPSRVPPPPPCCHPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFCGCYSVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPEVHLLPPSQEEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKTISLSLGK제4 제조예SEQ_5(서열번호5)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTTVPSRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDCWGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSEQ_6(서열번호 6)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTTVPSRVPPPPPCCHPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQPYGCYSVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPEVHLLPPSQEEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKTISLSLGK제5 제조예SEQ_7(서열번호7)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTTVPSRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDSWGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSEQ_6(서열번호 6)위와 같음제6 제조예SEQ_8(서열번호 8)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTGSAESKYGPPCPPCPAPEAAGGPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSYSEQ_9(서열번호9)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTGSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPEVHLLPPSQEEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKTISLSLGK제7 제조예SEQ_10(서열번호 10)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTGSAESKYGPPCPPCPAPEAAGGPRLSLHRPALEDLLLGSEANGLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKSEQ_9(서열번호 9)위와 같음제1 핵심예SEQ_11(서열번호11)KHYTNSSQDVTVPSRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSEQ_12(서열번호 12)KHYTNSSQDVTVPSRVPPPPPCCHPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFCGCYSVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPEVHLLPPSQEEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKTISLSLGK제2 핵심예SEQ_13(서열번호 13)TVPSRVPPPPPCCHPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDCWGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSEQ_14(서열번호14)TVPSRVPPPPPCCHPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQPYGCYSVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPEVHLLPPSQEEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSRLTVDKSRWQEGNVFSCSVLHEALHSHYTQKTISLSLGK3rd Key Example SEQ_15(Sequence Number 15)FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVN APYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTGSAESKYGP PCPPCPAPEAAGGPRLSLHRPALEDLLLGSEANGLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKSEQ_14(Sequence No. 14) Same as above 4th Key Example SEQ_16(Sequence No.16)GSAESKYGPPCPPCPAPEAAGGPRLSLHRPALEDLLLGSEANLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLSKSGNTFRPEV YTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKPTHVNVSVVMAEVDGTSYSEQ_17 (SEQ ID NO: 17)GSAESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFDSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPEVHLLPPSQEEMTKNQVSLTCLARGFYPKDIAVEWESNGQPENNYKTTPSRQEPSQGTTTFAVTSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKTISLSLGK 5th Key Example SEQ_18(Sequence Number 18)GSAESKYGPPCPPCPAPEAAGGPRLSLHRPALEDLLLGSEANGLTCTLTGLRDASGVTFTWTPSSGKSAVQGPPERDLCGCYSVSSVLPGCAEPWNHGKTFTCTAAYPESKTPLTATLS KSGNTFRPEVYTLPPPSEELALNELVTLTCLVKGFYPSDVLVRWLQGSQELPREKYLTWAPVLDSDGSFFLYSILRVAAEDWKKGDTFSCMVGHEALPLAFTQKTIDRLAGKSEQ_17 (SEQ ID NO: 17)Same as above

[0145] Each sequence SEQ_1 and SEQ_2 of the first and second preparation examples above corresponds to the amino acid sequences of IgA1 and IgG4, and is used as a control group for the third to seventh preparation examples.

[0146] The above third manufacturing example is a heterodimer according to each sequence SEQ_3 and SEQ_4, and contains all amino acid sequences corresponding to hPD-L1, hinge, and CH2-CH3 domains. SEQ_3 is based on a sequence derived from IgA1, and SEQ_4 is based on a sequence derived from IgG4, but includes mutual substitution mutations (swapping mutations) and N-bond glycosylation, and in particular, SEQ_4 is an LS mutation (M428L / N434S) so that the binding affinity with FcRn is enhanced.

[0147] The above-described fourth preparation example is a heterodimer according to each sequence SEQ_5 and SEQ_6, containing all amino acid sequences corresponding to hPD-L1, hinge, and CH2-CH3 domains. SEQ_5 is based on a sequence derived from IgA1, and SEQ_6 is based on a sequence derived from IgG4, but includes mutual substitution mutations (swapping mutations) and N-bond glycosylation. In particular, SEQ_5 includes a CW mutation, and SEQ_6 includes a PY mutation and an LS mutation (M428L / N434S), thereby improving the binding affinity with FcRn.

[0148] The above 5th preparation example is a heterodimer according to each sequence SEQ_7 and SEQ_6, and includes all amino acid sequences corresponding to hPD-L1, hinge, and CH2-CH3 domains. SEQ_7 and SEQ_6 include mutual substitution variants (swapping variants) and N-bond glycosylation, and in particular, SEQ_7 is based on a sequence derived from IgA1 but includes a CW variant.

[0149] The above 6th preparation example is a heterodimer according to each sequence SEQ_8 and SEQ_9, and includes all amino acid sequences corresponding to hPD-L1, hinge, and CH2-CH3 domains. SEQ_8 is based on a sequence derived from IgA1, and SEQ_9 is based on a sequence derived from IgG4, and includes mutual substitution variants (swapping variants) and N-bond glycosylation.

[0150] The above 7th preparation example is a heterodimer according to each sequence SEQ_10 and SEQ_9, and includes all amino acid sequences corresponding to hPD-L1, hinge, and CH2-CH3 domains. SEQ_10 is based on a sequence derived from IgA1, and SEQ_9 is based on a sequence derived from IgG4, and includes mutual substitution variants (swapping variants) and N-bond glycosylation.

[0151] The first key example above is a heterodimer formed by SEQ_11 and SEQ_12, containing all amino acid sequences corresponding to the hinge and CH2-CH3 domains. SEQ_11 is based on a sequence derived from IgA1, and SEQ_12 is based on a sequence derived from IgG4, but includes mutual substitution variants (swapping variants) and N-bond glycosylation, and in the case of SEQ_12, the binding affinity with FcRn is enhanced by an LS variant (M428L / N434S).

[0152] The second key example above is a heterodimer formed by SEQ_13 and SEQ_14, containing all amino acid sequences corresponding to the hinge and CH2-CH3 domains. SEQ_13 is based on a sequence derived from IgA1, and SEQ_14 is based on a sequence derived from IgG4, but includes mutual substitution variations (swapping variations) and N-bond glycosylation, and in the case of SEQ_13, the binding affinity with FcRn is enhanced by a CW variation, and in the case of SEQ_14, by a PY variation and an LS variation (M428L / N434S).

[0153] The above third key example is a heterodimer formed by SEQ_15 and SEQ_14, containing all amino acid sequences corresponding to the hinge and CH2-CH3 domains. SEQ_15 is based on a sequence derived from IgA1, and SEQ_14 is based on a sequence derived from IgG4, but includes mutual substitution variations (swapping variations) and N-bond glycosylation, and in the case of SEQ_15, the binding affinity with FcRn is enhanced by CW variations, and in the case of SEQ_14, by PY variations and LS variations (M428L / N434S).

[0154] The above-mentioned fourth key example is a heterodimer based on SEQ_16 and SEQ_17, containing all amino acid sequences corresponding to the hinge and CH2-CH3 domains. SEQ_16 is based on a sequence derived from IgA1, and SEQ_17 is based on a sequence derived from IgG4, but includes mutual substitution variants (swapping variants) and N-bond glycosylation.

[0155] The above-mentioned fifth core example is a heterodimer based on SEQ_18 and SEQ_17, containing all amino acid sequences corresponding to the hinge and CH2-CH3 domains. SEQ_16 is based on a sequence derived from IgA1, and SEQ_17 is based on a sequence derived from IgG4, but includes mutual substitution variants (swapping variants) and N-bond glycosylation.

[0156] As such, Manufacturing Examples 1 through 7 represent hPD-L1 in the Fab region being bound to the Fc region, while Key Examples 1 through 5 describe the parts excluding the Fab region. Here, Fab is a part that can be changed variably, and the core of the present invention concerns the Fc region—that is, Key Examples 1 through 5. In other words, considering the difficulty of performing experiments with the Fc region alone, hPD-L1 corresponding to Fab serves as a so-called "carrier protein," and for future use as a pharmaceutical and for pharmacological effects, substances that replace it can be introduced at any time.

[0157] IV. Manufacturing Method

[0158] 1. Method for preparing immunoglobulin Fc fusion protein

[0159] Routine recombinant processes were used to generate directed mutations in the sequences of the Fc domains of IgA (original) and IgG (original), which are used as starting points for the generation and testing of the immunoglobulin Fc fusion protein according to the present invention. It will be recognized by those skilled in the art that various techniques for generating changes in coding sequences can be used to produce vectors suitable for the expression of desired amino acid sequences in various host cells for recovery and testing.

[0160] 2. Host cell selection or host cell manipulation

[0161] As described herein, host cells selected for the expression of recombinant Fc-containing proteins or monoclonal antibodies are, without limitation, significant contributors to the final composition, including variations in the composition of the oligosaccharide moiety decorating the protein in the immunoglobulin CH2 domain. Accordingly, one aspect of the present invention includes the use of production cells expressing a desired therapeutic protein and / or the selection of host cells suitable for development.

[0162] 3. Example - Production and Testing of Immunoglobulin Fc Fusion Protein

[0163] A series of fusion proteins listed in Table 1 were constructed using standard recombinant methods. Regarding fusion proteins having fully variable domains, Fc fusion proteins and isotypes were constructed with or without fusing a known sequence of human PD-L1 to the present heteromer Fc, as described above. The fusion proteins according to the present invention were transiently expressed in CHO cells using standard cloning and expression procedures. The fusion proteins were purified using a Protein A column to achieve homogeneity of over 90%, after which further experimental analysis was performed.

[0164] V. Experimental Example

[0165] 1. Purification of Immunoglobulin Fc Fusion Protein and Verification of Productivity

[0166] 1.1.PAGE

[0167] To confirm the purification and productivity of the immunoglobulin Fc fusion protein candidate group according to the present invention, PAGE (polyacrylamide gel electrophoresis) was performed, and the results for each of lanes 1 to 21 are as shown in [Table 2] and Figure 2 below.

[0168] It is organized by lane, with conditions classified as NR (non-reducing condition) and R (reducing condition), and samples.

[0169] NR conditions refer to electrophoresis performed without reduction conditions, in which the disulfide bonds of proteins are maintained and proteins with dimers or higher-order structures appear as they are, and R conditions refer to electrophoresis performed under reduction conditions, in which the disulfide bonds of proteins are reduced—generally using beta-mercaptoethanol or DTT, etc.—and separated to appear as monomers.

[0170] Referring to Figure 2, the state of the protein, such as dimerization, can be confirmed by providing the molecular weights under two conditions. That is, under non-reducing conditions, a single band appears as the disulfide bonds are maintained, while under reducing conditions, the disulfide bonds are broken, resulting in two different molecular weights or a molecular weight value relatively close to 1 / 2, which leads to the appearance of bands with different positions or numbers, thereby confirming that it is composed of a reduced dimer.

[0171] Lane Number Condition Sample 1 - Marker (M) 2NR Negative control (NC) 3R Negative control (NC) 4NR Preparation Example 1 5R Preparation Example 1 6NR Preparation Example 2 7R Preparation Example 2 8NR Preparation Example 3 9R Preparation Example 3 10NR Preparation Example 4 11R Preparation Example 4 12NR Preparation Example 5 13R Preparation Example 5 14NR Preparation Example 6 15R Preparation Example 6 16NR Preparation Example 7 17R Preparation Example 7

[0172] 1.2. Purification of Fusion Protein Candidates

[0173] The fusion protein candidates were purified using SEC / Hig-tag purification / AEX purification / Affinity Chromatography methods, and the results shown in [Table 3] below were obtained.

[0174] SEC (Size-Exclusion Chromatography) refers to size exclusion chromatography, which separates proteins based on their size. His-tag purification is metal-affinity chromatography using histidine tags, which involves purifying histidine-tagged proteins using metal ions. AEX (Anion Exchange Chromatography) is anion exchange chromatography, which separates proteins based on their charge. PA (Protein A Affinity Chromatography) is affinity chromatography using Protein A, which involves purifying antibodies using Protein A that binds to the Fc region of IgG. Additionally, Q FF (Q Fast Flow) is a purification method using a strong anion exchange column, which separates proteins based on their charge differences.

[0175] The purification results are summarized in [Table 3]. As shown in [Table 3] below, purification was performed on the 1st to 7th preparation examples using different methods, and the values ​​were organized by concentration, total amount, and purity. The 1st preparation example yielded the largest amount of final product (58 mg), and the 2nd preparation example showed the highest purity (99.4%). Among the 3rd to 7th preparation examples, excluding the 7th preparation example, all showed a purity of 90% or higher based on the final product. Thus, it can be seen that the 1st to 7th preparation examples can be purified as designed.

[0176] Planned Preparation Method Concentration (mg / mL) Total Amount (mg) Purity (%) Preparation Example 1 Ni-NTA Affinity Chromatography 1.18 48 8.1 Size Exclusion Chromatography 2.65 49 9.7 Final Product 12.05 89 9.2 Preparation Example 2 Protein A Chromatography 7.25 14 8.8 Size Exclusion Chromatography 1.01 69 9.4 Final Product 14.6 14 99.4 Preparation Example 3 Protein A Chromatography 3.64 33 1.0 Size Exclusion Chromatography 0.4 69 1.1 Final Product 11.65 91.6 Preparation Example 4 Protein A Chromatography 1.04 18 7.4 Strong Anion Exchange Chromatography 1.72 49 6.4 Final Product 12.12 59 5.0 Preparation Example 5 Protein A Chromatography 1.0 449 0.3 Strong anion exchange chromatography 1.8 319 4.1 Final product 11.3 309 4.1 Preparation Example 6 Protein A chromatography 5.4 81 14.7 Size exclusion chromatography 0.2 48 9.0 Final product 10.8 39 0.6 Preparation Example 7 Protein A chromatography 3.0 52 22.2 Size exclusion chromatography 0.2 36 8.5 Final product 9.4 36 8.5

[0177] 2. Analysis of Fc candidates with increased FcαR binding affinity

[0178] 2.1. Analysis of binding affinity with FcαRI

[0179] In order to analyze the binding strength between the fusion protein candidate group according to the present invention and FcαRI, the binding strength of 9 test substances and 2 target proteins (FcαRI, FcRn) was measured using the following Direct immobilization method.

[0180] Each flow cell was activated by flowing a mixture of 100 mM N-hydroxysuccinimide (NHS) and 400 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) in a 1:1 ratio (v / v) through a Series S CM5 Chip for 420 seconds. Target proteins diluted with 10 mM sodium acetate (pH 5.0) were flowed at a flow rate of 10 μl / min to immobilize them in the sample flow cell. Excess carboxyl groups remaining on the surface of the Series SCM5 chip were inactivated by flowing 70 microliters (70 μl) of 1 M ethanolamine-HCl (pH 8.0). After setting the analyte saturation concentration to the maximum concentration, the sample was prepared by performing five three-fold serial dilutions using running buffer. The analyte was injected and regenerated using a multi-cycle kinetics method, and sensorgrams were measured. Injection time and flow rate were controlled using Biacore T200 control software. The measured sensorgrams were analyzed using a fitting model with BIAevaluation software (Version 3.1). The reliability of the sensorgram fitting was evaluated using Chi2 and U-value according to the manufacturer's manual.

[0181] The experimental conditions for each ligand were as shown in the following [Table 4].

[0182] Ligand Analysis Physical Ligand Immobilization Level Analyst Concentration (nM) Buffer Injection Conditions Dynamic-Fitting Model FcαRI Preparation Example 1 Preparation Example 2 Preparation Example 3 Preparation Example 4 Preparation Example 5 Preparation Example 6 Preparation Example 7 Preparation Example 157RU 1000 500 250 125 62.5 31.25 15.6 37.81 Running buffer: HBS-EP+pH 7.4 Regenerating Buffer: 10mM Glycine (pH 2.0) Contact: 80s Dissociation: 60s Flow Rate: 30ul / min Multi-cycle kinetics (MCK)-Steady state affinity FcRn Preparation Example 1 Preparation Example 2 Preparation Example 3 Preparation Example 4 Preparation Example 5 Preparation Example 6 Preparation Example 7 Preparation Example 325 RU12562.531.2515.637.813.911.950.980.49Running buffer: 50mM Sodium phosphate (pH 6.0), 150mM NaCl, 0.05% P20Regeneration buffer: 100mM Tris-HCl (pH 8.0)Contact: 240sDissociation: 300sFlow Rate: 30ul / minMulti-cycle kinetics (MCK)-1:1 binding model

[0183] As a result of the experiment, the bond rate constant (Ka), dissociation rate constant (Kd), and dissociation constant (KD) were obtained in [Table 5] below. Each value is expressed according to exponential notation (such exponential notation is not limited to [Table 5] and may be used throughout the description of the present invention).

[0184] Figures 4a to 4g show Surface Plasmon Resonance (SPR) data for each sample, which monitored the binding of the protein sample to FcαRI in real time.

[0185] The vertical axis (Resonance Unit, RU) represents the reaction unit indicating the degree of binding between the protein and the ligand; a higher value indicates stronger binding, and the binding curve shows a pattern of starting, reaching a maximum value, and then gradually decreasing. The horizontal axis represents time (Time, T), indicating the time taken for the binding analysis in seconds, and shows the entire process over time.

[0186] The association phase represents the point at which the protein binds to FcαRI; it is the rising section of the graph and reflects the binding strength between the ligand and the analyte. Additionally, the dissociation phase indicates the point at which the bound protein separates from FcαRI; it is the falling section of the graph and demonstrates the stability of the binding.

[0187] The above explanation regarding SPR data applies equally to Fig. 5a et al. or Fig. 6a et al. by changing only the ligand.

[0188] [Table 5] shows that ka is the binding rate constant, indicating how quickly the analyte binds to FcαRI, and kd is the dissociation rate constant, indicating how quickly the bound protein separates from FcαRI. A low kd value means that the binding is stable and dissociates slowly. KD is the equilibrium dissociation constant, representing the binding strength between the protein and FcαRI, and a lower value indicates a stronger binding.

[0189] Preparation Example ka (1 / Ms)kd (1 / s)KD (M) Preparation Example 1 4.15E+043.00E-027.21E-07 Preparation Example 2 NDND (non-detectable or Not determined)ND Preparation Example 3 3.66E+041.41E-013.85E-06 Preparation Example 4 5.73E+041.37E-012.39E-06 Preparation Example 5 6.22E+041.23E-011.97E-06 Preparation Example 6 1.57E+041.98E-011.26E-05 Preparation Example 7 NDNDND

[0190] In Preparation Examples 2 and 7, no data values ​​could be obtained (no binding or dissociation occurred), whereas in Preparation Examples 1 and 3 through 6, data values ​​were obtained; this indicates that binding and dissociation with the target protein FcαRI occurred in Preparation Examples 3 through 6. Preparation Example 1 showed binding to FcαRI via the Fc domain of IgA (control group), while Preparation Example 2 confirmed that the Fc domain of IgG4 (control group) did not bind well to FcRn. The binding rate constant (ka) was highest in the order of Preparation Example 5, Preparation Example 4, Preparation Example 3, and Preparation Example 6, while the dissociation rate (kd) was highest in the order of Preparation Examples 6, 3, 4, and 5. In particular, Preparation Examples 4 and 5 showed higher binding rate constants than Preparation Example 1 (control group). Meanwhile, the equilibrium dissociation constant (KD) was highest in the order of Preparation Example 6, Preparation Example 3, Preparation Example 4, and Preparation Example 5. Meanwhile, the 7th manufacturing example shows that it does not combine with FcαRI.

[0191] 2.2. Analysis of binding affinity with FcRn

[0192] The binding strength with FcRn was analyzed in the same manner as in 2.1., and the results are summarized in Figures 5a to 5g and [Table 6] below, and the meaning of each row and column is as in [Table 5].

[0193] Figure 4a, etc. is an SPR graph analyzing the ability of a sample to bind to FcRn, which monitors the binding of a protein sample to FcRn in real time.

[0194] Preparation Example 1 demonstrated that the Fc domain of IgA (control) does not bind to FcRn, while Preparation Example 2 confirmed that the Fc domain of IgG4 (control) binds well to FcRn. Preparation Example 3 showed a faster binding rate constant compared to Preparation Example 2; although the dissociation rate constant is slightly higher, the equilibrium constant indicates strong binding strength. Preparation Examples 4 and 5 were confirmed to have equilibrium constants equal to or greater than those of Preparation Example 2. Preparation Example 6 exhibits a very high binding rate constant, but dissociation occurs very rapidly, indicating that the binding strength is relatively low in relation to the KD value. Preparation Example 7 demonstrates that it does not bind to FcRn.

[0195] To summarize, the strongest bond strength is found in Preparation Example 3 and Preparation Example 4, the intermediate bond strength is found in Preparation Example 2 and Preparation Example 5, and the lowest bond strength is found in Preparation Example 6. It can be seen that the bond is relatively unstable and the bond strength is low due to the high dissociation rate (kd).

[0196] Preparation Example ka (1 / Ms)kd (1 / s)KD (M) 1st Preparation Example NDNDND 2nd Preparation Example 4.82E+031.14E-022.36E-06 3rd Preparation Example 2.03E+045.63E-022.77E-06 4th Preparation Example 1.21E+041.15E-029.50E-07 5th Preparation Example 1.29E+041.61E-021.25E-06 6th Preparation Example 2.15E+051.43E+006.65E-06 7th Preparation Example NDNDND

[0197] 3. Analysis of immunoglobulin Fc fusion protein candidates with reduced FcγR binding affinity

[0198] The binding affinity between nine test substances and one target protein (FcγR) was analyzed using the His Capture Kit, and the results are summarized in Figures 6a to 6g and [Table 7] below (as noted above, the method of interpreting the graphs and tables is the same as in [Table 5] and [Table 6]).

[0199] In [Table 7], ka1 and ka2 represent the binding rate constants of the first and second binding steps, respectively, kd1 and kd2 represent the dissociation rates of the first and second binding steps, respectively, and KD1 and KD2 are the equilibrium dissociation constants representing the binding strength of each step. Rmax1 and Rmax2 are the maximum reaction unit values, representing the signal strength at saturation.

[0200] The second preparation example showed relatively high ka1 values ​​(6.51E+04 and 1.17E+06) compared to other Fc candidates, indicating that these candidates have the characteristic of rapidly binding to FcγRI. It was confirmed that the remaining preparation examples did not bind to FcγRI.

[0201] Preparation Example ka1 (1 / Ms)kd1 (1 / s)KD1 (M)ka2 (1 / Ms)kd2 (1 / s)KD2 (M)Rmax1(RU)Rmax2(RU) Preparation Example 1 Preparation Example 2 Preparation Example 6.51E+041.11E-021.70E-077.23E+056.16E-018.51E-070.581.52E+01 Preparation Example 3 Preparation Example 4 Preparation Example 5 Preparation Example 6 Preparation Example 7 Preparation Example

[0202] 4. Analysis of Fc candidates with increased Protein A binding affinity

[0203] To test the protein A binding affinity of the fusion protein candidate, recombinant protein A-high performance liquid chromatography (rPA-HPLC) was performed, and the results are summarized in the following [Table 8].

[0204] Preparation Example 1 2.6 37 0.018 -0.027 Preparation Example 2.6 17 0.8 34 0.547 Preparation Example 3 2.6 08 0.4 30.244 Preparation Example 4 2.6 43 0.3 36 0.256 Preparation Example 5 2.6 42 0.4 27 0.319 Preparation Example 6 2.6 05 0.4 54 0.28 Preparation Example 7 2.6 13 0.2 44 0.132

[0205] In the first example, it was observed that the protein did not bind to Protein A, whereas in the case of the second example, it bound well to Protein A. Meanwhile, it was confirmed that all from the third to the seventh example bound to Protein A, which suggests that it is possible to purify the protein into Protein A after protein production.

[0206] 5. Analysis of candidate groups dissociating at increased pH

[0207] FcRn affinity chromatography was performed to test FcRn affinity, which has a significant effect on the half-life of candidate substances, and the results are summarized in the following [Table 9] and Figure 7.

[0208] FcRn binds to the Fc portion of immunoglobulin at low pH (endosome environment) and dissociates at high pH (neutral environment such as blood); therefore, through this mechanism, it can be reused, and its half-life in the body may be extended.

[0209] Preparation Example Retention Time (RT) Area (AU) Percentage of Area (% Area) pH Preparation Example 1 1.78 340 912 31100 5.6 Preparation Example 2 38.36 544 518 869 9.98 6.67 Preparation Example 3 41.26 738 278 078 0.48 6.9 Preparation Example 4 40.06 147 56 226 100 6.8 Preparation Example 5 40.05 44 717 96 4100 6.8 Preparation Example 6 21.27 838 395 6569 58 5.91 Preparation Example 7 27.43 3349 467 967 03 6.1

[0210] According to this, the first preparation example was shown to dissociate at pH 5.6 from an FcRn column, and the second preparation example was shown to dissociate at pH 6.67.

[0211] Preparation Examples 6 and 7 showed that the substance dissociated from the FcRn column at a pH lower than that of Preparation Example 2, whereas Preparation Examples 3 to 5 showed that it dissociated at a pH higher than that of Preparation Example 2, which served as the positive control.

[0212] This demonstrates that the molecules can dissociate at low pH within intracellular endosomes, bind to lysosomes, and be reused without degradation for secretion outside the cell. In other words, since the 3rd, 4th, and 5th preparation examples dissociate at a higher pH than the positive control, they exhibit the characteristic of dissociating at an appropriate pH while maintaining a strong binding affinity with FcRn.

Claims

1. Including the first Fc domain and the second Fc domain, One or more selected from the group consisting of the first Fc domain and the second Fc domain comprises at least one amino acid modification, and The first Fc domain above consists of a portion of the Fc amino acid sequence of IgA as the corresponding amino acid of IgG, The above second Fc domain consists of a portion of the Fc amino acid sequence of IgG as the corresponding amino acid of IgA, Including mutually substituted variants Immunoglobulin heteromer Fc-fusion protein.

2. Including the first Fc domain and the second Fc domain, One or more selected from the group consisting of the first Fc domain and the second Fc domain comprises at least one amino acid modification, and The first Fc domain is a sequence derived from the Fc amino acid sequence of IgA, and the second Fc domain is a sequence derived from the Fc amino acid sequence of IgG, A wherein one or more variations among SW variation, CW variation, and PY variation are processed on any one or more selected from the group consisting of the first Fc domain and the second Fc domain. Immunoglobulin heteromer Fc-fusion protein.

3. In Paragraph 1 or 2, Including that the LS mutation has been processed in the second Fc domain above, Immunoglobulin heteromer Fc-fusion protein.

4. In Paragraph 1 or 2, Comprising those treated with N-link glycosylation, Immunoglobulin heteromer Fc-fusion protein.

5. In Paragraph 1 or 2, Containing an IgA tailpiece domain, Immunoglobulin heteromer Fc-fusion protein.

6. In Paragraph 1 or 2, Comprising a reduced binding affinity to FcγRI compared to wild-type human antibody IgG4, Immunoglobulin heteromer Fc-fusion protein.

7. In Paragraph 1 or 2, including binding to Protein A Immunoglobulin heteromer Fc-fusion protein.

8. In Paragraph 1 or 2, The above fusion protein has no binding affinity with FcγRI at pH 7.4, or 6.65 x 10⁻⁶ -6 including having a dissociation constant (Kd) of less than Immunoglobulin heteromer Fc-fusion protein.

9. In Paragraph 1 or 2, With enhanced binding strength, the dissociation constant (Kd) value is 9.66 x 10⁻⁶ -7 including that which is less than Immunoglobulin heteromer Fc-fusion protein.

10. In Paragraph 4, The above-mentioned N-bond glycosylated is, Comprising enhanced binding affinity to FcαRI in contrast to non-N-linked glycosylated, Immunoglobulin heteromer Fc-fusion protein.

11. In Paragraph 1 or 2, The above-mentioned first Fc domain includes sequence number 11, and The above second Fc domain comprises sequence number 12, Immunoglobulin heteromer Fc-fusion protein.

12. In Paragraph 1 or 2, The above-mentioned first Fc domain includes sequence number 13, and The above second Fc domain comprises SEQ ID NO. 14, Immunoglobulin heteromer Fc-fusion protein.

13. In Paragraph 1 or 2, The above-mentioned first Fc domain includes sequence number 15, and The above second Fc domain comprises SEQ ID NO. 14, Immunoglobulin heteromer Fc-fusion protein.

14. In Paragraph 1 or 2, The above-mentioned first Fc domain includes sequence number 16, and The above second Fc domain comprises SEQ ID NO. 17, Immunoglobulin heteromer Fc-fusion protein.

15. In Paragraph 1 or 2, The above-mentioned first Fc domain includes sequence number 18, and The above second Fc domain comprises SEQ ID NO. 17, Immunoglobulin heteromer Fc-fusion protein.

16. In Paragraph 5, The above IgA tailpiece domain is, Comprising either SEQ ID NO. 20 or SEQ ID NO. 21, Immunoglobulin heteromer Fc-fusion protein.

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