Human antibody FC domain mutants and uses thereof

Abstract

The present invention relates to human antibody Fc domain mutants and uses thereof, and more specifically to human antibody Fc domain mutants in which amino acid 235 of the wild-type human antibody Fc domain is substituted with proline (P), and which have no or substantially no binding ability to human FcγRs and C1q, and therefore do not exhibit antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), or complement-dependent cytotoxicity (CDC), and uses thereof as antibody-drug conjugates.

Description

[Technical Field]

[0001] The present invention relates to human antibody Fc domain variants and uses thereof. [Background technology]

[0002] Protein therapeutics have been rapidly replacing non-specific small molecule therapeutics due to their highly specificity for disease targets and low side effects and toxicity, and are now widely used in clinical settings. Currently, the majority of protein therapeutics in clinical use are Fc fusion protein therapeutics, which are antibody therapeutics fused with the Fc region of an antibody.

[0003] Therapeutic antibodies are considered one of the most effective cancer treatments because they have extremely high target specificity compared to conventional small molecule drugs, low biotoxicity and few side effects, and an excellent half-life in the blood of approximately three weeks.In fact, major pharmaceutical companies and research institutions around the world are accelerating research and development of therapeutic antibodies that specifically bind to cancer cells, including cancer-causing factors, and effectively eliminate them.

[0004] Antibodies play a role in linking the humoral immune system and the cellular immune system. The Fab region of an antibody recognizes the antigen, while the Fc domain binds to receptors (Fc receptors or FcRs) for antibodies (immunoglobulins) on cells that are differentially expressed by all immune cells, with different mechanisms depending on the type of FcγR expressed on the surface of the immune cell to which it binds. When the Fc receptor-binding site on the Fc region of an antibody binds to the FcR on the cell surface via the Fc region, it leads to the phagocytosis and destruction of antibody-coated particles, the removal of immune complexes, and the lysis of antibody-coated target cells by killer cells (antibody-dependent cellular cytotoxicity). This triggers a variety of important biological responses, including the induction of immune cytotoxicity (ADCC), release of inflammatory mediators, and regulation of placental transfer and immunoglobulin production.

[0005] The Fc domain plays an important role in immune cell recruitment, antibody-dependent cellular cytotoxicity (ADCC), and antibody-dependent cellular phagocytosis (ADCP). In particular, the effector functions of antibodies, ADCC and ADCP, depend on interactions with Fc receptors present on the surface of many cells. Human Fc receptors are classified into five types, and the type of immune cell recruited depends on which Fc receptor an antibody binds to. For example, the Fc domain of an antibody binds to FcγRIIIa to induce ADCC, FcγRI or FcγRIIa to induce ADCP, C1q to activate the complement-dependent cytotoxicity (CDC) mechanism, and Fab region-bound target antigens, thereby providing the primary therapeutic effect of therapeutic antibodies.

[0006] The mechanism of action of this antibody Fc region is to induce the killing mechanism of defective cells (e.g., cancer or infected cells) and is an important mechanism of action for therapeutic antibodies. However, when the target antigen of a therapeutic antibody is expressed on immune cells rather than defective cells (e.g., cancer or infected cells), such as in immune checkpoint inhibitor antibodies and bispecific immune cell-engaging bispecific antibodies, if the Fc mechanism of action remains, it may destroy normal immune cells that are beneficial to the immune function bound by the antibody or activate the host's immune defenses, leading to safety issues and undesirable side effects.

[0007] For example, some therapeutic antibodies, such as immune checkpoint inhibitors that bind to immune cells and immune cell-inducing dual antibodies, have the problem of inducing an immune mechanism against target immune cells and causing the side effect of destroying the immune cells. Furthermore, immune checkpoint inhibitors (e.g., Keytruda, Opdivo, Libtayo, etc.) that target immune checkpoint proteins expressed on the surface of immune cells containing T cells have the drawback of destroying the immune cells themselves, which is intended to kill cancer cells, if the Fc-mediated mechanism of action remains, resulting in a side effect that reduces the antibody's inherent effectiveness.

[0008] In addition, immune cell-inducing double antibodies, which are antibody therapeutics that bind to antigens on the surface of cancer cells on one side and to immune cells on the other side, attracting immune cells to cancer cells for more effective cancer cell elimination, can have side effects such as immune cell destruction and ineffective cancer cell elimination if the antibody has an Fc-mediated immune mechanism. Agonist antibodies, which bind to target cells and induce their activation, and antagonist antibodies, which inhibit the interaction between target antigens and ligands, also exhibit toxicity toward target cells and antigens due to the Fc-mediated immune mechanism, reducing the antibody's inherent efficacy. Furthermore, when developing Fc fusion proteins by fusing an Fc region to an active substance such as a protein or compound to extend its half-life for therapeutic, diagnostic, or research purposes, toxicity can occur due to the Fc-mediated immune mechanism.

[0009] Therefore, to maintain the excellent efficacy of antibodies through target antigen-specific binding while resolving the side effects of immune cell death and off-target toxicity to normal cells, it is essential to eliminate the Fc-mediated mechanism. For this reason, IgG2 antibodies, which have the lowest FcγR binding affinity among human IgG subclasses and have a very low immune-mediated mechanism, are being considered in antibody development. However, IgG2 antibodies have physical properties such as the existence of multiple allotypes due to disulfide bond exchange in the hinge region and aggregation due to reduced stability. Therefore, IgG4 antibodies, which have the next lowest binding affinity, are being investigated and are currently being used in clinical development. However, IgG4 antibodies also suffer from the drawback of Fab arm exchange due to the lack of stability in the hinge region. Furthermore, their strong binding affinity to FcγRs, especially to FcγRI (with an equilibrium dissociation constant of several nM), can activate various Fc-mediated mechanisms. Therefore, to prevent destruction of target cells by the immune mechanism of antibodies, an Fc that has been deprived of its binding ability to FcγRs is required. Summary of the Invention [Problem to be solved by the invention]

[0010] An object of the present invention is to provide novel human antibody Fc domain mutants.

[0011] An object of the present invention is to provide a novel antibody with reduced effector function or a fragment thereof having immunological activity.

[0012] An object of the present invention is to provide novel antibody-drug conjugates.

[0013] An object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer.

[0014] An object of the present invention is to provide a method for treating cancer. [Means for solving the problem]

[0015] 1. A human antibody Fc domain mutant in which amino acid 235 of the human antibody Fc domain is substituted with proline (P).

[0016] 2. A human antibody Fc domain mutant according to item 1 above, wherein the human antibody Fc domain consists of the amino acid sequence of SEQ ID NO: 15.

[0017] 3. A human antibody Fc domain mutant according to item 1 above, wherein amino acid 234 of the human antibody Fc domain is further substituted with alanine (A).

[0018] 4. A human antibody Fc domain mutant according to item 1 above, wherein amino acid 265 of the human antibody Fc domain is further substituted with leucine (L), methionine (M), or asparagine (N).

[0019] 5. The human antibody Fc domain mutant according to item 1 above, wherein amino acid 329 of the human antibody Fc domain is further substituted with valine (V), leucine (L), or glycine (G).

[0020] 6. The human antibody Fc domain mutant according to item 1, wherein amino acid 268 of the human antibody Fc domain is further substituted with glutamine (Q).

[0021] 7. The human antibody Fc domain mutant according to item 1, wherein amino acid 296 of the human antibody Fc domain is further substituted with phenylalanine (F).

[0022] 8. The human antibody Fc domain mutant according to item 1, wherein amino acid 300 of the human antibody Fc domain is further substituted with phenylalanine (F).

[0023] 9. The human antibody Fc domain mutant according to item 1, wherein amino acid 333 of the human antibody Fc domain is further substituted with valine (V).

[0024] 10. The human antibody Fc domain mutant according to item 1, wherein the human antibody is IgG1.

[0025] 11. An antibody comprising the human antibody Fc domain mutant according to any one of items 1 to 10 above, or a fragment thereof having immunological activity.

[0026] 12. An antibody-drug conjugate in which the antibody or immunologically active fragment thereof according to item 11 is conjugated to one or more drugs.

[0027] 13. In the above item 12, the drug is a chimeric antigen receptor (CAR) cell therapy agent, an oncolytic drug, or the like. drug), immunotherapy, cytotoxic agent, angiogenesis inhibitor, kinase inhibitor, costimulatory molecule blocker, adhesion molecule blocker, anti-cytokine agent, nucleic acid therapeutic, anti-CTLA-4 agent, anti-PD-1 agent, anti-PD-L1 agent, anti-PD-L2 agent, TNF-α cross-linker, TRAIL cross-linker, anti-CD27 agent, anti-CD30 agent, anti-CD40 agent, anti-4-1BB agent, anti-GITR agent, anti-OX40 agent, anti-TRAILR1 agent, anti-TRAILR2 agent, targretin, interferon-α, clobetasol, pegylated interferon, prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, anti-chemokine, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable label or reporter, TNF antagonist, anti-rheumatic an antibody-drug conjugate, which is any one selected from the group consisting of a drug, a muscle relaxant, a narcotic, a nonsteroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antibacterial agent, an antipsoriatic agent, a corticosteroid, an anabolic steroid, erythropoietin, immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta-agonist, an inhaled steroid, epinephrine or an analog thereof, a cytokine, a cytokine antagonist, a PD-1 antagonist, an adenosine A2AR antagonist, a CD73 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG-3 inhibitor, an anthracycline, an antisense oligonucleotide (ASO), an siRNA, and an aptamer.

[0028] 14. A pharmaceutical composition for treating or preventing cancer, comprising the human antibody Fc domain variant according to any one of items 1 to 10; an antibody containing the variant or a fragment thereof having immunological activity; or the antibody-drug conjugate according to item 12 or 13.

[0029] 15. The pharmaceutical composition for treating or preventing cancer according to item 14, wherein the cancer is any one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, esophageal cancer, lymphatic gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system malignant lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0030] 16. A method for treating cancer, comprising the step of administering to a subject the human antibody Fc domain variant described in any one of items 1 to 10; an antibody containing the variant or a fragment thereof having immunological activity; or the antibody-drug conjugate described in item 12 or 13.

[0031] 17. The method for treating cancer according to item 16, wherein the cancer is any one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, gastric cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, esophageal cancer, lymphatic gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system malignant lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0032] 18. A composition for target detection, comprising a conjugate of the antibody or a fragment thereof having immunological activity according to item 11 and a functional substance.

[0033] 19. The target detection composition according to item 18, wherein the functional substance is one or more selected from the group consisting of a contrast agent, a radioisotope, an inorganic particle, a fluorescent marker, a staining substance, an enzyme, a polypeptide, a nucleic acid, a carbohydrate, and a lipid.

[0034] 20. A method for diagnosing a disease, comprising the step of administering the target detection composition according to item 18 to an individual.

[0035] 21. The method for diagnosing a disease according to item 20, wherein the disease is any one selected from the group consisting of cancer, cardiovascular disease, neuropathy, diabetes, autoimmune disease, inflammatory disease, viral infection, and allergy. [Effects of the Invention]

[0036] The human antibody Fc domain variants of the present invention do not bind or do not substantially bind to human FcγRs and C1q.

[0037] The human antibody Fc domain variants of the present invention do not exhibit effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).

[0038] The human antibody Fc domain mutants of the present invention have pH-dependent FcRn-binding activity and thermal stability.

[0039] The human antibody Fc domain mutants of the present invention can reduce drug toxicity and enhance efficacy.

[0040] The human antibody Fc domain variants of the present invention can maintain the half-life of drugs and eliminate target toxicity. [Brief explanation of the drawings]

[0041] [Figure 1] 1a and 1b are schematic diagrams of mammalian cell display technology for glycosylated Fc. [Figure 2]Figures 2a to 2e show the results of SDS-PAGE analysis after expression and purification of highly purified tetrameric FcγRI-streptavidin and tetrameric FcγRIIIa-158V-streptavidin, FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, FcγRIIIa-158F-GST, and FcRn-GST. [Figure 3] 3a and 3b show the results of analyzing the binding activity of fluorescently labeled tetrameric FcγRIIIa-Alexa647 and protein A-FITC to wild-type Fc (WT Fc) expressed in CHO cells. [Figure 4] FIG. 4 shows the results of aligning the goat IgG Fc sequence and the human IgG Fc sequence and comparing the amino acids. [Figure 5] Figures 5a and 5b show the results of SDS-PAGE analysis of the glycosylated Fc variants after expression and purification, and the mutation sequences of each glycosylated Fc variant. [Figure 6] Figures 6a and 6b show the results of ELISA analysis of the binding ability of the prepared glycated Fc variants to FcγRI and FcγRIIIa-158V. [Figure 7] FIG. 7 is a schematic diagram of a site-directed mutation library for glycosylated Fc engineering. [Figure 8] FIG. 8 is a schematic diagram of glycosylated Fc engineering using CHO cell display. [Figure 9] Figure 9 shows the results of SDS-PAGE analysis of selected glycosylated Fc variants (LP1, LP5, LP6, LP7 and LP8) after expression and purification. [Figure 10]Figures 10a to 10c show the results of ELISA analysis of the binding affinity of glycated trastuzumab Fc mutants containing selected glycated Fc mutants (LP1, LP5, LP6, LP7, and LP8) to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F. [Figure 11] FIG. 11 shows the results of SDS-PAGE analysis of glycosylated Fc variants (LALPs) with reduced binding affinity to FcγRs after expression and purification, and their mutant sequences. [Figure 12] Figures 12a to 12c show the results of ELISA analysis of the binding ability of a glycated Fc mutant (LALP) with reduced binding ability to FcγRs to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F. [Figure 13] FIG. 13 shows SDS-PAGE analysis of selected glycosylated Fc variants (LALP1, LALP5, LALP6, LALP7 and LALP8) and their mutant sequences after expression and purification. [Figure 14] Figures 14a to 14c show the results of ELISA analysis of the binding affinity of glycated trastuzumab Fc mutants containing selected glycated Fc mutants (LALP1, LALP5, LALP6, LALP7, and LALP8) to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F. [Figure 15] Figure 15 shows the results of DSF analysis of the thermal stability of the discovered glycated trastuzumab Fc variants (LALP, LALP1, LALP5, LALP6, LALP7, and LALP8). [Figure 16] FIG. 16 shows the results of DSF analysis of the thermal stability of the discovered glycated trastuzumab Fc variants (LP1, LP5, LP6, LP7, and LP8). [Figure 17]Figure 17 shows the results of ELISA analysis of the FcRn binding ability of the discovered glycated trastuzumab Fc variants (LALP, LALP1, LALP5, LALP6, and LALP7). [Figure 18] Figures 18a and 18b show the results of ELISA analysis of the FcRn binding ability of the discovered glycosylated trastuzumab Fc variants (LP1, LP5, and LP6). [Figure 19] Figure 19 shows the results of ELISA analysis of the C1q binding ability of the discovered glycated trastuzumab Fc variants (LALP, LALP1, LALP5, LALP6, and LALP7). [Figure 20] FIG. 20 shows the results of ELISA analysis of the C1q binding ability of the discovered glycosylated trastuzumab Fc variants (LP1, LP5, and LP6). [Figure 21] Figure 21a shows the results of SDS-PAGE analysis of the conventional glycosylated trastuzumab Fc variant (LALAPG) after expression and purification, and its mutant sequence. Figure 21b shows the results of a comparative analysis of the FcγRI binding ability of the discovered glycosylated trastuzumab Fc variants (LP1, LP5, LP6, and LP7) with that of LALA on HER2-expressing target cells. [Figure 22] FIG. 22 shows the results of analyzing the binding affinity of the conventional glycated trastuzumab Fc variant (LALAPG) and the newly discovered glycated trastuzumab Fc variants (LALP1, LALP5, and LALP6) to hFcγRI. [Figure 23] FIG. 23 shows the results of analyzing the binding affinity of a conventional glycated trastuzumab Fc variant (LALAPG) and the newly discovered glycated trastuzumab Fc variants (LALP1, LALP5, and LALP6) to hFcγRIIIa-158F. [Figure 24] FIG. 24 shows the results of SDS-PAGE analysis of a glycosylated trastuzumab Fc variant (LALPPG) produced by combining P329G with the mutations discovered in the present invention, after expression and purification, and the mutant sequences. [Figure 25]Figures 25a to 25c show the results of ELISA analysis of the binding affinity of the glycated trastuzumab Fc variants (LALP and LALPPG) discovered in the present invention to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F. [Figure 26] FIG. 26 shows the results of analyzing the reduced PBMC-mediated ADCC function of the glycated trastuzumab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention by real-time cell killing assay. [Figure 27] Figures 27a and 27d show the results of analyzing the reduced PBMC-mediated ADCC function of the glycated trastuzumab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention by counting the number of dead cells using real-time cell imaging. [Figure 28] Figure 28 shows the results of analyzing the reduced NK92-mediated ADCC function of the glycated rituximab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention. [Figure 29] FIG. 29 shows the results of analyzing the reduced ADCP function of the glycosylated trastuzumab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention. [Figure 30] FIG. 30 shows the results of analyzing the reduced CDC function of glycated rituximab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention. [Figure 31] 31a and 31b show the results of ELISA analysis of the FcRn binding ability in mice of the glycosylated trastuzumab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention and the results of in vivo PK analysis. [Figure 32] Figures 32a and 32b show the results of ELISA analysis of the binding ability of the glycosylated trastuzumab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention to mFcγRI, mFcγRIIb, mFcγRIII, and mFcγRIV in mice. [Figure 33]Figures 33a and 33b show the results of ELISA analysis of the binding affinity of the glycated trastuzumab Fc variants (LALP1, LALP5, and LALP6) discovered in the present invention to cFcγRI, cFcγRIIa, cFcγRIIb, and cFcγRIII in cynomolgus monkeys. DETAILED DESCRIPTION OF THE INVENTION

[0042] The present invention provides human antibody Fc domain variants and uses thereof.

[0043] The present invention provides human antibody Fc domain mutants in which amino acid 235 of the wild-type human antibody Fc domain is substituted with proline (P), and which have no or substantially no binding ability to human FcγRs and C1q and therefore do not exhibit antibody-dependent cellular cytotoxicity (ADCC) or antibody-dependent cellular phagocytosis (ADCP), as well as uses thereof as antibody-drug conjugates.

[0044] In the present invention, the usual one-letter and three-letter codes for natural amino acids are used, as well as the commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc.

[0045] The amino acids abbreviated in the present invention are written according to the IUPAC-IUB nomenclature as follows: alanine: A, arginine: R, asparagine: N, aspartic acid: D, cysteine: C, glutamic acid: E, glutamine: Q, glycine: G, histidine: H, isoleucine: I, leucine: L, lysine: K, methionine: M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.

[0046] In the present invention, the position of a specific amino acid in an amino acid sequence refers to the position numbered according to the Kabat numbering system.

[0047] As used herein, the term "amino acid modification / mutation" refers to the substitution, insertion, and / or deletion, preferably substitution, of an amino acid in a polypeptide sequence. As used herein, the term "amino acid substitution" or "substitution" refers to the replacement of an amino acid at a specific position in the polypeptide sequence of a wild-type human antibody Fc domain with another amino acid. For example, a human antibody Fc domain mutant containing an L235P substitution means that the 235th amino acid residue in the amino acid sequence of a wild-type human antibody Fc domain, leucine (L), is replaced with proline (P).

[0048] The human antibody Fc domain mutant of the present invention has amino acid 235 (L) of the wild-type human antibody Fc domain substituted with proline (P).

[0049] In one embodiment, the human antibody Fc domain variant of the present invention may comprise, in addition to L235P, any one or more amino acid substitutions selected from the group consisting of L234A, D265N, D265M, D265L, P329V, P329L, P329G and P331L.

[0050] In one embodiment, the human antibody Fc domain variants of the present invention may comprise amino acid substitutions L235P and L234A of the wild-type human antibody Fc domain.

[0051] In one embodiment, the human antibody Fc domain variants of the present invention may comprise the amino acid substitutions D265N, D265M or D265L in addition to L235P and L234A of the wild-type human antibody Fc domain.

[0052] In one embodiment, the human antibody Fc domain variants of the present invention may comprise amino acid substitutions of P329V, P329L or P329G in addition to L235P and L234A of the wild-type human antibody Fc domain.

[0053] In one embodiment, the human antibody Fc domain mutant of the present invention may further have a substitution of amino acid 268 with glutamine (Q) in addition to L235P and L234A of the wild-type human antibody Fc domain.

[0054] In one embodiment, the human antibody Fc domain mutant of the present invention may further have a substitution of amino acid 296 with phenylalanine (F) in addition to L235P and L234A of the wild-type human antibody Fc domain.

[0055] In one embodiment, the human antibody Fc domain mutant of the present invention may further have a substitution of amino acid 300 with phenylalanine (F) in addition to L235P and L234A of the wild-type human antibody Fc domain.

[0056] In one embodiment, the human antibody Fc domain mutant of the present invention may further have a substitution of amino acid 333 with valine (V) in addition to L235P and L234A of the wild-type human antibody Fc domain.

[0057] In one embodiment, the human antibody Fc domain variant of the present invention may be a human antibody Fc domain variant LALP comprising the amino acid substitutions L234A and L235P. The human antibody Fc domain variant LALP may comprise the amino acid sequence of SEQ ID NO: 1 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 2.

[0058] In one embodiment, the human antibody Fc domain mutant of the present invention may be the human antibody Fc domain mutant LALP1, which contains the amino acid substitutions L234A, L235P, and D265N. The human antibody Fc domain mutant LALP1 may contain the amino acid sequence of SEQ ID NO: 3 and may be encoded by a nucleic acid molecule containing the base sequence of SEQ ID NO: 4.

[0059] In one embodiment, the human antibody Fc domain variant of the present invention may be the human antibody Fc domain variant LALP5, which contains the amino acid substitutions L234A, L235P, and P329V. The human antibody Fc domain variant LALP5 may comprise the amino acid sequence of SEQ ID NO:5 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO:6.

[0060] In one embodiment, the human antibody Fc domain variant of the present invention may be the human antibody Fc domain variant LALP6, which contains the amino acid substitutions L234A, L235P, and P329L. The human antibody Fc domain variant LALP6 may comprise the amino acid sequence of SEQ ID NO:7 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO:8.

[0061] In one embodiment, the human antibody Fc domain variant of the present invention may be the human antibody Fc domain variant LALP7, which comprises the amino acid substitutions L234A, L235P, and D265M. The human antibody Fc domain variant LALP7 may comprise the amino acid sequence of SEQ ID NO:9 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO:10.

[0062] In one embodiment, the human antibody Fc domain variant of the present invention may be the human antibody Fc domain variant LALP8, which contains the amino acid substitutions L234A, L235P, D265L, and P331L. The human antibody Fc domain variant LALP8 may comprise the amino acid sequence of SEQ ID NO: 11 and may be encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 12.

[0063] In one embodiment, the human antibody Fc domain variant of the present invention may be the human antibody Fc domain variant LALPPG, which contains the amino acid substitutions L234A, L235P, and P329G. The human antibody Fc domain variant LALPPG may comprise the amino acid sequence of SEQ ID NO: 13 and may be encoded by a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 14.

[0064] In one embodiment, the human antibody (immunoglobulin) may be IgA, IgM, IgE, IgD, or IgG, or a variant thereof. The human antibody may be IgG1, IgG2, IgG3, or IgG4, preferably IgG1, more preferably an anti-HER2 antibody, and most preferably trastuzumab. Papain digestion of the antibody produces two Fab domains and one Fc domain. In human IgG molecules, the Fc region is generated by papain digestion of the N-terminus of Cys 226.

[0065] In one embodiment, the human antibody (immunoglobulin) may be IgG1 or a variant thereof. The Fc domain of wild-type IgG1 may comprise the amino acid sequence of SEQ ID NO: 15 and may be encoded by a nucleic acid molecule comprising the base sequence of SEQ ID NO: 16.

[0066] In one embodiment, the human antibody Fc domain variants of the present invention may have reduced binding avidity to Fc gamma receptors (FcγRs) compared to wild-type human antibody Fc domains, where the Fcγ receptor may be FcγRI, FcγRIIa, FcγRIIb, or FcγRIIIa.

[0067] In one embodiment, the human antibody Fc domain variants of the present invention may have reduced binding avidity to C1q compared to the wild-type human antibody Fc domain.

[0068] In one embodiment, the human antibody Fc domain variants of the present invention may have a reduced effector function compared to a wild-type human antibody Fc domain.

[0069] In one embodiment, the effector function may be an Fc-mediated effector function selected from C1q binding, complement activation, complement dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), Fc receptor binding including Fc gamma receptor binding, protein A binding, protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement dependent cellular cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, Fc-containing polypeptide internalization, target downregulation, ADC uptake, induction of apoptosis, cell killing, cell cycle arrest, and any combination thereof.

[0070] In one embodiment, the human antibody Fc domain variants of the present invention can have a pH-dependent FcRn-binding affinity similar to that of the wild-type human antibody Fc domain, and can have an in vivo half-life and thermal stability similar to that of the wild-type human antibody Fc domain.

[0071] In one embodiment, the human antibody Fc domain variants of the present invention can be used to prevent killing of cells to which they bind.

[0072] In one embodiment, the human antibody Fc domain variants of the present invention can be used in immune checkpoint inhibitor antibodies or bispecific immune cell engaging bispecific antibodies.

[0073] The human antibody Fc domain variants of the present invention can be produced by any method known in the art. In one embodiment, the human antibody Fc domain variants of the present invention encode a polypeptide sequence containing specific amino acid modifications, and are then used to form nucleic acids that can be cloned into host cells, expressed, and assayed, as necessary. Various methods for this purpose are described in "Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons."

[0074] Nucleic acids encoding the Fc domain variants of the present invention can be inserted into an expression vector for protein expression. Expression vectors typically contain a protein operably linked, i.e., in a functional relationship, with regulatory or control sequences, selection markers, optional fusion partners, and / or additional elements. The Fc variants of the present invention can be produced by culturing a host cell transformed with a nucleic acid, preferably an expression vector containing a nucleic acid encoding the Fc domain variant of the present invention, under appropriate conditions to induce protein expression. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and may vary depending on the host cell used. Preferably, the Fc domain variants of the present invention can be produced using Escherichia coli as a host cell, which has low production costs and high industrial value.

[0075] The present invention provides a method for producing an Fc domain variant, comprising the steps of culturing host cells into which nucleic acid encoding the Fc domain variant has been introduced under conditions suitable for protein expression, and purifying or isolating the expressed Fc domain variant from the host cells.

[0076] "FcRn" or "neonatal Fc receptor" refers to a protein that binds to the Fc region of an IgG antibody and is encoded, at least in part, by the FcRn gene. FcRn can be from any organism, including, but not limited to, human, mouse, rat, rabbit, and monkey.

[0077] As is known in the art, a functional FcRn protein often comprises two polypeptides, referred to as a light chain and a heavy chain. The light chain is β-2-microglobulin, and the heavy chain is encoded by the FcRn gene. Unless otherwise specified herein, FcRn or an FcRn protein refers to the complex of the FcRn heavy chain and β-2-microglobulin.

[0078] The present invention provides antibodies comprising a human antibody Fc domain mutant or fragments thereof having immunological activity.

[0079] In one embodiment, the antibody or immunologically active fragment thereof may have ablated binding to FcγRs and may have reduced binding to Fc gamma receptors (FcγRs) or C1q compared to a wild-type human antibody.

[0080] In one embodiment, the Fc gamma receptor may be FcγRI, FcγRIIa, FcγRIIb, or FcγRIIIa.

[0081] In one embodiment, the antibody or immunologically active fragment thereof may have reduced effector function compared to a wild-type human antibody.

[0082] In one embodiment, the antibody may be a polyclonal antibody, a monoclonal antibody, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a chimeric antibody, or an antibody conjugate.

[0083] In one embodiment, a human antibody comprises a humanized antibody.

[0084] The immunologically active fragment may be an antibody Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer, complementarity determining region fragment, or diabody.

[0085] Antibodies can be isolated or purified by various methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunoprecipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As known in the art, various natural proteins, such as bacterial proteins A, G, and L, can bind to antibodies and be used for purification. Often, purification is possible via specific fusion partners.

[0086] Antibodies include not only whole antibody forms but also functional fragments of antibody molecules. Whole antibodies have a structure consisting of two full-length light chains and two full-length heavy chains, with each light chain linked to a heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment that retains antigen-binding function. Examples of antibody fragments include (i) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and the first heavy chain constant region (CH1); (ii) an Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of the VH domain; (v) separated CDR regions; (vi) an F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer (PCT / US92 / 09965), and (ix) a diabody (WO94 / 13804), which is a multivalent or multispecific fragment produced by gene fusion.

[0087] The antibody or immunologically active fragment thereof of the present invention can be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The antibody can be produced recombinantly or synthetically.

[0088] The antibody or immunologically active fragment thereof may be isolated from a living organism (not present in a living organism) or may be non-naturally occurring, for example, synthetically or recombinantly produced.

[0089] "Antibody" refers to a substance produced by the immune system in response to antigen stimulation. Its type is not particularly limited, and it can be naturally or non-naturally produced (e.g., synthetically or recombinantly). Antibodies are highly stable in vitro and in vivo, with long half-lives, making them ideal for mass expression and production. Furthermore, their inherent dimeric structure gives them exceptionally high avidity. A complete antibody consists of two full-length light chains and two full-length heavy chains, each linked to a heavy chain by a disulfide bond. Antibody constant regions are divided into heavy-chain and light-chain constant regions. Heavy-chain constant regions include gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses including gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). Light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0090] The term "heavy chain" refers to a full-length heavy chain comprising a variable region domain VH containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, three constant region domains CH1, CH2, and CH3, and a hinge, as well as fragments thereof. The term "light chain" refers to a full-length light chain comprising a variable region domain VL containing an amino acid sequence with sufficient variable region sequence to confer antigen specificity, as well as a constant region domain CL, as well as fragments thereof.

[0091] The "Fc domain," "Fc fragment," or "Fc region" together with the Fab domain / fragment form an antibody. The Fab domain / fragment consists of the variable region of the light chain (VL) and the variable region of the heavy chain (VH), the constant region of the light chain (CL) and the first constant region of the heavy chain (CH1). The Fc domain / fragment consists of the second constant region (CH2) and the third constant region (CH3) of the heavy chain.

[0092] In one aspect, the present invention relates to a nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same, or an immunologically active fragment thereof.

[0093] In one aspect, the invention relates to a vector comprising the nucleic acid molecule, and a host cell comprising the vector.

[0094] Nucleic acid molecules of the present invention may be isolated or recombinant and include DNA and RNA in single- and double-stranded forms, as well as corresponding complementary sequences. In the case of nucleic acids isolated from naturally occurring sources, the isolated nucleic acid is one that is separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid is isolated. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid produced by these procedures can be understood as an isolated nucleic acid molecule. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. A nucleic acid is operably linked when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein, the form in which the polypeptide is secreted; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the polypeptide sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in the same reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0095] Isolated nucleic acid molecules encoding the Fc domain variants of the present invention, antibodies containing the variants, or immunologically active fragments thereof can be modified in various ways in the coding region without altering the amino acid sequence of the Fc domain variants, antibodies containing the variants, or immunologically active fragments thereof, taking into account codon degeneracy or the codons preferred in the organism in which they are to be expressed. Furthermore, those skilled in the art will understand that even portions excluding the coding region can be modified in various ways without affecting gene expression, and that such modified genes are also within the scope of the present invention. That is, the nucleic acid molecules of the present invention may contain mutations in one or more nucleic acid bases by substitution, deletion, insertion, or a combination thereof, as long as they encode proteins with equivalent activity, and these mutations are also within the scope of the present invention. The sequences of such nucleic acid molecules may be single-stranded or double-stranded, and may be DNA or RNA (mRNA) molecules.

[0096] An isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or an antibody comprising the same, or an immunologically active fragment thereof, can be inserted into an expression vector for protein expression. Expression vectors typically contain a protein operably linked, i.e., in a functional relationship, with regulatory or control sequences, a selection marker, any fusion partners, and / or additional elements. The Fc domain variant of the present invention, or an antibody comprising the same, or an immunologically active fragment thereof, can be produced by culturing a host cell transformed with the nucleic acid under appropriate conditions, preferably an expression vector containing an isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or an antibody comprising the same, or an immunologically active fragment thereof, and inducing protein expression. Various suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and vary depending on the host cell used. Preferably, Escherichia coli, which has low production costs and high industrial value, can be used as the host cell.

[0097] Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. Suitable vectors contain expression regulatory elements such as a promoter, operator, initiation codon, termination codon, polyadenylation signal, and enhancer, as well as a signal sequence or leader sequence for membrane targeting or secretion, and can be constructed in a variety of ways depending on the purpose. The promoter of the vector may be constitutive or inducible. Examples of signal sequences that can be used include the PhoA signal sequence, OmpA signal sequence, etc. when the host is an Escherichia sp. bacterium; the α-amylase signal sequence, subtilisin signal sequence, etc. when the host is a Bacillus sp. bacterium; the MFα signal sequence, SUC2 signal sequence, etc. when the host is a yeast; and the insulin signal sequence, α-interferon signal sequence, and antibody molecule signal sequence when the host is an animal cell, but are not limited to these. Furthermore, the vector may contain a selection marker for selecting host cells containing the vector, and if it is a replicable expression vector, it contains an origin of replication.

[0098] As used herein, the term "vector" refers to a delivery vehicle into which a nucleic acid sequence can be inserted for introduction into a cell capable of replicating the nucleic acid sequence. The nucleic acid sequence may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (e.g., bacteriophages). Those skilled in the art can construct vectors using standard recombinant techniques.

[0099] In one embodiment, when preparing the vector, expression regulatory sequences such as promoters, terminators, and enhancers, sequences for membrane targeting or secretion, and the like can be appropriately selected and combined in a variety of ways depending on the purpose, depending on the type of host cell in which the Fc domain mutant, or an antibody containing the same, or a fragment thereof having immunological activity is to be produced.

[0100] "Expression vector" refers to a vector containing a nucleic acid sequence that encodes at least a portion of a transcribed gene product. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors can contain various regulatory sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors can also contain nucleic acid sequences that serve other functions.

[0101] "Host cell" refers to any transformable organism, including eukaryotes and prokaryotes, that can replicate the vector or express genes encoded by the vector. A host cell may be transfected or transformed by the vector, which refers to the process by which an exogenous nucleic acid molecule is delivered or introduced into a host cell.

[0102] In one embodiment, the host cell may be a bacterium or an animal cell, the animal cell line may be a CHO cell, a HEK cell or an NSO cell, and the bacterium may be E. coli.

[0103] In one aspect, the invention relates to a fusion protein in which the Fc domain variant of the invention, or an antibody, or an immunologically active fragment thereof, is linked to a cargo molecule.

[0104] In one embodiment, the cargo molecule may be a detection agent, a therapeutic agent, a drug, a peptide, a growth factor, a cytokine, a receptor trap, a chemical compound, a carbohydrate moiety, an enzyme, an antibody or fragment thereof, a DNA-based molecule, a viral vector, or a cytotoxic agent; one or more liposomes or nanocarriers loaded with a detection agent, a therapeutic agent, a drug, a peptide, an enzyme, an antibody or fragment thereof, a DNA-based molecule, a viral vector, or a cytotoxic agent; or one or more nanoparticles, nanowires, nanotubes, or quantum dots.

[0105] In one embodiment, the fusion protein may be an agonist antibody, an antagonist antibody, or an antibody therapeutic.

[0106] The present invention provides antibody-drug conjugates in which the antibody of the present invention or a fragment thereof having immunological activity is conjugated to one or more drugs.

[0107] In one embodiment, the drug may be an immune checkpoint inhibitor or a bispecific immune cell engager and may have reduced effector function.

[0108] In one embodiment, the drug is a chimeric antigen receptor (CAR) cell therapy, an oncolytic drug, an immunotherapy, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a costimulatory molecule blocker, an adhesion molecule blocker, an anti-cytokine agent, a nucleic acid therapy, an anti-CTLA-4 agent, an anti-PD-1 agent, an anti-PD-L1 agent, an anti-PD-L2 agent, a TNF-α cross-linking agent, a TRAIL cross-linking agent, an anti-CD27 agent, an anti-CD30 agent, an anti-CD40 agent, an anti-4-1BB agent, an anti-GITR agent, an anti-OX40 agent, an anti-TRAILR1 agent, an anti-TRAILR2 agent, a TAGR agent, an anti-TRAILR3 agent, an anti-TRAILR4 agent, an anti-TRAILR5 agent, an anti-TRAILR6 agent, an anti-TRAILR7 agent, an anti-TRAILR8 agent, an anti-TRAILR9 agent, an anti-TRAILR10 agent, an anti-TRAILR11 agent, an anti-TRAILR12 agent, an anti-TRAILR12 agent, an anti-TRAILR13 agent, an anti-TRAILR14 agent, an anti-TRAILR15 agent, an anti-TRAILR16 agent, an anti-TRAILR17 agent, an anti-TRAILR18 agent, an anti-TRAILR19 agent, an anti-TRAILR19 agent, an anti-TRAILR19 agent, an anti-TRAILR19 agent, an anti-TRAILR110 agent, an anti-TRAILR120 agent, an anti-TRAILR111 agent, an anti-TRAILR121 agent, an anti-TRAILR131 agent, an anti-TRAILR142 agent, an anti-TRAILR152 agent, an anti-TRAILR162 agent, an anti-TRAILR173 agent, an anti-TRAILR1 tin, interferon-α, clobetasol, peginterferon, prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, antichemokines, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable labels or reporters, TNF antagonists, antirheumatic drugs, muscle relaxants, narcotics, non-steroidal anti-inflammatory drugs (NSAIDs) anti-inflammatory drug), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antibacterial, antipsoriatic agent, corticosteroid, anabolic steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormone replacement, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma medication, beta agonist, inhaled steroid, epinephrine or analog thereof, cytokine, cytokine antagonist, PD-1 antagonist, adenosine A2AR antagonist, CD73 inhibitor, CTLA-4 inhibitor, TIM-3 inhibitor, LAG-3 inhibitor, anthracycline, antisense oligonucleotide (ASO), siRNA, aptamer, or combinations thereof.

[0109] In one aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient a human antibody Fc domain variant, an antibody comprising the variant, or a fragment thereof having immunological activity, or an antibody therapeutic agent comprising the variant.

[0110] In one embodiment, the cancer may be any one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, esophageal cancer, lymphatic gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system malignant lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0111] In one embodiment, the composition of the present invention may further comprise an immunogenic cell death inducer, which may be any one or more selected from the group consisting of anthracycline anticancer agents, taxane anticancer agents, anti-EGFR antibodies, BK channel agonists, bortezomib, cardiac glycosides, cyclophosmid anticancer agents, GADD34 / PP1 inhibitors, LV-tSMAC, measles virus, bleomycin, mitoxantrone, and oxaliplatin. The anthracycline anticancer drug may be daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, sabarubicin, or valrubicin. The taxane anticancer drug may be paclitaxel or docetaxel.

[0112] When the pharmaceutical composition for treating or preventing cancer of the present invention is administered together with a chemical anticancer drug (anticancer agent), it can enhance the cancer therapeutic effect of the conventional anticancer agent by killing cancer cells. The combined administration may be performed simultaneously or sequentially with the anticancer agent. Examples of anti-cancer drugs include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin, and carboplatin; anti-cancer antibiotics such as dactinomycin (actinomycin D), plicamycin, and mitomycin C; and plant alkaloids. Alkaloids include, but are not limited to, vincristine, vinblastine, etoposide, teniposide, topotecan, and iridotecan.

[0113] "Prevention" refers to any action that inhibits or delays the onset, spread, and recurrence of cancer by administering the pharmaceutical composition according to the present invention.

[0114] "Treatment" refers to any action that kills cancer cells or improves or beneficially changes the symptoms of cancer by administering the composition of the present invention. Those skilled in the art will be able to determine the exact criteria for diseases for which the composition of the present invention is effective and the degree of improvement, enhancement, and treatment by referring to materials provided by the Korean Medical Association, etc.

[0115] The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to the amount of a pharmaceutically acceptable salt of the composition that is effective for preventing or treating a target disease. The therapeutically effective amount of the composition of the present invention varies depending on various factors, such as the administration method, the target site, and the patient's physical condition. Therefore, when used in humans, the dosage must be determined as an appropriate amount taking into consideration both safety and efficacy. It is also possible to extrapolate the dosage for humans from the effective amount determined through animal experiments. Such considerations when determining the effective amount are described, for example, in Hardman and Limbird, eds., Goodman and Gilman's *The Pharmacological Basis of Therapeutics*, 10th ed. (2001), Pergamon Press; and E.W. Martin, ed., *Remington's Pharmaceutical Sciences*, 18th ed. (1990), Mack Publishing Co.

[0116] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount," as used herein, refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to any medical treatment without causing adverse effects. The effective dose level can be determined based on factors including the patient's health status, the type and severity of cancer, the activity of the drug, its sensitivity to the drug, the method, time, and route of administration, the excretion rate, the duration of treatment, and other factors well known in the medical field. The compositions of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents. They may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer the minimum amount necessary to achieve maximum efficacy without adverse effects, and this amount can be easily determined by one of ordinary skill in the art.

[0117] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable additive. Examples of pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, candy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carbonauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive of the present invention is preferably contained in an amount of 0.1 to 90 parts by weight of the composition, but is not limited thereto.

[0118] The compositions of the present invention may also contain carriers, diluents, excipients, or combinations of two or more thereof commonly used in biological preparations. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition. For example, compounds listed in the Merck Index, 13th ed., Merck & Co. Inc., saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components may be used. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to the compositions to formulate them into injection forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets. Furthermore, suitable formulations may be prepared according to the disease or component using appropriate methods in the art or the methods disclosed in Remington's Pharmaceutical Sciences (Mack Publishing Company, Easton PA, 18th, 1990).

[0119] The composition of the present invention may be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically in the form of an injection) or orally, depending on the intended method. The dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and disease severity. The daily dosage of the composition of the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and more preferably administered once or in divided doses per day.

[0120] Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and may contain, in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.

[0121] The present invention provides a method for treating cancer, comprising administering to a subject a human antibody Fc domain variant; an antibody or an immunologically active fragment thereof comprising the variant; an antibody-drug conjugate; or the pharmaceutical composition.

[0122] In one aspect, the present invention provides a method for producing a human antibody Fc domain variant, comprising the steps of: a) culturing a host cell containing a vector comprising a nucleic acid molecule encoding a human antibody Fc domain variant of the present invention; and b) recovering the polypeptide expressed by the host cell.

[0123] In one aspect, the invention provides a method for producing an antibody with reduced effector function, comprising the steps of: a) culturing a host cell containing a vector comprising a nucleic acid molecule encoding an antibody of the invention or an immunologically active fragment thereof; and b) purifying the expressed antibody from the host cell.

[0124] In one embodiment, antibody purification includes filtration, HPLC, anion or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably affinity chromatography using Protein A.

[0125] The antibodies of the present invention or immunologically active fragments thereof can be used for target detection and imaging.

[0126] The present invention provides a composition for target detection, which comprises a conjugate in which an antibody or a fragment thereof having immunological activity is bound to a functional substance.

[0127] The target may be an antigen, antibody, drug, biomarker, etc.

[0128] Target detection and imaging can diagnose a condition or disease in an individual.

[0129] The functional substance may be one or more selected from the group consisting of a contrast agent, a radioisotope, a fluorescent marker, a stain, an enzyme, a polypeptide, a nucleic acid, a carbohydrate, and a lipid.

[0130] Contrast agents include lipiodol, ioxithalamic acid, iohexol, iopamidol, ioversol, iodixanol, gadobutrol, gadoterate meglumine, gadodiamide, gadobenate meglumine, barium sulfate, and the like.

[0131] Radioactive isotopes include: 3 H, 18 F, 11 C. 14 C. 15 O. 13 N, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Includes Re etc.

[0132] Fluorescent markers include green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), and the like.

[0133] Dyeing substances include acridine dyes, cyanine dyes, fluorine dyes, oxazine dyes, phenanthridine dyes, rhodamine dyes, and the like.

[0134] Enzymes include β-glucuronidase, β-D-glucosidase, urease, peroxidase, alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, and the like.

[0135] In one embodiment, the presence or absence and stage of disease in an individual can be diagnosed by detecting an antigen using the target detection composition of the present invention.

[0136] In one embodiment, the target detection compositions of the present invention can be used to detect antigens, thereby monitoring the therapeutic effectiveness of drugs administered to an individual.

[0137] The target detection composition of the present invention can be applied to various analytical methods using immune responses. Examples of analytical methods include immunoassays and immunodetection methods. Immunoassays and immunodetection methods include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassay (FIA), chemiluminescent immunoassay, and Western blotting.

[0138] The target detection compositions of the present invention can be used for in vivo detection (imaging) methods, such as scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), near-infrared (NIR) optical imaging, and magnetic resonance imaging (MRI).

[0139] Diseases that can be diagnosed include, for example, cancer, cardiovascular disease, neurological disorders, diabetes, autoimmune diseases, inflammatory diseases, viral infections, and allergies.

[0140] The target detection composition of the present invention can include a reagent for carrying out an antigen-antibody reaction or a reagent for detecting the reaction. The reagent for carrying out the antigen-antibody reaction can include a buffer, salts, etc. The reagent for detecting the reaction can include preparations commonly used in immunoassays or immunodetection methods, such as a monoclonal antibody, its antibody fragment, or a labeled secondary antibody that recognizes the conjugate, and a substrate corresponding to the label.

[0141] The present invention will now be described in more detail with reference to examples.

[0142] Example Example 1. Construction of a mammalian cell display system for screening of glycosylated Fc variants To discover novel glycosylated Fc variants capable of ablation of immune mechanisms, we constructed an Fc library. Specifically, we utilized the FLP-FRT gene recombination system, which is used to generate stable cell lines, to display glycosylated Fc domains on the surface of mammalian cells and stably screen them. Furthermore, for cell membrane display, the platelet-derived growth factor receptor (PDGFR) transmembrane domain was fused to the C-terminus of the Fc. The Fc-PDGFR gene was prepared by cloning into the pcDNA5 / FRT plasmid (Invitrogen, V601020) containing an FRT site. This plasmid was co-transfected with pOG44 plasmid (Invitrogen, V600520), which expresses the FLP enzyme responsible for genetic recombination, into CHO cells (Invitrogen, R75807) containing an FRT locus. Genetic recombination was induced, resulting in integration of the Fc-PDGFR DNA into the chromosomal DNA of the CHO cells, resulting in the generation of a CHO cell line stably expressing glycated Fc. To select CHO cells in which genetic integration had occurred after transfection, a hygromycin B resistance gene was also integrated, and the cells were treated with 500 μg / ml hygromycin B (Invitrogen, 10687010) to select and generate CHO cell lines stably expressing glycated Fc (Figures 1a and 1b).

[0143] Example 2. Expression and purification of FcγRs Fc gamma receptors (FcγRs) were produced to select Fc variants that completely eliminate the antibody-mediated immune mechanism by FACS screening of cell lines stably expressing the glycosylated Fc library (stable cell lines) using the glycosylated Fc CHO cell display system established in Example 1. Specifically, streptavidin was fused to the C-terminus of each receptor to prepare a tetrameric FcγRIIIa-158V-streptavidin-His, which improves the visible binding affinity with Fc and enables efficient FACS screening. Furthermore, to analyze the binding affinity of the discovered glycated Fc variants to FcγRs by ELISA, we produced the necessary tetrameric FcγRI-streptavidin-His, FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, and FcγRIIIa-158F-GST. We also produced FcRn-GST, which is required to analyze the FcRn binding affinity, which is involved in the in vivo half-life of antibodies. To produce each receptor protein, each protein produced above was cloned into an animal cell expression vector and then transfected into Expi293F cells using PEI and cultured for 7 days at 37°C, 125 rpm, and 8% CO2. After incubation, the supernatant was collected, equilibrated with PBS, and purified by Ni-NTA (Anti-His) or anti-GST affinity chromatography. SDS-PAGE gel analysis confirmed that highly purified tetrameric FcγRI-streptavidin, tetrameric FcγRIIIa-158V-streptavidin, FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, FcγRIIIa-158F-GST, and FcRn-GST were obtained (Figures 2a-e).

[0144] Example 3. Labeling and validation of FcγRs For FACS screening of the produced proteins, tetrameric FcγRIIIa-streptavidin was labeled with Alexa647 (Invitrogen, A20173) fluorescent dye. To confirm the expression and level of the displayed Fc variants, FITC (Invitrogen, F6434) was conjugated to protein A (Amicogen, 1070020), which does not overlap with the FcγR binding site. The conjugation of fluorescent dyes (Alexa647 and FITC) was performed according to the manufacturer's instructions. The fluorescently labeled tetrameric FcγRIIIa-streptavidin-Alexa647 and protein A-FITC were then induced to bind to wild-type Fc displayed on CHO cells, and their activity was confirmed. The fluorescent signal from Protein A-FITC conjugation confirmed that Protein-FITC had normal activity and that wild-type Fc displayed in CHO cells was stably expressed (Figure 3a). Furthermore, tetrameric FcγRIIIa-streptavidin-Alexa647 also showed normal binding to wild-type Fc (Figure 3b). These results demonstrate that Alexa647-labeled FcγRIIIa also exhibited excellent activity, verifying that the Fc displayed in CHO cells was normally expressed and functioning properly.

[0145] Example 4. Preparation of glycated Fc variants based on goat IgG Fc sequence Among IgG antibodies derived from various animals, goat IgG has very low cross-reactivity with human FcγRs (ST Jung et al. (2010) "Aglycosylated IgG variants expressed in bacteria that selectively bind FcγRI potentiate tumor cell killing by monocyte-dendritic cells", Proceedings of the National Academy of Sciences USA (PNAS), 107:2, 604-609; ST Jung, et al. (2013) "Effective phagocytosis of low Her2 tumor cell lines with engineered, aglycosylated IgG displaying high FcγRIIa affinity and selectivity", ACS Chemical Biology, 8:2, 368-375; M. Jo et al. (2018) "Engineered (See "Aglycosylated full-length IgG Fc variants exhibiting improved FcγRIIIa binding and tumor cell clearance," mAbs, 2:10, 278-289; HW Yoon et al. (2019) "Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies," Molecular Immunology, 114, 62-71.) Therefore, we compared the sequences of goat IgG Fc and human IgG Fc and introduced mutations mainly in four regions of IgG antibody Fc predicted to interact with human FcγR (lower hinge, B / C loop, C' / E loop, and F / G loop) to create human glycosylated Fc variants derived from the goat IgG Fc sequence (Figure 4).

[0146] Example 5. Animal cell expression and purification of trastuzumab Fc variants, including glycated Fc variants The glycosylated Fc variants prepared in Example 4 were cloned into the heavy chain gene of the model antibody trastuzumab to prepare an expression vector. First, the heavy chain gene and light chain gene of the variants were mixed at a 1:1 ratio in Freestyle 293 expression medium (Gibco, 12338-018). Next, PEI (polyethylenimine, Polyscience, 23966) was mixed with the expression vector gene at a 4:1 ratio and incubated at room temperature for 20 minutes. The mixture was then diluted with 2 x 10 6 The transfection was performed on Expi293F animal cells cultured at a density of 1000 cells / ml. The cells were cultured at 37°C, 125 rpm, and 8% CO2 for 7 days, then centrifuged to collect the supernatant. The supernatant was equilibrated with 25x PBS and filtered through a 0.2 μm syringe filter. Protein A resin was added to the filtered culture medium containing the trastuzumab Fc variants and stirred at 4°C for 16 hours. The resin was then spun down and washed with 2 ml of 1x PBS. The resin was eluted with 300 μl of 100 mM glycine (pH 2.7) and neutralized with 100 μl of 1 M Tris-HCl (pH 8.0). Amicon Ultra-4 centrifugal filter units 30K (Merck Millipore, UFC503096) were used to exchange the buffer, and highly purified glycated antibody trastuzumab Fc variants were obtained by SDS-PAGE analysis (Figures 5a and 5b).

[0147] Example 6. Analysis of human FcγR binding ability of glycated Fc variants ELISA analysis was performed to confirm the FcγRs binding ability of the glycated trastuzumab Fc variants purified in Example 5. Specifically, 50 μl of each FcγRs-GST (FcγRI-GST and FcγRIIIa-158V-GST) diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) and immobilized at 4°C for 16 hours. After blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour, the plate was washed four times with 180 μl of 0.05% PBST. Then, 50 μl of each of the glycated trastuzumab Fc variants serially diluted with 1% skim milk was dispensed into each well and incubated at room temperature for 1 hour. After washing, 50 μl of HRP-Protein L (GenScript, M00098) was added to each well, and the plates were incubated at room temperature for 1 hour. After washing, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to develop color. The reaction was terminated by adding 50 μl of 2M H2SO4 to each well, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0148] As a result, it was confirmed that among the glycated Fc mutants, only the Fc mutant with L235P introduced had eliminated FcγRI (Figure 6a) binding ability and had reduced binding ability to FcγRIIIa-158V compared to wild-type IgG1 (Figure 6b).

[0149] Example 7. Construction of a glycosylated Fc mutant library to eliminate FcγRs binding ability Based on the L235P mutant constructed in Example 6, glycosylated Fc engineering was performed using a CHO cell Fc display system established to completely eliminate binding to FcγRIIIa-158V. Specifically, to select glycosylated Fc mutants with eliminated FcγRs binding, a CHO cell-based glycosylated antibody Fc mutant library was constructed by introducing mutations into four Fc regions (lower hinge, B / C loop, C' / E loop, and F / G loop) that have a significant impact on FcγRs binding to L235P-introduced human IgG1 Fc (Figure 7). The library genes were co-transfected with an FLP expression plasmid, as in the construction of the cell display system described in Example 1, and then subjected to a selection process using hygromycin B medium to generate CHO cell lines stably expressing the glycosylated Fc mutant library.

[0150] Example 8. Selection and production of FcγRIIIa binding-depleted Fc variants using CHO cell display To select glycated Fc variants with ablated FcγRIIIa binding, the CHO cell Fc display system and CHO cell lines stably expressing the Fc library established in the previous example were induced to bind FcγRIIIa-Alexa647 and protein A-FITC. Protein A-FITC expression levels and FcγRIIIa binding were then simultaneously monitored, and populations predicted to exhibit ablated FcγRIIIa binding were gated and selected by FACS screening (Figure 8). The selected glycated Fc variant-expressing CHO cells were recovered by genomic DNA prep, and the nucleotide sequences of the selected engineered glycated Fc variants were confirmed (Table 1). Highly purified glycated antibody trastuzumab Fc variants were produced and purified by the method described in Example 5 to analyze the FcγRs binding of the selected engineered glycated Fc variants (LP1, LP5, LP6, LP7, and LP8) by ELISA (Figure 9).

[0151] [Table 1]

[0152] Example 9. Analysis of human FcγR binding ability of glycated trastuzumab Fc variants ELISA analysis was performed to confirm the FcγRs binding ability of the glycosylated trastuzumab Fc variants (LP1, LP5, LP6, LP7, and LP8) purified in Example 8. Specifically, 50 μl of each of the FcγRs-GSTs (FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, and FcγRIIIa-158F-GST) diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-bind 96-well microplate (Costar, 3590) and immobilized at 4°C for 16 hours. After washing, the wells were blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. Each well was washed four times with 180 μl of 0.05% PBST. After four washes, 50 μl of glycated trastuzumab Fc variants (LP1, LP5, LP6, LP7, and LP8) serially diluted in 1% skim milk were dispensed into each well and incubated at room temperature for 1 hour. After washing, the wells were incubated with 50 μl of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, followed by further washing. Color development was achieved by adding 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) to each well. The reaction was terminated by adding 50 μl of 2M H2SO4 to each well, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0153] The results showed that the glycated trastuzumab Fc mutants discovered in the above Examples, unlike the LALA(L234A / L235A) Fc mutant, significantly reduced binding to FcγRI and FcγRIIIa-158V. The glycated trastuzumab Fc mutants in the above Examples were also confirmed to have reduced binding to FcγRIIa-131H and FcγRIIa-131R compared to the LALA(L234A / L235A) Fc mutant (Figures 10a to 10c).

[0154] Example 10. Construction of glycated Fc mutants with reduced FcγRs binding ability In the previous example, because the L235P mutant retained binding to FcγRIIIa-158V, additional mutations were introduced to reduce binding to all FcγRs. To this end, the L234A mutant was combined with L235P to generate the glycosylated Fc mutant LALP (L234A and L235P) (Table 2).

[0155] [Table 2]

[0156] Example 11. Production of glycated Fc mutants with reduced FcγRs binding ability and analysis of human FcγRs binding ability In order to analyze the binding ability of the glycated Fc mutants LALP (L234A / L235P) and LALA (L234A / L235A) Fc mutants prepared in Example 10 to FcγRs by ELISA, highly purified glycated antibody trastuzumab Fc mutants were produced and purified using the method described in Example 5 (Figure 11). To confirm the FcγR binding ability of the purified glycosylated trastuzumab Fc variants, 50 μl of each FcγRs-GST (FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, and FcγRIIIa-158F-GST) diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-binding 96-well microplate (Costar, 3590) and immobilized for 16 hours at 4°C. After blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour, the wells were washed four times with 180 μl of 0.05% PBST. 50 μl of glycated trastuzumab Fc variants serially diluted in 1% skim milk were added to each well and incubated at room temperature for 1 hour. After washing, 50 μl of HRP-Protein L (GenScript, M00098) was added to each well and incubated at room temperature for 1 hour, followed by washing. Color development was achieved by adding 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028). The reaction was terminated by adding 50 μl of 2M H2SO4 to each well, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0157] The results showed that the LALP (L234A / L235P) Fc mutant exhibited significantly reduced FcγR binding compared to wild-type IgG1 Fc, and was significantly less potent than the LALA (L234A / L235A) Fc mutant, which is currently widely used in clinical settings to reduce the side effects of immune cell destruction. It was confirmed that the binding affinity to FcγRIIa-131H, FcγRIIa-131R, and FcγRIIIa-158V was reduced (FIGS. 12a to 12c).

[0158] Example 12. Construction of a glycosylated Fc mutant library to completely eliminate FcγRs binding ability The glycated Fc variant LALP (L234A and L235P) prepared in Example 10 exhibited reduced FcγRIIIa-158V binding affinity compared to the LALA (L234A / L235A) Fc variant, but some binding affinity remained. Therefore, to completely eliminate binding affinity, we performed glycated Fc engineering using an established CHO cell display system. Specifically, to select glycated Fc variants with eliminated FcγRs binding affinity, we constructed a CHO cell-based glycated antibody Fc variant library by introducing mutations into four Fc regions (lower hinge, B / C loop, C' / E loop, and F / G loop) that have a significant impact on FcγRs binding to LALP (L234A / L235P)-introduced human IgG1 Fc (Figure 7). The library genes were co-transfected with an FLP expression plasmid in the same manner as in the cell display system construction method described in Example 1, and then a selection process using hygromycin B medium was carried out to generate a CHO cell line stably expressing the glycated Fc variant library.

[0159] Example 13. Selection and production of Fc mutants that completely eliminate FcγRs binding ability using CHO cell display To select glycated Fc mutants with abolished FcγRIIIa binding, the CHO cell Fc display system and CHO cell lines stably expressing the Fc library established in the previous examples were induced to bind FcγRIIIa-Alexa647 and protein A-FITC. Protein A-FITC expression levels and FcγRIIIa binding were then simultaneously monitored, and populations predicted to exhibit abolished FcγRIIIa binding were gated and selected by FACS screening (Figure 8). The selected glycated Fc mutant-expressing CHO cells were recovered by genomic DNA prep, and the nucleotide sequences of the selected engineered glycated Fc mutants were confirmed (Table 3). To analyze the FcγRs binding ability of the selected engineered glycated Fc variants (LALP1, LALP5, LALP6, LALP7, and LALP8) by ELISA, highly purified glycated antibody trastuzumab Fc variants were produced and purified by the method described in Example 5 (Figure 13).

[0160] [Table 3]

[0161] Example 14. Analysis of human FcγRs binding ability of glycated trastuzumab Fc mutants that completely eliminate FcγRs binding ability ELISA analysis was performed to confirm the FcγRs binding ability of the glycated trastuzumab Fc variants (LALP1, LALP5, LALP6, LALP7, and LALP8) purified in Example 13. Specifically, 50 μl of FcγRs-GST (FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, and FcγRIIIa-158F-GST) diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-binding 96-well microplate (Costar, 3590) and immobilized at 4 °C for 16 hours. After washing, 100 μl of 4% skim milk (GenomicBase, SKI400) was added and blocked at room temperature for 1 hour. The wells were washed four times with 180 μL of 0.05% PBST. After four washes, 50 μL of glycated trastuzumab Fc variants (LALP1, LALP5, LALP6, LALP7, and LALP8) serially diluted in 1% skim milk were added to each well and incubated at room temperature for 1 hour. After washing, the wells were incubated with 50 μL of HRP-Protein L (GenScript, M00098) for 1 hour at room temperature and washed again. Color development was achieved by adding 50 μL of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) to each well. The reaction was terminated by adding 50 μL of 2M H2SO4 to each well, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0162] As a result, it was confirmed that the glycated trastuzumab Fc mutant discovered in the above example completely eliminated binding to all FcγRs compared to wild-type IgG1 and the conventional mutant LALA (L234A / L235A) (Figures 14a to 14c).

[0163] Example 15. Analysis of thermal stability of glycated trastuzumab Fc mutants that completely eliminate FcγRs binding ability To assess the thermostability of glycated trastuzumab Fc variants (LALA, LP1, LP5, LP6, LP7, LP8, LALP, LALP1, LALP5, LALP6, LALP7, and LALP8), differential scanning fluorimetry (DSF) analysis was performed. Specifically, 45 μl of each glycated trastuzumab Fc variant diluted to 5 μM in 1x PBS was mixed with 5 μl of SYPRO Orange (Invitrogen, S6651) dye diluted at 200x, and the mixture was dispensed into a PCR plate (Thermo Scientific, AB0900W). A control, 1x PBS, was also prepared in the same manner (all samples were run in triplicate). An optically clear sealing film (Thermo Scientific, AB1170) was attached to the plate containing the sample, and the fluorescence intensity was measured using a QuantStudio 3 Real-Time PCR System (Applied Biosystems, A28567) while increasing the temperature from 25°C to 99.9°C at a rate of 0.03°C per second. The fluorescence values ​​at each temperature were fitted with a Boltzmann model using OriginPro software, and the midpoint of the sigmoidal transition curve was calculated.

[0164] As a result, it was confirmed that the discovered glycated trastuzumab Fc mutants, except for the LP8 (L235P / D265L / P331L) Fc mutant and the LALP8 (L234A / L235P / D265L / P331L) Fc mutant, had decomposition temperatures similar to that of wild-type IgG1 (Figures 15 and 16).

[0165] Example 16. Analysis of human FcRn binding ability of glycated trastuzumab Fc mutants that completely eliminate FcγRs binding ability To determine the FcRn binding affinity of glycated trastuzumab Fc variants, which contributes to their in vivo half-life, we performed a pH-dependent ELISA. Specifically, 50 μl of each glycated trastuzumab Fc variant diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-binding 96-well microplate (Costar, 3590) and immobilized at 4°C for 16 hours. Then, the plate was blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST (pH 6.0 / pH 7.4), 50 μl of serially diluted FcRn-GST in 1% skim milk (pH 6.0 / pH 7.4) was added to each well and incubated at room temperature for 1 hour. After washing, the plates were incubated with 50 μl of anti-GST-HRP conjugate (GE Healthcare, RPN1236V) at room temperature for 1 hour and washed again. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each plate to develop color. 50 μl of 2M H2SO4 was added to each plate to terminate the reaction, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0166] As a result, it was confirmed that the glycated trastuzumab Fc variants of the present invention, LALP, LALP1, LALP5, and LALP6, maintain the FcRn binding properties of wild-type IgG1 (FIG. 17).

[0167] Furthermore, it was confirmed that the glycosylated trastuzumab Fc variants LP1, LP5, LP6 and LP7 of the present invention maintain the FcRn binding properties of wild-type IgG1 (FIGS. 18a and 18b).

[0168] Example 17. Analysis of human C1q binding ability of glycated trastuzumab Fc mutants that completely eliminate FcγRs binding ability To confirm the C1q binding ability of the trastuzumab Fc variants of the present invention, ELISA analysis was performed. Specifically, 50 μl of each glycated trastuzumab Fc variant diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-binding 96-well microplate (Costar, 3590) and immobilized at 4°C for 16 hours. The plate was then blocked with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour. After washing four times with 180 μl of 0.05% PBST, 50 μl of C1q (Quidel, A400) protein serially diluted in 1% skim milk was dispensed into each well and incubated at room temperature for 1 hour. After washing the plate, 50 μl of anti-Clq-HRP (Invitrogen, PA1-84324) was added to each well, incubated at room temperature for 1 hour, and washed again. After adding 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) to each well to develop color, the reaction was terminated by adding 50 μl of 2 M H2SO4 to each well, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0169] As a result, it was confirmed that the binding ability to C1q was also effectively eliminated for all of the discovered glycosylated trastuzumab Fc variants (LALP, LALP1, LALP5, LALP6, and LALP7) (FIG. 19).

[0170] It was also confirmed that the binding ability of LP1, LP5, LP6, LP7 and LP8 to C1q was effectively eliminated (FIG. 20).

[0171] Example 18. Analysis of human FcγRs binding ability of glycated trastuzumab Fc mutants that completely eliminate FcγRs binding ability 18-1. Construction and production of control glycosylated trastuzumab Fc mutant To compare the binding activity of the LALAPG (L234A / L235A / P329G) Fc mutant (Reference: WO2012130831A1), which is currently under clinical development, to FcγRs with the glycosylated Fc mutants of the present invention, the LALAPG (L234A / L235A / P329G) Fc mutant was constructed and produced and purified by the method described in Example 5 (Figure 21a).

[0172] 18-2. Analysis of human FcγRI binding affinity based on HER2-expressing target cells To confirm and compare the FcγRI binding ability of the glycosylated trastuzumab Fc variants (LP1, LP5, LP6, and LP7) and the LALA prepared in Example 18-1 by reproducing the in vivo mechanism of action, a binding analysis was performed using SKOV-3 cells expressing HER2, the target antigen of the trastuzumab Fc variants. 6 SKOV-3 cells were treated with 100 nM each of trastuzumab WT, trastuzumab LALA mutant, and glycosylated trastuzumab Fc variants of the present invention (LP1, LP5, LP6, and LP7), and then induced with 50 nM tetrameric FcγRI-Alexa647 and Protein A-FITC at a 1:1000 ratio or 100 nM tetrameric FcγRI-Alexa647 and Protein A-FITC at a 1:1000 ratio, and incubated for 1 hour. After washing with 1x PBS / EDTA, expression levels and removed FcγRI binding capacity were analyzed by FACS using Protein A-FITC.

[0173] As a result, it was confirmed that the glycated trastuzumab Fc variant of the present invention not only has lower FcγRI binding affinity than the conventional Fc variant LALA (L234A / L235A), but also has a binding strength similar to that of human serum IgG used as a negative control (Figure 21b).

[0174] 18-3. Analysis of human FcγR binding affinity using Octet The FcγRs binding ability of glycated trastuzumab Fc variants (LALP1, LALP5, and LALP6) and LALAPG prepared in Example 18-1 was analyzed using Octet to confirm and compare the in vivo mechanism of action.

[0175] OCTET is a method of measuring the binding between biological molecules by applying the phenomenon of light interference with a BLI (Bio-Layer Interferometry) system, and compares the difference in the interference pattern of white light reflected from the surface of a biocenter chip with and without a ligand. In other words, the binding and dissociation of two molecules was measured, and the difference in binding between molecules was analyzed.

[0176] Specifically, a FAB2G sensor (a sensor chip that binds to the CH1 domain of human IgG) was loaded with an Fc mutant-introduced trastuzumab model antibody diluted in 1x kinetic buffer or 1x PBS. Monomeric FcγRs diluted in 1x kinetic buffer or 1x PBS were run at different concentrations to measure the affinity between the antibody and FcγR. For FcγRI analysis, the antibody and FcγRI were diluted in 1x kinetic buffer, and the FcγRI was analyzed at a concentration of 62.5 nM in two-fold increments starting from 500 nM. For other FcγRs analysis, both the antibody and FcγRs were diluted in 1x PBS, and the FcγRs were analyzed at a concentration of 625 nM in two-fold increments starting from 5000 nM.

[0177] The results of the analysis are reported in R eq The calculated response at equilibrium was confirmed by the difference in the interference pattern of white light when a signal was present. eq is the signal when the binding and dissociation rates of the two molecules are in equilibrium, and R eq The difference in the white light interference pattern in the presence of a signal is shown on the Y axis as Binding (nm).

[0178] As a result, the trastuzumab Fc variants of the present invention not only had lower FcγRI binding affinity than the conventional Fc variant LALA (L234A / L235A), but also lower FcγRI binding affinity than LALAPG (L234A / L235A / P329G), confirming the excellent effect of completely eliminating hFcγRI binding (Figure 22).

[0179] Furthermore, it was confirmed that the trastuzumab Fc variants of the present invention have significantly reduced binding affinity compared to FcγRIIIa-158F, which still retains strong binding affinity in LALA (L234A / L235A) and LALAPG (L234A / L235A / P329G) (Figure 23).

[0180] The trastuzumab Fc variants of the present invention had completely eliminated binding to FcγRI, FcγRIIa-131H, FcγRIIa-131R, FcγRIIb, FcγRIIIa-158V, and FcγRIIIa-158F. In particular, it was confirmed that the trastuzumab Fc variants of the present invention also had completely eliminated binding to FcγRI and FcγRIIIa-158F, for which the LALAPG variant had binding affinity (Table 4).

[0181] [Table 4]

[0182] Example 19. Production of combinatorial glycated Fc variants and analysis of human FcγRs binding avidity To analyze whether the combination of the known P329G mutation with the LALP (L234A / L235P) mutation of the present invention reduces FcγR binding, we created and produced an LALPPG (L234A / L235P / P329G) Fc mutant (Table 5). Subsequently, we produced and purified a trastuzumab LALPPG mutant using the method described in Example 5 (Figure 24). ELISA analysis was performed to compare the FcγR binding ability of the purified trastuzumab LALPPG mutant and trastuzumab LALP mutant with that of the conventional trastuzumab LALA mutant and trastuzumab LALAPG mutant.

[0183] Specifically, 50 μl of each FcγRs-GST (FcγRI-GST, FcγRIIa-131H-GST, FcγRIIa-131R-GST, FcγRIIb-GST, FcγRIIIa-158V-GST, and FcγRIIIa-158F-GST) diluted to 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was placed in a flat-bottom polystyrene high-binding 96-well microplate (Costar, 3590) and immobilized for 16 hours at 4°C. After immobilization, 100 μl of 4% skim milk (GenomicBase, SKI400) was added and blocked for 1 hour at room temperature. Each well was washed four times with 180 μl of 0.05% PBST. After washing four times, 50 μl of glycated trastuzumab Fc variants serially diluted in 1% skim milk were added to each well and incubated at room temperature for 1 hour. After washing, 50 μl of HRP-Protein L (GenScript, M00098) was added and incubated at room temperature for 1 hour. The wells were then washed again. 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to each well to develop color. The reaction was terminated by adding 50 μl of 2M H2SO4 to each well, and the absorbance was analyzed using an Epoch microplate spectrophotometer (BioTek).

[0184] [Table 5]

[0185] The results confirmed that the LALPPG(L234A / L235P / P329G) Fc mutant did not bind to any FcγRs (Figures 25a to 25c). This demonstrates that the LALP(L234A / L235P) Fc mutant effectively eliminates FcγR binding activity even when combined with the known P329G mutant.

[0186] Example 20. Confirmation of PBMC-mediated ADCC depletion of glycated trastuzumab Fc variants The reduced effector function of the glycated Fc variants of the present invention was confirmed. Using real-time cell analysis, the glycated Fc variants were analyzed for hPBMC (human peripheral blood mononuclear cell)-mediated antibody-dependent cellular cytotoxicity (ADCC). Cell death was measured by passing an electric current through a cell culture vessel with a gold sensor attached to the bottom. Cell death was measured by taking advantage of the fact that when adherent cells cover the sensor surface, resistance is generated and current generation is low, whereas when cells are absent or dead, resistance is low and current generation is high. The cell death assay was performed using SKBR-3 cells expressing HER2, the target antigen of the trastuzumab Fc variants. SKBR-3 cells were seeded at 10,000 cells / well in RPMI (Gibco, 11875093) medium containing 10% FBS (Gibco, 16000044) onto an E-Plate (Agilent, 300600900) and then placed in an xCELLigence RTCA SP (Agilent, 300600900) for cell adhesion and proliferation. PBMCs were isolated from whole blood obtained from healthy donors using Histopaque 1077 (Sigma Aldrich, 10771), washed with 1x PBS, and diluted in RPMI medium containing 10% FBS. PBMCs and antibodies were added to the E-Plate and incubated at room temperature for 30 minutes to allow cell precipitation. The E-plate was placed in the RTCA instrument and monitored in real time over time using RTCA software 1.2 (Agilent) at 37°C and 5% CO2. Cytolysis was analyzed at various time points. hPBMC and antibody-mediated cell killing was analyzed by measuring the decrease in resistance, and the results were expressed as % cytolysis using the following formula:

[0187] Cytolysis (%) = (resistance value of well administered with target cells alone - resistance value of sample well) / resistance value of well administered with target cells alone × 100

[0188] As a result, the glycosylated trastuzumab Fc variants of the present invention exhibited significantly lower cytolysis rates than the conventional Fc variant LALA (L234A / L235A), confirming a significant reduction in ADCC activity and demonstrating a more effective elimination of ADCC than the LALAPG (L234A / L235A / P329G) Fc variant (Figure 26).

[0189] Real-time cell imaging was also used to confirm cell death by ADCC. SKBR-3 cells were mixed with 2 μM Red CMTPX dye (Invitrogen, C34552) diluted in phenol red-free RPMI-1640 (Gibco, 11835030) medium and incubated at 37°C and 5% CO for 30 minutes. After labeling, cells were washed with 1× PBS and plated at 1× 10 SKBR-3 cells in phenol red-free RPMI medium containing 10% FBS and 1× Antibiotic-Antimycotic (Gibco, 15240062) on a black plate (Greiner, 655090). 4 Cells / well were added and incubated overnight in a 37°C, 5% CO2 incubator. The next day, PBMCs were isolated from whole blood obtained from healthy donors using Histopaque 1077. PBMCs were labeled with 0.5 μg / ml Hoechst 33342 (Invitrogen, H3570) diluted with 1×PBS for 5 minutes at room temperature and washed three times with 1×PBS. 1×10 PBMCs were added to each well. 5 After treating the cells / well with antibodies and Caspase-3 / 7 detection reagents (Invitrogen, C10423), the plate was placed in a LionheartFX (Agilent) instrument and cell death was imaged over time using a fluorescence microscope at 37°C and 5% CO2.

[0190] When cancer cells died (apoptosis) mediated by hPBMC (human peripheral blood mononuclear cell), such death was imaged as green dots, and ADCC activity was confirmed by counting the number of green dots (Figures 27a to 27c).

[0191] As a result, the glycated trastuzumab Fc variants of the present invention killed significantly fewer cells than the conventional Fc variant LALA (L234A / L235A) and LALAPG (L234A / L235A / P329G) Fc variants, confirming the reduced ADCC activity of the glycated trastuzumab Fc variants of the present invention ( Figure 27d ).

[0192] Example 21. Confirmation of NK92-mediated ADCC elimination of glycated rituximab Fc variants The glycosylated Fc variants of the present invention were introduced into a rituximab model antibody to analyze NK92-mediated antibody-dependent cellular cytotoxicity (ADCC). Ramos, a cancer cell line expressing the rituximab target antigen, and the NK92 cell line (ATCC, PTA8836) expressing GFP-FcγRIIIa were harvested and then subjected to cell down at 300 × g for 3 minutes and 125 × g for 10 minutes, respectively, followed by resuspension and washing in PBS. The prepared target and effector cells were plated in a V-bottom 96-well plate (Corning, 3894) at 20,000 cells / well for Ramos, 200,000 cells / well for GFP-FcγRIIIa-NK92 (E:T = 10:1), and antibody samples. The ADCC response was induced by incubating the cells at 37°C in a 5% CO2 incubator for 3 hours. After incubation, 5 nM Sytox Red (Invitrogen, S34859) dye was added and incubated at room temperature for 15 minutes to stain dead cells. After the reaction was completed, the plate was spun down at 200 × g for 5 minutes to collect the pellet, which was then washed twice with PBS and analyzed using a BD FACS Lyric (BioTek) instrument.

[0193] After staining the NK92-mediated dead cells, the number of dead cells was analyzed by FACS.

[0194] The results were expressed as cytotoxicity (%) using the following formula: Cytotoxicity (%) = (number of dead cells / total number of cells) × 100 - background

[0195] As a result, the glycated rituximab Fc variants of the present invention exhibited significantly lower cytotoxicity values ​​than the conventional Fc variant LALA (L234A / L235A), confirming a significant reduction in ADCC activity. The values ​​were lower than those of the LALAPG (L234A / L235A / P329G) Fc variant and human serum IgG used as a negative control, confirming that ADCC was effectively eliminated (Figure 28).

[0196] Example 22. Confirmation of ADCP reduction by glycated trastuzumab Fc variants To confirm the reduced effector function of the glycosylated Fc variants of the present invention, antibody-dependent cellular phagocytosis (ADCP) was analyzed. PBMCs were isolated from whole blood obtained from healthy donors using Histopaque 1077, followed by MACS (Miltenyi, 130-118-906) CD14 positive sorting to isolate monocytes. The isolated monocytes were treated with 50 ng / ml GM-CSF (PeproTech, 300-03) in RPMI medium containing 15% FBS and differentiated into macrophages at 37°C and 5% CO2 for one week. SKBR-3 cells labeled with 2 μM PKH67 (Sigma Aldrich, MIDI67-1KT) were incubated at 2 × 10 in RPMI medium diluted with 10% human serum. 4 The differentiated macrophages were harvested with Accutase (Innovative Cell Technologies, AT104) and then cultured at 1 × 10 5Cells were plated at 1000 cells / well and incubated with antibodies in each well at 37°C and 5% CO2 for 4 hours. They were then harvested with Accutase, washed with RPMI medium diluted with 10% human serum (Sigma Alrich, H6914-20ML), and fixed with 1% formaldehyde for 10 minutes at room temperature in the dark. They were then washed with 1x PBS and labeled with APC-anti-human CD14 antibody (BioLegend, 301807) and APC-CD11b antibody (BioLegend, 301309) for 30 minutes at room temperature in the dark. After washing, fluorescence was measured using a BD FACS Lyric instrument and expressed as % phagocytosis using the following formula:

[0197] Phagocytosis(%)=double positive / (target cell+double positive)×100

[0198] As a result, the glycated trastuzumab Fc variant of the present invention showed a lower phagocytosis rate than the wild-type trastuzumab Fc, confirming that ADCP was removed (FIG. 29).

[0199] Example 23. Confirmation of reduced CDC of glycated rituximab Fc variants To confirm the reduced effector function of the glycosylated Fc variants of the present invention, complement-dependent cytotoxicity (CDC) was analyzed using Daudi, a cancer cell line expressing the target antigen of rituximab. In a V-bottom 96-well plate, 5 × 10 cells of Daudi were cultured in phenol red-free RPMI-1640 (Gibco, 11835030) medium containing 20% ​​human complement serum (Sigma-Aldrich, S1764). 5Cells / well and each antibody were added and incubated for 1 hour at 37°C and 5% CO2. After incubation, lysed cells were labeled with FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, 640922) in Annexin V binding buffer (Biolegend, 422201) for 20 minutes in the dark at room temperature. After washing with Annexin V binding buffer, the cells were fixed with 3% formaldehyde for 10 minutes at room temperature and then washed in the same manner. The diluted samples were analyzed for fluorescence intensity and cytotoxicity (%) using a BD FACS Lyric system.

[0200] Cytotoxicity(%)=(pro-apoptotic cell+dead cell) / (total cell)×100-background

[0201] As a result, the glycosylated rituximab Fc variants of the present invention exhibited significantly lower cytotoxicity values ​​than the conventional Fc variants LALA (L234A / L235A) and LALAPG (L234A / L235A / P329G), confirming their superior effect in reducing CDC (Figure 30).

[0202] Example 24. Analysis of activity of glycated trastuzumab Fc variants in mice To confirm the FcRn binding ability of the glycated trastuzumab Fc variants of the present invention, which is involved in the in vivo half-life in mice, ELISA analysis was performed by the method described in Example 16 above using mouse FcRn-GST.

[0203] As a result, it was confirmed that the trastuzumab Fc mutants of the present invention maintain the FcRn binding properties of wild-type IgG1 (FIG. 31a).

[0204] Additionally, in vivo PK analysis was performed to understand the interactions of the glycosylated trastuzumab Fc variants of the present invention in vivo. Antibodies were intravenously injected into C57BL / 6 mice at 2 mg / kg, using five mice per variant. Blood was collected from each mouse at 1, 24, 168, 312, 504, 672, 840, 1,008, 1,176, and 1,344 hours post-injection, and the supernatant was centrifuged at 1,000 × g for 15 minutes to obtain serum. The serum was stored at -80°C and then subjected to ELISA analysis to measure the antibody concentration in the serum. HER2 diluted at 4 μg / ml in 0.05 M Na2CO3 (pH 9.6) was coated onto a high-binding plate and immobilized at 4°C for 16 hours. After blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 1 hour, the wells were washed four times with 180 μl of 0.05% PBST. Standards and serum serially diluted in 1% skim milk were then added to each well and incubated at room temperature for 1 hour. After washing, 50 μl of goat human IgG H+L (Jackson Immunoresearch, 109-036-003) was added to each well and incubated at room temperature for 1 hour, followed by washing. Color development was achieved by adding 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028). The reaction was terminated by adding 50 μl of 2M H2SO4 to each well, and the absorbance at 450 nm was measured using an Epoch microplate spectrophotometer (BioTek). The antibody concentrations were analyzed using a standard curve to confirm the concentrations of trastuzumab and glycated trastuzumab Fc variants over time. The results confirmed that the glycated trastuzumab Fc variants of the present invention maintain concentrations similar to those of wild-type trastuzumab and may have an in vivo half-life ( Figure 31b ).

[0205] Example 25. Analysis of mouse FcγR binding ability of glycated trastuzumab Fc variants The use of glycated trastuzumab Fc variants of the present invention in animal experiments was observed. ELISA analysis was performed using mouse FcγRs-GST according to the method described in Example 11 above to confirm the binding ability of the glycated trastuzumab Fc variants to mouse FcγRs.

[0206] As a result, it was confirmed that the glycosylated trastuzumab Fc variants of the present invention (LALP1, LALP5, and LALP6) completely eliminated binding to mFcγRI, mFcγRIIb, and mFcγRIII. Furthermore, binding to mFcγRIV, to which the LALA variant (L234A / L235A) has strong binding affinity, was also completely eliminated (Figures 32a and 32b).

[0207] Example 26. Analysis of binding affinity of glycated trastuzumab Fc variants to cynomolgus monkey FcγRs The use of glycated trastuzumab Fc variants of the present invention in animal experiments was observed. ELISA analysis was performed using cynomolgus monkey FcγRs-GST according to the method described in Example 11 above to confirm the binding ability of the glycated trastuzumab Fc variants to cynomolgus monkey FcγRs.

[0208] As a result, it was confirmed that the glycosylated trastuzumab Fc variants of the present invention (LALP1, LALP5, and LALP6) completely eliminated their binding to cFcγRIIa, cFcγRIIb, and cFcγRIII. Furthermore, the LALA variant (L234A / L235A) also completely eliminated its binding to cFcγRI and cFcγRIII, for which the LALA variant (L234A / L235A) has strong binding affinity (Figures 33a and 33b).

[0209] The glycated trastuzumab Fc variants of the present invention (LALP1, LALP5, LALAP6) were compared with wild-type IgG1, the LALA variant, and the LALAPG variant in terms of binding avidity to human FcγRs, mouse FcγRs, cynomolgus monkey FcγRs, and human C1q, binding avidity to FcRn, thermal stability (Tm), ADCC activity, ADCP activity, and CDC activity (Tables 6 and 7).

[0210]

Table 6

[0211]

Table 7

Claims

1. A human antibody Fc domain mutant in which amino acid 235 of the human antibody Fc domain is substituted with proline (P).

2. The human antibody Fc domain mutant of claim 1, wherein the human antibody Fc domain consists of the amino acid sequence of SEQ ID NO:

15.

3. The human antibody Fc domain mutant according to claim 1, wherein amino acid 234 of the human antibody Fc domain is further substituted with alanine (A).

4. The human antibody Fc domain variant of claim 1, wherein amino acid 265 of the human antibody Fc domain is further substituted with leucine (L), methionine (M), or asparagine (N).

5. The human antibody Fc domain variant of claim 1, wherein amino acid 329 of the human antibody Fc domain is further substituted with valine (V), leucine (L), or glycine (G).

6. The human antibody Fc domain mutant according to claim 1, wherein amino acid 268 of the human antibody Fc domain is further substituted with glutamine (Q).

7. The human antibody Fc domain mutant according to claim 1, wherein amino acid 296 of the human antibody Fc domain is further substituted with phenylalanine (F).

8. The human antibody Fc domain mutant according to claim 1, wherein amino acid 300 of the human antibody Fc domain is further substituted with phenylalanine (F).

9. The human antibody Fc domain mutant according to claim 1, wherein amino acid 333 of the human antibody Fc domain is further substituted with valine (V).

10. The human antibody Fc domain mutant of claim 1, wherein the human antibody is IgG1.

11. An antibody or a fragment thereof having immunological activity, comprising the human antibody Fc domain mutant according to any one of claims 1 to 10.

12. An antibody-drug conjugate in which the antibody or immunologically active fragment thereof according to claim 11 is conjugated to one or more drugs.

13. The drugs include chimeric antigen receptor (CAR) cell therapeutic agents, oncolytic drugs, immunotherapeutic agents, cytotoxic agents, angiogenesis inhibitors, kinase inhibitors, costimulatory molecule blockers, adhesion molecule blockers, anti-cytokine agents, nucleic acid therapeutic agents, anti-CTLA-4 agents, anti-PD-1 agents, anti-PD-L1 agents, anti-PD-L2 agents, TNF-α cross-linking agents, TRAIL cross-linking agents, anti-CD27 agents, anti-CD30 agents, anti-CD40 agents, anti-4-1BB agents, anti-GITR agents, anti-OX40 agents, and anti-TRAIL R1 agents, anti-TRAILR2 agents, targretin, interferon-α, clobetasol, peginterferon, prednisone, romidepsin, bexarotene, methotrexate, triamcinolone cream, anti-chemokines, vorinostat, gabapentin, cyclosporine, rapamycin, FK506, detectable labels or reporters, TNF antagonists, anti-rheumatic drugs, muscle relaxants 13. The antibody-drug conjugate of claim 12, wherein the conjugate is any one selected from the group consisting of drugs, narcotics, nonsteroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blockers, antibacterial agents, antipsoriatic agents, corticosteroids, anabolic steroids, erythropoietin, immunization, immunoglobulins, immunosuppressants, growth hormones, hormone replacement drugs, radiopharmaceuticals, antidepressants, antipsychotics, stimulants, asthma medications, beta-agonists, inhaled steroids, epinephrine or analogs thereof, cytokines, cytokine antagonists, PD-1 antagonists, adenosine A2AR antagonists, CD73 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG-3 inhibitors, anthracyclines, antisense oligonucleotides (ASOs), siRNA, and aptamers.

14. A pharmaceutical composition for treating or preventing cancer, comprising a human antibody Fc domain variant described in any one of claims 1 to 10; an antibody containing the variant or a fragment thereof having immunological activity; or an antibody-drug conjugate described in claim 12 or 13.

15. The cancers include brain cancer, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, 15. The pharmaceutical composition for treating or preventing cancer according to claim 14, wherein the cancer is any one selected from the group consisting of esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system malignant lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

16. A method for treating cancer, comprising the step of administering to a subject a human antibody Fc domain variant described in any one of claims 1 to 10; an antibody containing the variant or a fragment thereof having immunological activity; or an antibody-drug conjugate described in claim 12 or 13.

17. 17. The method of claim 16, wherein the cancer is any one selected from the group consisting of brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer, small intestine cancer, rectal cancer, fallopian tube cancer, perianal cancer, endometrial cancer, vaginal cancer, vulva cancer, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, central nervous system tumor, primary central nervous system malignant lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

18. A composition for target detection, comprising a conjugate of the antibody or a fragment thereof having immunological activity according to claim 11 bound to a functional substance.

19. The target detection composition according to claim 18, wherein the functional substance is one or more selected from the group consisting of contrast agents, radioactive isotopes, inorganic particles, fluorescent markers, staining substances, enzymes, polypeptides, nucleic acids, carbohydrates, and lipids.

20. 20. A method for diagnosing a disease, comprising administering to an individual the target detection composition of claim 18.

21. The method for diagnosing a disease according to claim 20, wherein the disease is any one selected from the group consisting of cancer, cardiovascular disease, neurological disorder, diabetes, autoimmune disease, inflammatory disease, viral infection, and allergy.

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