Enhanced effector functionality of glycosylated Fc mutant polypeptides

Abstract

This disclosure provides one or more oligomannose-type N-glycans and glycosylated Fc domain variants containing Fc domain mutations. The disclosure also provides nucleic acids encoding Fc domain variants and host cells for constructing Fc domain variants. Methods for increasing the yield of Fc domain variants and methods for treating diseases using Fc domain variants are also provided.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 419,188 (filed October 25, 2022), the disclosure of which is included in its entirety by reference herein. [Background technology]

[0002] Therapeutic antibodies are widely used in clinics to treat patients with many difficult diseases, including cancer. See Redman JM et al. (2015), Mechanisms of action of therapeutic antibodies for cancer. Mol Immunol. vol. 67 (2 Pt A): 28-45. Many of them have been demonstrated to act through effector functions, along with antibody-dependent cell-mediated cytotoxicity (ADCC) as the primary mechanism. See Bournazos S et al. (2017), Signaling by Antibodies: Recent Progress. Annual Review of Immunology, vol. 35: 285-311.

[0003] In ADCC, antibody-antigen interactions result in increased affinity of the IgG-Fc domain to FcγRIIIa expressed on natural killer (NK) cells, leading to signal transduction, cell degranulation, and subsequent target cell death. In addition to cytotoxicity against pathogens and cancer cells, ADCC has recently been demonstrated to be involved in the immunomodulation of several checkpoint inhibitors. See Ingram JR et al. (2018), Proc. Natl. Acad. USA, vol. 115(15): 3912-7 and Goletz C et al. (2018), Frontiers in Immunology, vol. 9: 1614. ADCC is also required for high efficacy of antibodies against certain autoimmune diseases. See Bloemendaal FM et al. (2017), Gastroenterology, vol. 153(5): 1351-62.e4.

[0004] Numerous efforts have been made to enhance ADCC to improve the effectiveness of disease treatments. For example, protein modification of the FC-CH2 domain using site-directed mutagenesis can significantly increase FcγRIIIa binding and ADCC activity. See Lazar GA et al. (2006), Proc. Natl. Acad. USA, vol. 103(11):4005-10. Further modification of the Fc domain can further enhance FcγRIIIa binding and ADCC activity. Nevertheless, despite these efforts, there is still a need to create new Fc variant antibodies and other binding polypeptides with improved efficacy and manufacturability for use in the treatment of various diseases. [Overview of the Initiative] [Means for solving the problem]

[0005] This disclosure is partly based on the finding that the manufacturability and effector function of certain glycosylated Fc domain variants (e.g., oligomannose-modified Fc variants) are improved compared to conventional Fc variant domains. Accordingly, this disclosure is partly based on the finding that glycosylation of Fc variant polypeptides (e.g., using kifunensin and related glycosylation inhibitors) is to improve their manufacturability and utility as therapeutic agents.

[0006] In one embodiment, a composition comprising a group of isolated glycosylated polypeptides, each containing an N-glycan-containing Fc domain, wherein the Fc domain is subjected to the following EU numbering mutations: (i)~(ix): (i) Aspartic acid (D) at amino acid position 239, (ii) Aspartic acid (D) at amino acid position 267, (iii) Aspartic acid (D) or glutamic acid (E) at amino acid position 268, (iv) Alanine (A) or cysteine ​​(C) at amino acid position 298, (v) Isoleucine (I), methionine (M), glutamine (Q), or tryptophan (W) at amino acid position 314, (vi) Phenylalanine (F) or methionine (M) at amino acid position 330, (vii) Glutamic acid (E) at amino acid position 332, (viii) Aspartic acid (D), isoleucine (I), proline (P), or threonine (T) at amino acid position 339, (ix) Phenylalanine (F) or tryptophan (W) at amino acid position 373 It further includes at least one of the following: A composition is provided that contains Man5-9(GlcNAc)2N-glycan in a molar ratio of at least 50% relative to the total N-glycan.

[0007] In a particular exemplary embodiment, Man8 and Man9 together represent Man in the composition. 5-9(GlcNAc)2N-glycan is a major species.

[0008] In some exemplary embodiments, the composition contains Man9(GlcNAc)2N-glycan in a molar ratio of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% relative to the total N-glycan.

[0009] In yet another exemplary embodiment, the composition contains at least 97% Man9(GlcNAc)2N-glycan in molar ratio relative to the total N-glycan.

[0010] In certain exemplary embodiments, at least 80% of the N-glycans in the composition are defucosylated in molar ratio relative to the total N-glycans.

[0011] In other exemplary embodiments, the conjugated polypeptide of the composition is produced by culturing cells expressing the conjugated polypeptide in the presence of a mannosidase inhibitor. In some embodiments, the mannosidase inhibitor is kifunensin. In certain embodiments, the concentration of kifunensin is about 60 ng / mL to about 2500 ng / mL. In one exemplary embodiment, the concentration of kifunensin is about 2000 ng / mL.

[0012] In certain exemplary embodiments, Man 5-9 The bound polypeptide of the composition containing (GlcNAc)2N-glycan is Man 5-9 Compared to a reference polypeptide that is otherwise identical but does not contain (GlcNAc)2N-glycan, it exhibits improved affinity for binding to the Fcγ receptor. In some exemplary embodiments, the Fcγ receptor is human FcγRIIIa. In particular exemplary embodiments, Man 5-9 The binding polypeptide of the composition containing (GlcNAc)2N-glycan has an increased affinity for binding to human FcγRIIIa, at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher compared to the reference polypeptide. In some exemplary embodiments, Man 5-9The conjugated polypeptide of a composition containing (GlcNAc)2N-glycan exhibits increased antibody-dependent cell-mediated cytotoxicity (ADCC) activity compared to the reference polypeptide. In certain exemplary embodiments, the ADCC activity of the conjugated polypeptide is at least 1, 2, 3, 4, or 5 times higher than that of the reference polypeptide. In certain exemplary embodiments, the reference polypeptide has a wild-type (WT)Fc domain. In another exemplary embodiment, the reference polypeptide is not prepared by culturing cells expressing the reference polypeptide in the presence of kifunensin.

[0013] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239.

[0014] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamic acid (E) at amino acid position 332.

[0015] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

[0016] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 267.

[0017] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 268.

[0018] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamic acid (E) at amino acid position 268.

[0019] In some exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains alanine (A) at amino acid position 298.

[0020] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

[0021] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains cysteine ​​(C) at amino acid position 298.

[0022] In some exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains isoleucine(I) at amino acid position 314.

[0023] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains methionine (M) at amino acid position 314.

[0024] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamine (Q) at amino acid position 314.

[0025] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains tryptophan (W) at amino acid position 314.

[0026] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains phenylalanine (F) at amino acid position 330.

[0027] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains methionine (M) at amino acid position 330.

[0028] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 339.

[0029] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains isoleucine(I) at amino acid position 339.

[0030] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains proline (P) at amino acid position 339.

[0031] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains threonine (T) at amino acid position 339.

[0032] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains phenylalanine (F) at amino acid position 373.

[0033] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains tryptophan (W) at amino acid position 373.

[0034] In another embodiment, a composition comprising a group of isolated glycosylated polypeptides, each containing an Fc domain containing an N-glycan, This further includes mutations that increase the binding of the Fc domain to the Fc receptor, At least 50% Man in molar ratio for all N-glycans 5-9 (GlcNAc)2N-glycan is included, The Fc domain, according to EU numbering, further contains cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302. A composition is provided.

[0035] In a particular exemplary embodiment, Man8 and Man9 together are Man 5-9 (GlcNAc)2N-glycan is a major species.

[0036] In some exemplary embodiments, the composition contains Man9(GlcNAc)2N-glycan in a molar ratio of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% relative to the total N-glycan.

[0037] In yet another exemplary embodiment, the composition comprises at least 97% Man9(GlcNAc)2 N-glycan on a molar basis relative to total N-glycans.

[0038] In certain exemplary embodiments, at least 80% of the N-glycans are defucosylated on a molar basis relative to total N-glycans in the composition.

[0039] In other exemplary embodiments, the binding polypeptide of the composition is made by culturing cells that express the binding polypeptide in the presence of a mannosidase inhibitor. In some embodiments, the mannosidase inhibitor is kifunensine. In certain embodiments, the concentration of kifunensine is from about 60 ng / mL to about 2500 ng / mL. In one exemplary embodiment, the concentration of kifunensine is about 2000 ng / mL.

[0040] In certain exemplary embodiments, the binding polypeptide of a composition comprising Man 5-9 (GlcNAc)2 N-glycan has an improved affinity for binding to Fcγ receptors as compared to a reference polypeptide that does not contain Man 5-9 (GlcNAc)2 N-glycan but is otherwise identical. In an exemplary embodiment, the Fcγ receptor is human FcγRIIIa. In a particularly exemplary embodiment, the binding polypeptide of a composition comprising Man 5-9 (GlcNAc)2 N-glycan has an affinity for binding to human FcγRIIIa that is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 times higher and improved as compared to the reference polypeptide. In some exemplary embodiments, the Man 5-9 (GlcNAc)2 N-glycan-containing binding polypeptide of the composition has improved antibody-dependent cell-mediated cytotoxicity (ADCC) activity as compared to the reference polypeptide. In certain exemplary embodiments, the ADCC activity of the binding polypeptide is at least 1, 2, 3, 4 or 5 times higher as compared to the reference polypeptide. In certain exemplary embodiments, the reference polypeptide has a wild-type (WT) Fc domain.

[0041] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239.

[0042] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamic acid (E) at amino acid position 332.

[0043] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

[0044] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 267.

[0045] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 268.

[0046] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamic acid (E) at amino acid position 268.

[0047] In some exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains alanine (A) at amino acid position 298.

[0048] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

[0049] In certain exemplary embodiments, the Fc domain of the bound polypeptide contains cysteine ​​(C) at amino acid position 298.

[0050] In some exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains isoleucine(I) at amino acid position 314.

[0051] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains methionine (M) at amino acid position 314.

[0052] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamine (Q) at amino acid position 314.

[0053] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains tryptophan (W) at amino acid position 314.

[0054] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains phenylalanine (F) at amino acid position 330.

[0055] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains methionine (M) at amino acid position 330.

[0056] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 339.

[0057] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains isoleucine(I) at amino acid position 339.

[0058] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains proline (P) at amino acid position 339.

[0059] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains threonine (T) at amino acid position 339.

[0060] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains phenylalanine (F) at amino acid position 373.

[0061] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains tryptophan (W) at amino acid position 373.

[0062] In certain exemplary embodiments, the Fc domain of the binding polypeptide further comprises aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307.

[0063] In further exemplary embodiments, Man 5-9 The conjugated polypeptide of the composition containing (GlcNAc)2N-glycan has a Tm of 10°C or less of the reference polypeptide having a WT Fc domain. In some embodiments, the reference polypeptide having a WT Fc domain is expressed by cells cultured in the absence of kifunensin, and Man 5-9 The conjugated polypeptide containing (GlcNAc)2N-glycan is expressed by cells cultured in the presence of kifunensin. In some embodiments, Man 5-9 The conjugated polypeptide containing (GlcNAc)2N-glycan has a Tm of 5°C or less than the reference polypeptide having a WT Fc domain. In some embodiments, the reference polypeptide having a WT Fc domain is expressed by cells cultured in the presence of kifunensin, and Man 5-9 The (GlcNAc)2N-glycan-containing conjugated polypeptide is expressed by cells cultured in the presence of kifunensin.

[0064] In yet another embodiment, a composition comprising a group of isolated glycosylated polypeptides, each containing an Fc domain containing an N-glycan, This further includes mutations that increase the binding of the Fc domain to the Fc receptor, Man, in molar ratio, of at least 50% of total N-glycans 5-9 (GlcNAc)2N-glycan is included, The Fc domain further contains aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, according to EU numbering. A composition is provided.

[0065] In certain exemplary embodiments, Man8 and Man9 together are Man in the composition. 5-9 (GlcNAc)2N-glycan is a major species.

[0066] In some exemplary embodiments, the composition contains Man9(GlcNAc)2N-glycan in a molar ratio of more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, or more than 95% relative to the total N-glycan.

[0067] In yet another exemplary embodiment, the composition contains at least 97% Man9(GlcNAc)2N-glycan in molar ratio relative to the total N-glycan.

[0068] In certain exemplary embodiments, at least 80% of the N-glycans in the composition are defucosylated by molar ratio compared to the total N-glycans.

[0069] In other exemplary embodiments, the conjugated polypeptide of the composition is produced by culturing cells expressing the conjugated polypeptide in the presence of a mannosidase inhibitor.

[0070] In certain exemplary embodiments, Man 5-9 The bound polypeptide of the composition containing (GlcNAc)2N-glycan is Man 5-9 Compared to a reference polypeptide that is otherwise identical but does not contain (GlcNAc)2N-glycan, it exhibits improved affinity for binding to the Fcγ receptor. In exemplary embodiments, the Fcγ receptor is human FcγRIIIa. In particular exemplary embodiments, Man 5-9The bound polypeptide of the composition containing (GlcNAc)2N-glycan has at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher affinity for binding to human FcγRIIIa compared to the reference polypeptide. In some exemplary embodiments, the Man of the present composition 5-9 The conjugated polypeptide containing (GlcNAc)2N-glycan exhibits enhanced antibody-dependent cytotoxicity (ADCC) activity compared to the reference polypeptide. In certain exemplary embodiments, the ADCC activity of the conjugated polypeptide is at least 1, 2, 3, 4, or 5 times higher than that of the reference polypeptide. In certain exemplary embodiments, the reference polypeptide has a wild-type (WT) Fc domain.

[0071] In other exemplary embodiments, the conjugated polypeptide of the composition is produced by culturing cells expressing the conjugated polypeptide in the presence of a mannosidase inhibitor. In some embodiments, the mannosidase inhibitor is kifunensin. In certain embodiments, the concentration of kifunensin is about 60 ng / mL to about 2500 ng / mL. In one exemplary embodiment, the concentration of kifunensin is about 2000 ng / mL.

[0072] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239.

[0073] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamic acid (E) at amino acid position 332.

[0074] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

[0075] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 267.

[0076] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 268.

[0077] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamic acid (E) at amino acid position 268.

[0078] In some exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains alanine (A) at amino acid position 298.

[0079] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

[0080] In certain exemplary embodiments, the Fc domain of the bound polypeptide contains cysteine ​​(C) at amino acid position 298.

[0081] In some exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains isoleucine(I) at amino acid position 314.

[0082] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains methionine (M) at amino acid position 314.

[0083] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains glutamine (Q) at amino acid position 314.

[0084] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains tryptophan (W) at amino acid position 314.

[0085] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains phenylalanine (F) at amino acid position 330.

[0086] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains methionine (M) at amino acid position 330.

[0087] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains aspartic acid (D) at amino acid position 339.

[0088] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains isoleucine(I) at amino acid position 339.

[0089] In yet another exemplary embodiment, the Fc domain of the bound polypeptide in the composition contains proline (P) at amino acid position 339.

[0090] In certain exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains threonine (T) at amino acid position 339.

[0091] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains phenylalanine (F) at amino acid position 373.

[0092] In further exemplary embodiments, the Fc domain of the bound polypeptide in the composition contains tryptophan (W) at amino acid position 373.

[0093] In certain exemplary embodiments, Man 5-9 The binding polypeptide in the composition containing (GlcNAc)2N-glycan exhibits a higher binding affinity to the neonatal Fc receptor (FcRn) compared to the binding polypeptide having a WT Fc domain.

[0094] In other exemplary embodiments, the Fc domain of the bound polypeptide in the composition further comprises cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302, according to EU numbering.

[0095] In certain exemplary embodiments, one or more of the conjugated polypeptides in the composition are antibodies. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In some embodiments, the antibody is a multispecific antibody. In some embodiments, the multispecific antibody is a CODV-based formula selected from the group consisting of: DVD-Ig, optionally CODV-Ig, CrossMab, CrossMab-Fab, and Tandem Fab. In some embodiments, the multispecific antibody is a T cell engager. In some embodiments, the multispecific antibody is an NK cell engager.

[0096] In some embodiments, one or more of the binding polypeptides include an immunoglobulin single variable domain (ISV).

[0097] In some embodiments, one or more of the binding polypeptides of the present disclosure comprises one or more VHHs.

[0098] In further exemplary embodiments, one or more of the bound polypeptides in the composition include antigen-binding fragments.

[0099] In yet another exemplary embodiment, one or more of the bound polypeptides in the composition include a single-chain variable region (ScFv) sequence.

[0100] In further exemplary embodiments, one or more of the bound polypeptides in the composition contain an IgG Fc domain. In some embodiments, the Fc domain is an IgG1 domain. In some embodiments, the Fc domain is a human Fc domain.

[0101] In yet another exemplary embodiment, one or more of the conjugated polypeptides in the composition include a lysosome-targeted chimera (LYTAC).

[0102] In other embodiments, one or more binding polypeptides further include an Fc domain containing a mutation that increases binding to the Fc receptor, where the Fc receptor is the human FcγRIIIa receptor.

[0103] In certain embodiments, the composition is a pharmaceutical composition.

[0104] In a further embodiment, a method for producing the conjugated polypeptide of the composition is provided, comprising culturing cells expressing the conjugated polypeptide in the presence of kifunensin. In some embodiments, the concentration of kifunensin in the cell culture is about 60 ng / mL to about 2500 ng / mL. In some embodiments, the concentration of kifunensin in the cell culture is about 2000 ng / mL.

[0105] In further embodiments, isolated nucleic acid molecules are provided, comprising nucleic acids capable of expressing one or more of the binding polypeptides of the composition. In some embodiments, a vector comprising the isolated nucleic acid molecule is provided. In some embodiments, the vector is an expression vector. In further embodiments, a host cell comprising the vector is provided.

[0106] In another embodiment, a method is provided for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising administering an effective amount of a pharmaceutical composition to the subject. In some embodiments, the disease or disorder is cancer. In some embodiments, the disease or disorder is an inflammatory disease. In some embodiments, the disease or disorder is an autoimmune disease.

[0107] The aforementioned and other features and advantages of the present invention will be better understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings. [Brief explanation of the drawing]

[0108] [Figure 1]Figure 1 is a schematic diagram showing antibody production using inhibitors such as kifunensin, which inhibit enzymes in the glycosylation pathway. This figure shows that the addition of kifunensin stops the glycan process, prevents the addition of fucose, and prevents the disruption of the oligomannose structure. [Figure 2A-2B] Figures 2A and 2B show SDS-PAGE analysis of wild-type (WT) antibodies and DE (S239D / I332E) antibodies specific to protein 1 with different glycan modifications. Figure 2A shows the antibodies under non-reducing conditions, and Figure 2B shows the antibodies under reducing conditions. In both Figures 2A and 2B, the lane contents are as follows: Lane 1: Protein molecular weight marker, Lane 2: WT, Lane 3: Highly galactosylated WT, Lane 4: WT containing oligomannose, Lane 5: Defucosylated WT, Lane 6: DE, Lane 7: Highly galactosylated DE, Lane 8: DE containing oligomannose, and Lane 9: Defucosylated DE. [Figure 3A-3B] Figures 3A-H show the structures and MALDI spectra of N-linked glycans from antibodies specific to protein 1 with different glycan modifications. N-linked glycans were released from the antibodies using PNGase F and analyzed using MALDI-TOF MS. MALDI spectra are presented for WT (Figure 3A) and DE (Figure 3B) antibodies; highly galactosylated WT (Figure 3C) and DE (Figure 3D) antibodies; oligomannose-containing WT (Figure 3E) and DE (Figure 3F) antibodies; and defucosylated WT (Figure 3G) and DE (Figure 3H) antibodies. [Figure 3C-3D] Same as above. [Figure 3E-3F] Same as above. [Figure 3G-3H] Same as above. [Figure 4A-4B] Figures 4A and 4B graphically show the FcγRIIIa binding of antibodies specific to protein 1 with different glycan modifications. The interaction between FcγRIIIa and antibodies was measured by SPR using Biacore®. Figure 4A shows receptor binding of WT and DE antibodies containing high galactose or oligomannose. Figure 4B shows receptor binding of defucose WT and DE antibodies. [Figure 5A-5B]Figures 5A and 5B graphically show the ADCC activity of antibodies specific to protein 1 with different glycan modifications. The effector function of the antibodies was determined using an ADCC reporter gene assay. Figure 5A shows the ADCC activity of WT and DE antibodies containing high galactose or oligomannose. Figure 5B shows the ADCC activity of defucose-free WT and DE antibodies. [Figure 6A-6B] Figures 6A and 6B graphically show the correlation between FcγRIIIa binding and ADCC and various percentages of defucosylated glycans in DE antibodies specific to protein 1. Figure 6A shows FcγRIIIa binding in DE antibodies with different percentages of defucosylated glycans. Figure 6B shows ADCC in DE antibodies with different percentages of defucosylated glycans. [Figure 7A] Figures 7A and 7B show SDS-PAGE of Fc variants of antibodies specific to protein 2, with and without kifunensin treatment. Figure 7A shows the non-reducing condition. Figure 7B shows the reducing condition. [Figure 7B] Figures 7A and 7B show SDS-PAGE of Fc variants of antibodies specific to protein 2, with and without kifunensin treatment. Figure 7A shows the non-reducing condition. Figure 7B shows the reducing condition. [Figure 8A] Figures 8A-8F graphically show the MALDI-TOF glycan analysis of variants of protein 2-specific antibodies with and without kifunensin treatment. Figure 8A shows that the wild-type antibody mainly contains G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. [Figure 8B] Figures 8A-8F graphically show the MALDI-TOF glycan analysis of variants of protein 2-specific antibodies with and without kifunensin treatment. Figure 8B shows that the S298A antibody mainly contains G0F and G1F glycans without kifunensin treatment, and Man9 glycan with kifunensin treatment. [Figure 8C]Figures 8A-F graphically show the MALDI-TOF glycan analysis of variants of protein 2-specific antibodies with and without kifunensin treatment. Figure 8C shows that the S239D antibody mainly contains G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. [Figure 8D] Figures 8A-8F graphically show the MALDI-TOF glycan analysis of variants of antibodies specific to protein 2 with and without kifunensin treatment. Figure 8D shows that the S239D / S298A antibody mainly contains G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man9 glycan with kifunensin treatment. [Figure 8E] Figures 8A-F graphically show the MALDI-TOF glycan analysis of variants of protein 2-specific antibodies with and without kifunensin treatment. Figure 8E shows that the I332E antibody mainly contains G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. [Figure 8F] Figures 8A-8F graphically show the MALDI-TOF glycan analysis of variants of protein 2-specific antibodies with and without kifunensin treatment. Figure 8F shows that the S239D / I332E antibody mainly possesses G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. [Figure 9A]Figures 9A-G show the results of human FcγRIIIa binding affinity for various Fc variants of protein 2-specific antibodies with and without kifunensin treatment. Figure 9A is a table containing the measured binding affinity metrics. These results show that kifunensin treatment increases the affinity for hFcγRIIIa by 2.4 to 7 times for all Fc variants. Figure 9B shows sensograms of binding to wt antibody with and without kifunensin treatment. Figure 9C shows sensograms of binding to S239D(D) antibody with and without kifunensin treatment. Figure 9D shows sensograms of binding to S239D / S298A(DA) antibody with and without kifunensin treatment. Figure 9E shows sensograms of binding to S298A(A) antibody with and without kifunensin treatment. Figure 9F shows sensorgrams of binding to I332E antibody with and without kifunensin treatment. Figure 9G shows sensorgrams of binding to S239D / I332E(DE) antibody with and without kifunensin treatment. [Figure 9B-9C] Same as above. [Figure 9D-9E] Same as above. [Figure 9F-9G] Same as above. [Figure 10] Figure 10 graphically shows the tm of protein 2-specific antibodies using nanoDSF analysis, including WT without kifunensin, WT with kifunensin, S239D / I332E without kifunensin, and S239D / S298A with kifunensin. All variants showed lower Tm compared to WT without kifunensin. [Figure 11A-11C]Figures 11A-F graphically show the mass spectrometry glycan analysis of various antibodies specific to protein 3, with and without kifunensin treatment. Figure 11A shows WT antibodies specific to protein 3, with and without kifunensin treatment. Figure 11B shows LS antibodies specific to protein 3, with and without kifunensin treatment. Figure 11C shows YTE antibodies specific to protein 3, with and without kifunensin treatment. Figure 11D shows YD antibodies specific to protein 3, with and without kifunensin treatment. Figure 11E shows DQ antibodies specific to protein 3, with and without kifunensin treatment. Figure 11F shows DW antibodies specific to protein 3, with and without kifunensin treatment. All kifunensin-treated antibodies have an oligomannose content of >97% Man9(GlcNAc)2. All untreated antibodies are >80% defucosylated. [Figure 11D-11F] Same as above. [Figure 12] Figure 12 graphically shows the FcγRIIIa binding affinity response to WT, DQ, DW, LS, YD, and YTE antibodies specific to protein 3, with and without kifunensin treatment. Enhanced FcγRIIIa binding was observed for all variants expressed using kifunensin. [Figures 13A-13B] Figures 13A-C graphically show human FcRN binding to various antibodies specific to protein 3 at pH 6.0 (Figure 13A) and pH 7.4 (Figure 13B). Both Figure 13A (pH 6.0) and Figure 13B (pH 7.4) show the binding results for WT, LS, YTE, DQ, DW, and YD antibodies specific to protein 3, with and without kifunensin treatment. Figure 13C shows a scattering plot of the results at pH 6.0. At pH 6.0, no significant changes in on or off binding rates were observed. DQ, DW, and YD all exhibited faster on and off binding rates compared to LS. At pH 7.0, a slight decrease in the human FcRN binding response was observed in the kifunensin-treated samples. [Figure 13C] Same as above. [Figure 14]Figure 14 graphically shows the thermal stability, as determined by DSF, for protein 3-specific WT, LS, YTE, DQ, DW, and YD antibodies with and without kifunensin treatment. The solid black curve represents the case without kifunensin treatment, and the dotted curve represents the case with kifunensin treatment. Kifunensin treatment makes all antibody variants even more unstable by 4–8°C (compared to Fc mutation alone). DW with kifunensin treatment shows a 16°C decrease in thermal stability compared to WT. [Figure 15] Figure 15 graphically shows the FcRn affinity determined by chromatography against protein 3-specific WT, LS, YTE, DQ, DW, and YD antibodies with and without kifunensin treatment. The solid black curve represents the untreated sample, and the dotted curve represents the treated sample. The kifunensin-treated sample shows a similar pH elution profile to the untreated sample. This data supports the results of FcRN binding, which shows little effect on overall binding affinity upon kifunensin treatment. [Figure 16] Figure 16 is a table providing metrics related to the human FcγRIIIa binding affinity of human IgG1 antibodies specific to protein 4, including various combinations of S239D(D), S239D / S298A(DA), S239D / I332E(DE), R292C / V302C(SEFL2.2), T256D / T307Q(DQ), and M428L / N434S(LS), with and without kifunensin treatment. These results show that kifunensin treatment increases the affinity for hFcγRIIIa by 1.6 to 7.7 times for all Fc variants tested. DE showed the highest affinity with kifunensin treatment. R292C / V302C, DQ, DQ+R292C / V302C, and LS maintained binding affinity similar to that of the WT antibody. [Figure 17A-17C]Figures 17A-E show sensorgrams of human FcγRIIIa binding affinity for the following antibodies specific to protein 4: WT (Figure 17A), DE (Figure 17B), DA treated with kifunensin (Figure 17C), DQ+D+R292C / V302C (SEFL2.2) (Figure 17D), and DQ+DA+R292C / V302C (SEFL2.2) (Figure 17E). All tested variants showed stronger binding affinity than the WT antibody. [Figures 17D-17E] Same as above. [Figure 18] Figure 18 shows SDS-PAGE of antibodies specific to protein 5 under reducing and non-reducing conditions. For both the reducing and non-reducing gels, the lanes are as follows: Lane 1: DA + kifunensin, Lane 2: DE + R292C / V302C, Lane 3: defucosylated, and Lane 4: WT. [Figure 19A] Figures 19A-D show graphs of MALDI-TOF glycan analysis of antibodies specific to protein 5. Figure 19A shows the results for the WT antibody. The major glycans were determined to be G0F and G1F. The WT antibody was also determined to be 95.1% fucosylated and 4.9% defucosylated. Figure 19B shows the results for the DE+R292C / V302C antibody. The major glycans were determined to be G0F and G1F. Figure 19C shows the results for the DA+kyfunensin antibody. The major glycans were determined to be Man9(GlcNAc)2 and Man8(GlcNAc)2. Figure 19D shows the results for the defucosylated antibody. The major glycan was determined to be G0. [Figure 19B] Same as above. [Figure 19C] Same as above. [Figure 19D] Same as above. [Figures 20A-20B]Figures 20A-D show sensorgrams of binding analysis of various antibodies against protein 5. Figure 20A shows the WT antibody. Figure 20B shows the DA+ kifunensin antibody. Figure 20C shows the DE+ R292C / V302C (disulfide) antibody. Figure 20D shows the defucosylated antibody. All antibodies had similar binding affinity to protein 5. [Figures 20C-20D] Same as above. [Figures 21A-21B] Figures 21A-D show sensorgrams of binding affinity to human FcγRIIIa for various antibodies specific to protein 5. Figure 21A shows the WT antibody. Figure 21B shows the DA+ kifunensin antibody. Figure 21C shows the DE+ R292C / V302C (disulfide) antibody. Figure 21D shows the defucosylated antibody. All variants showed higher binding affinity to human FcγRIIIa than the WT. [Figures 21C-21D] Same as above. [Figures 22A-22C] Figures 22A-F show sensorgrams of human FcγRIIIa binding affinity for the following antibodies: WT (Figure 22A), WT with kifunensin treatment (Figure 22B), S298A (Figure 22C), S298A with kifunensin treatment (Figure 22D), H268D (Figure 22E), and H268D with kifunensin treatment (Figure 22F). All variants showed higher binding affinity to human FcγRIIIa than the WT. [Figures 22D-22F] Same as above. [Figure 23A] Figures 23A and 23B show the numerical values ​​of human FcγRIIIa binding affinity with and without kifunensin (Figure 23A) and with kifunensin (Figure 23B). The values ​​relative to the WT in Figures 23A and 23B are shown in bold. These figures show that for several variants tested, the binding affinity to human FcγRIIIa is improved compared to the WT. [Figure 23B]Figures 23A and 23B show the numerical values ​​of human FcγRIIIa binding affinity with and without kifunensin (Figure 23A) and with kifunensin (Figure 23B). The values ​​relative to the WT in Figures 23A and 23B are shown in bold. These figures show that for several variants tested, the binding affinity to human FcγRIIIa is improved compared to the WT. [Figure 24] Figure 24 shows the numerical values ​​for human FcγRIIIa binding affinity with and without kifunensin, as well as the Tm values ​​for the tested Fc variants and WT. This figure shows that for several of the tested variants, the binding affinity to human FcγRIIIa is increased compared to WT. [Modes for carrying out the invention]

[0109] This disclosure provides novel glycosylated Fc domain variants (e.g., binding polypeptides containing an Fc domain variant) containing mannose-rich glycans (e.g., oligomannose-type N-glycans). In exemplary embodiments, the Fc domain variant contains both an Fc mutation that enhances binding to the Fc receptor and one or more oligomannose-type N-glycans complexed in the Fc region. In some exemplary embodiments, the Fc variant is produced in host cells cultured in the presence of a mannosidase inhibitor (e.g., the α-mannosidase I inhibitor kifunensin). In exemplary embodiments, the present invention provides a composition comprising a population of Fc variant polypeptides in which at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%) of the Fc variant polypeptide contains an oligomannose-type N-glycan. In exemplary embodiments, the main oligomannose-type N-glycan is Man8(GlcNAc)2 or Man9(GlcNAc)2.

[0110] In certain embodiments, the glycosylated Fc domain variants of this disclosure exhibit improved antibody-dependent cytotoxicity (ADCC) activity compared to Fc domain variants that lack oligomannose-type N-glycans but are otherwise identical to the glycosylated Fc domain variants. In some exemplary embodiments, the glycosylated Fc domain variants include Fc mutations that result in improved thermal stability. This disclosure also provides nucleic acids encoding glycosylated Fc domain variants, recombinant expression vectors and host cells for producing glycosylated Fc domain variants, and pharmaceutical compositions comprising isolated glycosylated Fc domain variants. Methods using conjugated polypeptides comprising the glycosylated Fc domain variants disclosed herein to treat various diseases are also provided. The glycosylated Fc domain variants disclosed herein are useful in a variety of therapies where targeted killing of the Fc of target cells is desirable.

[0111] definition Please understand that the methods described herein are not limited to the specific methods and experimental conditions disclosed herein, as such methods and conditions may vary. Also, please understand that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them.

[0112] Furthermore, unless otherwise specified, the experiments described herein utilize conventional molecular and cell biological and immunological techniques within the scope of the art of the art. Such techniques are well known to those skilled in the art and are adequately described in the literature. See, for example, Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987–2008) (including all supplementary materials), Molecular Cloning: A Laboratory Manual (4th edition) by MR. Green, and J. Sambrook and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

[0113] Unless otherwise defined, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. In the event of potential ambiguity, the definitions provided herein take precedence over dictionary or external definitions. Unless otherwise required by context, singular terms include plural forms, and plural terms include singular forms. The use of "or" means "and / or" unless otherwise specified. The use of the term "including" and other forms such as "includes" and "included" is not limited.

[0114] In general, the nomenclature used in relation to cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein is well known and commonly used in the art. The methods and techniques provided herein are generally carried out, unless otherwise specified, according to conventional methods well known in the art, as described in the various general and more specific references cited and discussed throughout this specification. Enzyme reactions and purification techniques are carried out as commonly performed in the art or as described herein, according to the manufacturer's specifications. The nomenclature used in relation to analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry described herein, as well as the experimental procedures and techniques therein, are well known and commonly used in the art. Standard techniques are used in the chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

[0115] To facilitate easier understanding of this disclosure, the following terms are defined:

[0116] The term "polypeptide" refers to any polymer chain of amino acids and, unless otherwise inconsistent with the context, includes natural or artificial proteins, polypeptide analogs or variants of protein sequences, or fragments thereof. Polypeptides can be monomers or polymers. In the case of antigenic polypeptides, a polypeptide fragment optionally contains at least one sequential or nonlinear epitope of the polypeptide. The precise boundaries of at least one epitope fragment can be determined using the usual skills of the art. A polypeptide fragment may, for example, contain at least about 5 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, or at least about 20 consecutive amino acids.

[0117] The terms “isolated protein” or “isolated polypeptide” refer to a protein or polypeptide that, by its origin or source, does not associate with its naturally associated components in its natural state; substantially contains no other proteins from the same species; is expressed by cells of a different species; or does not occur in nature. Thus, a protein or polypeptide that is chemically synthesized or synthesized in a cell system different from the cells from which it originates is “isolated” from its naturally associated components. A protein or polypeptide may also be made substantially free of its naturally associated components by being isolated using protein purification techniques well known in the art.

[0118] As used herein, the terms “binding protein” or “binding polypeptide” refer to a protein or polypeptide (e.g., an antibody or a fragment thereof) that contains at least one binding site involved in selective binding to a target antigen of interest (e.g., a human antigen). Exemplary binding sites include antibody variable domains, ligand-binding sites of receptors, or receptor-binding sites of ligands. In certain embodiments, the binding protein or binding polypeptide may contain multiple (e.g., two, three, four, or more) binding sites. In certain embodiments, the binding protein or binding polypeptide is not a therapeutic enzyme.

[0119] As used herein, the term “natural residue” means an amino acid residue that occurs naturally at a specific amino acid position in a binding polypeptide (e.g., an antibody or fragment thereof) and has not been modified, introduced, or altered by human intervention. As used herein, the terms “modified binding protein,” “modified binding polypeptide,” “modified binding protein,” or “modified binding polypeptide” mean a binding polypeptide and / or binding protein (e.g., an antibody or fragment thereof) that includes a mutation resulting in at least one amino acid substitution, deletion, and / or addition to the natural (i.e., wild-type) amino acid sequence, and / or a change in glycosylation (e.g., hyperglycosylation, hypoglycosylation, and / or deglycosylation) at one or more amino acid positions relative to the natural (i.e., wild-type) amino acid sequence.

[0120] The term "ligand" refers to any substance that is capable of or can bind to another substance. Similarly, the term "antigen" refers to any substance to which an antibody can be produced. While "antigen" is generally used in relation to antibody-binding substrates and "ligand" is often used in relation to receptor-binding substrates, these terms are not distinguishative and encompass a wide range of overlapping chemical entities. To avoid ambiguity, antigen and ligand are used interchangeably throughout this specification. Antigens / ligands may be peptides, polypeptides, proteins, aptamers, polysaccharides, sugar molecules, carbohydrates, lipids, oligonucleotides, polynucleotides, synthetic molecules, inorganic molecules, organic molecules, and any combination thereof.

[0121] As used herein, the term "specifically binds" means that an antibody or its antigen-binding fragment binds to an area of ​​up to approximately 1 x 10⁶ times. -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 M or a smaller dissociation constant (K) D ) refers to the ability to bind to an antigen and / or the ability to bind to an antigen with an affinity at least twice as high as its affinity to a nonspecific antigen. Specific binding of an antibody can be binding to a target antigen through the CDR sequence. Antibodies can also specifically bind to FcRs such as FcRn or FcγRIIIa through the Fc region.

[0122] Dissociation constant of binding proteins (K D) can be determined, for example, by surface plasmon resonance. Generally, surface plasmon resonance analysis uses the Biacore system (Cytiva Life Sciences, Marlborough, MA) or the Carterra LSA platform (Carterra, Salt Lake City, UT) to measure the binding interaction between the ligand (target antigen on a biosensor matrix) and the analyte (binding protein in solution) in real time by surface plasmon resonance (SPR). Surface plasmon analysis can also be performed by immobilizing the analyte (binding protein on a biosensor matrix) and presenting the ligand (target antigen). Where used herein, "K" is used. D The term "dissociation constant" refers to the dissociation constant of the interaction between a specific binding protein and its target antigen.

[0123] As used herein, the term “immunoglobulin domain” may refer to immunoglobulin A, immunoglobulin D, immunoglobulin E, immunoglobulin G, or immunoglobulin M. An immunoglobulin domain may be an immunoglobulin heavy chain region or a fragment thereof. In some examples, an immunoglobulin domain is derived from an antibody (e.g., a mammalian antibody, recombinant antibody, chimeric antibody, modified antibody, human antibody, humanized antibody) or an antigen-binding fragment thereof.

[0124] As used herein, the term “antibody” refers to such an assembly (e.g., an intact antibody molecule, an antibody fragment, or a variant thereof) that has significant known specific immune response activity against an antigen of interest (e.g., a tumor-associated antigen). Antibodies and immunoglobulins include light and heavy chains, which may or may not have interchain covalent bonds between them. The basic immunoglobulin structures in vertebrate systems are relatively well understood.

[0125] As will be discussed in more detail below, the general term “antibody” encompasses five distinct classes of antibodies that can be biochemically distinguished. While all five classes of antibodies are clearly within the scope of this disclosure, the following discussion generally concerns the IgG class of immunoglobulin molecules. With respect to IgG, an immunoglobulin consists of two identical light chains with a molecular weight of approximately 23,000 daltons and two identical heavy chains with molecular weights of 53,000 to 70,000. The four chains are linked by disulfide bonds in a “Y” configuration, where the light chains begin at a “Y” entry point and support the heavy chains that continue through a variable region.

[0126] The light chains of immunoglobulins are classified as either kappa or lambda (κ, λ). Each heavy chain class can be bound to either a kappa or lambda light chain. Generally, when immunoglobulins are produced by hybridomas, B cells, or genetically engineered host cells, the light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus of the branched ends of the Y configuration to the C-terminus at the bottom of each chain. Those skilled in the art will understand that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε), and that these include several subclasses (e.g., γ1-γ4). It is the properties of this chain that determine the "class" of an antibody as IgG, IgM, IgA IgG, or IgE, respectively. Immunoglobulin isotype subclasses (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, etc.) are well-characterized and known to confer functional specialization. Modified versions of these classes and isotypes are readily identifiable to those skilled in the art in light of this disclosure and are therefore within the scope of this disclosure.

[0127] Both the light and heavy chains are divided into regions of structural homology and functional homology. The term “region” refers to a part or portion of an immunoglobulin or antibody chain, including constant regions or variable regions, and more distinct parts or portions of such regions. For example, a light chain variable region, as defined herein, includes “complementarity-determining regions” or “CDRs” scattered between “framework regions” or “FRs.”

[0128] Regions of immunoglobulin heavy or light chains can be defined as "constant" (C) regions or "variable" (V) regions based on the relative absence of sequence variants within the regions of various class members in the case of "constant regions," or on the significant variation within the regions of various class members in the case of "variable regions." The terms "constant region" and "variable region" can also be used functionally. In this regard, it will be understood that the variable region of an immunoglobulin or antibody determines antigen recognition and specificity. Conversely, the constant region of an immunoglobulin or antibody confers important effector functions such as secretion, transplacental migration, Fc receptor binding, and complement binding. The subunit structures and three-dimensional configurations of the constant regions of various immunoglobulin classes are well known.

[0129] The constant and variable regions of immunoglobulin heavy and light chains fold into domains. The term "domain" refers to a spherical region of a heavy or light chain that contains, for example, a β-pleated sheet and / or peptide loops stabilized by intrachain disulfide bonds (e.g., containing 3-4 peptide loops). The constant region domain on the light chain of an immunoglobulin is interchangeably referred to as the "light chain constant region domain," "CL region," or "CL domain." The constant domain on the heavy chain (e.g., hinge, CH1, CH2, or CH3 domain) is interchangeably referred to as the "heavy chain constant region domain," "CH region domain," or "CH domain." The variable domain on the light chain is interchangeably referred to as the "light chain variable region domain," "VL region domain," or "VL domain." The variable domain on the heavy chain is interchangeably referred to as the "heavy chain variable region domain," "VH region domain," or "VH domain."

[0130] Conventionally, variable constant region domains are numbered more rapidly because they are more distal to the antigen-binding site or the amino terminus of the immunoglobulin or antibody. The N-terminus of each immunoglobulin heavy chain and light chain is a variable region, while the C-terminus is a constant region; the CH3 domain and CL domain actually contain the carboxyl terminus of the heavy chain and light chain, respectively. Therefore, the domains of light chain immunoglobulins are arranged in a VL-CL orientation, and the domains of heavy chains are arranged in a VH-CH1-hinge-CH2-CH3 orientation.

[0131] The assignment of amino acids to each variable region domain follows the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat also provides a widely used numbering convention (Kabat numbering) that assigns the same number to corresponding residues between different heavy chain variable regions or between different light chain variable regions. CDR1, 2, and 3 of the VL domain are also referred herein as CDR-L1, CDR-L2, and CDR-L3, respectively. CDR1, 2, and 3 of the VH domain are also referred herein as CDR-H1, CDR-H2, and CDR-H3, respectively. If so, the assignment of CDRs may follow IMGT® (Lefranc et al., Developmental & Comparative Immunology 27:55-77; 2003) instead of Kabat. The numbering of the heavy chain constant regions follows the EU index described in Kabat (Kabat, Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD, 1987 and 1991).

[0132] The term "EU index" refers to the EU numbering convention for the constant region of antibodies, as described in Edelman GM et al. (1969), Proc. Natl. Acad. USA, 63, 78-85 and Kabat et al., Sequences of Proteins of Immunological Interest, USDept. Health and Human Services, 5th edition, 1991 (each of which is incorporated herein by reference in its entirety). Unless otherwise stated, all antibody Fc region numbering used herein corresponds to the EU numbering scheme as described in Edelman GM et al. (1969), Proc. Natl. Acad. USA, vol. 63(1): 78-85.

[0133] As used herein, the term "VH domain" includes the amino-terminal variable domain of an immunoglobulin heavy chain, and the term "VL domain" includes the amino-terminal variable domain of an immunoglobulin light chain.

[0134] As used herein, the term “CH1 domain” includes, for example, the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain extending from approximately 114–223 in the Kabat numbering system (EU positions 118–215). The CH1 domain is adjacent to the VH domain, is amino-terminal to the hinge region of the immunoglobulin heavy chain molecule, and does not form part of the Fc region of the immunoglobulin heavy chain.

[0135] As used herein, the term “hinge region” includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain. The hinge region comprises approximately 25 residues, is flexible, and thus allows the two N-terminal antigen-binding regions to move independently. The hinge region can be subdivided into three distinct domains: the upper, middle, and lower hinge domains (Roux KH et al. (1998), J.Immunol., vol.161:4083-90).

[0136] As used herein, the term “CH2 domain” includes, for example, a portion of a heavy-chain immunoglobulin molecule extending from a position approximately 244–360 (EU position 231–340) in the Kabat numbering system. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are sandwiched between two CH2 domains of an intact native IgG molecule. In one embodiment, the conjugated polypeptide of this disclosure includes a CH2 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0137] As used herein, the term “CH3 domain” includes a portion of a heavy-chain immunoglobulin molecule extending approximately 110 residues from the N-terminus of the CH2 domain, for example, from a position approximately 361–476 (EU position 341–445) in the Kabat numbering system. The CH3 domain typically forms the C-terminal portion of an antibody. However, in some immunoglobulins, further domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain in the μ-chain of IgM and the e-chain of IgE). In one embodiment, the conjugated polypeptide of this disclosure includes a CH3 domain derived from an IgG1 molecule (e.g., a human IgG1 molecule).

[0138] As used herein, the term “CL domain” includes, for example, the constant region domain of an immunoglobulin light chain extending from Kabat position approximately 107A to 216. The CL domain is adjacent to the VL domain. In one embodiment, the binding polypeptide of this disclosure includes a CL domain derived from a kappa light chain (e.g., human kappa light chain).

[0139] As shown above, the variable region of an antibody enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. Specifically, the VL and VH domains of the antibody combine to form a variable region (Fv) that defines the three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site located at the ends of each arm of the Y. More specifically, the antigen-binding site is defined by three complementary-determining regions (CDRs) in both the heavy-chain and light-chain variable regions. As used herein, the term “antigen-binding site” includes a site that specifically binds to (immunely reacts to) an antigen (e.g., a cell surface or soluble antigen). The antigen-binding site includes the immunoglobulin heavy-chain and light-chain variable regions, and the binding site formed by these variable regions determines the specificity of the antibody. The antigen-binding site is formed by different variable regions for each antibody. The modified antibodies of this disclosure include at least one antigen-binding site.

[0140] In certain embodiments, the binding polypeptide of the present disclosure comprises at least two antigen-binding domains that provide association between the binding polypeptide and a selected antigen. The antigen-binding domains do not need to originate from the same immunoglobulin molecule. In this regard, the variable region may originate from any type of animal that can be induced to initiate a humoral response and produce an immunoglobulin against the desired antigen. Thus, the variable region of the binding polypeptide may be of mammalian origin, for example, from humans, mice, rats, goats, sheep, non-human primates (e.g., cynomolgus macaques, macaques, etc.), lupine, or camelids (e.g., from camels, llamas, and related species).

[0141] In naturally occurring antibodies, the six CDRs present on each monomeric antibody are short, discontinuous sequences of amino acids specifically positioned to form antigen-binding sites when the antibody takes its three-dimensional configuration in an aqueous environment. The remaining portions of the heavy and light chain variable domains exhibit less intermolecular variability in their amino acid sequences and are called framework regions. These framework regions primarily adopt β-sheet higher-order structures, and the CDRs connect these β-sheet structures, forming loops that in some cases form parts of them. Thus, these framework regions act to form a scaffold that positions the six CDRs in the correct orientation through non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs defines a complementary surface to the epitope on the immunoreactive antigen. This complementary surface facilitates the non-covalent binding of the antibody to the immunoreactive antigen epitope.

[0142] Exemplary conjugated polypeptides include antibody variants. As used herein, the term “antibody variant” includes antibodies that have been modified to not exist in nature, such as synthetic and modified forms of antibodies that contain at least two heavy chain portions but do not contain two complete heavy chains (e.g., domain deletion antibodies or minibodies); multispecific forms of antibodies modified to bind to two or more different antigens or different epitopes on a single antigen (e.g., bispecific, tripspecific); and heavy chain molecules that can be linked to scFv molecules, etc. Furthermore, the term “antibody variant” includes multivalent forms of antibodies (e.g., trivalent, tetravalent antibodies that bind to three, four or more copies of the same antigen).

[0143] As used herein, the term “valence” refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds to one target molecule or specific site on a target molecule. If a polypeptide contains two or more target binding sites, each target binding site may specifically bind to the same or different molecules (e.g., different ligands or different antigens, or different epitopes on the same antigen). A target-binding polypeptide typically has at least one binding site specific to a human antigen molecule.

[0144] The term "specificity" refers to the ability to specifically bind (e.g., e.g., elicit an immune response) to a given target antigen (e.g., a human target antigen). A binding polypeptide may be monospecific and may contain one or more binding sites that specifically bind to a target, or a polypeptide may be multispecific and may contain two or more binding sites that specifically bind to the same or different targets. In certain embodiments, a binding polypeptide is specific to two different (e.g., non-overlapping) portions of the same target. In certain embodiments, a binding polypeptide is specific to two or more targets. Exemplary binding polypeptides (e.g., antibodies) containing antigen-binding sites that bind to antigens expressed on tumor cells are known in the Art, and one or more CDRs from such antibodies may be included in antibodies as described herein.

[0145] As used herein, the terms “antigen” or “target antigen” refer to a molecule or part of a molecule that can be bound by a binding site of a binding polypeptide. A target antigen may have one or more epitopes.

[0146] As used herein, “effector function” refers to a biochemical event resulting from the interaction between an antibody Fc region and an Fc receptor or ligand. A “functional Fc region” has “effector function” in its native sequence Fc region. Examples of “effector function” include antibody-dependent cell-mediated cytotoxicity (ADCC) or antibody-dependent cell-mediated phagocytosis (ADCP).

[0147] As used herein, “ADCC activity” refers to the ability of a binding polypeptide to induce an ADCC response. ADCC is a cell-mediated response in which antigen-nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a binding polypeptide bound to the surface of a target cell, which then causes the target cell to lyse (i.e., “die”). The primary mediator cell is the natural killer (NK) cell. NK cells express only FcγRIII, with FcγRIIIb being the activating receptor and FcγRIIIb being the inhibitory receptor; monocytes express FcγRI, FcγRII, and FcγRIII (Ravetch et al. (1991), Annu. Rev. Immunol., 9:457-92).

[0148] The terms "about" or "approximately" mean within approximately 20% of a given value or range, such as within approximately 10%, within approximately 5%, or within approximately 1% or less.

[0149] As used herein, “administer” or “dosage” means the act of physically delivering an extracorporeal substance (e.g., isolated bound polypeptides provided herein) to a patient by injection or other means, including, but not limited to, the lungs (e.g., inhalation), mucous membranes (e.g., nasal cavity), intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. If the disease or its symptoms are under control or treatment, administration of the substance is typically performed after the onset of the disease or its symptoms. If the disease or its symptoms are preventable, administration of the substance is typically performed before the onset of the disease or its symptoms and may be continued chronically to delay the appearance or reduce the severity of disease-related symptoms.

[0150] As used herein, the term “composition” is intended to encompass any product containing a specific component (e.g., an isolated bound polypeptide provided herein) in a specific amount, as well as any product resulting directly or indirectly from a combination of specific components in a specific amount.

[0151] "Effective dose" means the amount of active pharmaceutical ingredient (e.g., the isolated bound polypeptide of this disclosure) sufficient to achieve the desired physiological outcome in an individual requiring the drug. The effective dose may vary between individuals depending on the health and physical condition of the individual being treated, the taxonomic group of the individual being treated, the formulation of the composition, the assessment of the individual's medical condition, and other relevant factors.

[0152] As used herein, the terms “subject” and “patient” are interchangeable. As used herein, the subject may be a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a mammal (e.g., monkeys and humans). In certain embodiments, as used herein, the term “subject” refers to a vertebrate such as a mammal. Examples of mammals include, but are not limited to, humans, non-human primates, wild animals, wild animals, domesticated animals, sport animals, and pets.

[0153] As used herein, the term “treatment” means any protocol, method and / or agent that can be used to prevent, manage, treat and / or improve a disease or its associated symptoms. In some embodiments, the term “treatment” means any protocol, method and / or agent that can be used to modulate the immune response to infection or its associated symptoms in a subject. In some embodiments, the terms “therapy” and “treatment” mean biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare workers, that are useful for preventing, managing, treating and / or improving a disease or its associated symptoms. In other embodiments, the terms “therapy” and “treatment” mean biological therapies, supportive therapies and / or other therapies known to those skilled in the art, such as healthcare workers, that are useful for modulating the immune response to infection or its associated symptoms in a subject.

[0154] As used herein, the terms “to treat,” “treatment,” and “to treat” refer to a reduction or improvement in the progression, severity, and / or duration of a disease or associated symptoms resulting from the administration of one or more treatments (including, but not limited to, the administration of one or more prophylactic or therapeutic agents, e.g., isolated bound polypeptides provided herein). As used herein, the term “to treat” may also refer to altering the course of the disease being treated. The therapeutic effects of treatment include, but are not limited to, prevention of disease onset or recurrence, relief of symptoms, reduction of direct or indirect pathological consequences of the disease, slowing of disease progression, improvement or mitigation of the disease state, and remission or improvement of prognosis.

[0155] As used herein, the terms “N-glycan” or “N-linked glycan” refer to a glycan that is linked to the amide nitrogen of an asparagine or arginine residue in a protein via an N-acetylglucosamine residue. In certain embodiments, the binding polypeptide of interest herein uses a glycan that is “N-linked” via an asparagine residue to a glycosylation site in the polypeptide backbone of the binding polypeptide. The glycosylation site may be a natural or modified glycosylation site. These “N-linked glycosylation sites” occur, for example, in a peptide primary structure containing the amino acid sequence asparagine-X-serine / threonine, where X is any amino acid residue other than proline and aspartic acid. Such N-glycans are well described, for example, in Drickamer K, Taylor ME (2006) Introduction to Glycobiology, 2nd ed., (which is incorporated herein by reference in its entirety).

[0156] As used herein, the term “glycomorphization” refers to any technically recognized method for altering the glycoform profile of a binding protein composition to produce a “modified glycan.” In certain embodiments, glycomorphized binding proteins and / or binding polypeptides, and methods for producing glycomorphized binding proteins and / or binding polypeptides are provided.

[0157] As used herein, the terms “G0 glycoform” or “G0,” “G1 glycoform” or “G1,” and “G2 glycoform” or “G2” refer to glycoforms of N-glycans having 0, 1, or 2 terminal galactose residues, respectively. These terms include G0, G1, and G2 glycoforms containing fucosylated or bifacial N-acetylglucosamine residues.

[0158] As used herein, the terms "G0F glycoform," "G1F glycoform," and "G2F glycoform" refer to fucosylated "G0 glycoform," "G1 glycoform," and "G2 glycoform," respectively.

[0159] As used herein, the terms “oligomannose-type N-glycan” or “oligomannose glycan” refer to any one or a combination of the following mannose-rich structures: Man5(GlcNAc)2, Man6(GlcNAc)2, Man7(GlcNAc)2, Man8(GlcNAc)2, and Man9(GlcNAc)2. See Schachter et al. “Mannose Oligosaccharide”, 4.06.3.3.1, Comprehensive Glycoscience, 2007. As used herein, Man5 refers to the structure Man5(GlcNAc)2; Man6 refers to the structure Man6(GlcNAc)2; Man7 refers to the structure Man7(GlcNAc)2; Man8 refers to the structure Man8(GlcNAc)2; and Man9 refers to the structure Man9(GlcNAc)2. In certain exemplary embodiments, the oligomannose glycan is Man8(GlcNAc)2 or Man9(GlcNAc)2.

[0160] As defined herein, the terms “lysosome-targeted chimera” or “LYTAC” refer to a bifunctional molecule comprising a region capable of binding to a cell surface lysosome-targeted receptor and a region capable of binding to the extracellular domain of a target protein, including but not limited to secreted extracellular proteins and membrane-bound proteins. Thus, LYTACs are a useful alternative to the above-mentioned PROTACs when the target protein of interest is not intracellular. Further LYTAC disclosures and exemplary LYTACs are described in Banik SM et al. (2019), ChemRxiv., Banik SM et al. (2020), Nature, vol. 584(7820): 291-297, International Publication No. 2015 / 143091, and International Publication No. 2020 / 132100 (these are incorporated herein by reference, respectively). When used herein, the portion of LYTAC that can bind to the extracellular domain of the target protein corresponds to the antigen-binding protein or a fragment thereof of this disclosure.

[0161] Oligomannose-type N-glycan In exemplary embodiments, a conjugated polypeptide containing an oligomannose-type N-glycan is disclosed herein. In other exemplary embodiments, the oligomannose-type N-glycan is obtained by culturing cells modified to express the conjugated polypeptide in the presence of a mannosidase inhibitor (e.g., the α-mannosidase I inhibitor kifunensin or its derivatives or functional homologs). In these embodiments, treatment of cells with a mannosidase inhibitor prevents the formation of complex N-glycans while producing a conjugated polypeptide containing an oligomannose-type N-glycan.

[0162] In other embodiments, cells modified to express a binding polypeptide may lack one or more glycosidases required for the initial processing of N-glycans. In some embodiments, culture conditions may be such that the activity of one or more of these glycosidases is inhibited. As a result of one or both of these conditions, oligosaccharide synthesis shifts to oligomannose-type species. For example, cells may lack one or more glycosidases selected from the group consisting of α-glucosidase I, α-glucosidase II, and α-mannosidase I. Cells lacking the glycosidase of interest may be modified using methods such as those described in, for example, Tymms et al., Gene Knockout Protocols (Methods in Molecular Biology), Humana Press, 1st ed., 2001; and Joyner, Gene Targeting: A Practical Approach, Oxford University Press, 2nd ed., 2000. For example, glycosidase-deficient cells may be modified using lectin selection. See Stanley P et al. (1975), Proc. Natl. Acad. USA, vol. 72(9): 3323-3327.

[0163] In some embodiments, the conjugated polypeptide containing oligomannose-type N-glycan may be prepared by chemically binding a non-glycosylated antibody or Fc fusion protein with a separately synthesized oligosaccharide moiety.

[0164] In some embodiments, cells are modified not to express one or more glycosidases selected from the group consisting of α-glucosidase I, α-glucosidase II, and α-mannosidase I. In one embodiment, the glycosidase gene can be disrupted by targeted mutagenesis. In some embodiments, targeted mutagenesis can be achieved, for example, by targeting a CRISPR (clustered and regularly arranged short palindromic sequence repeat) site in the glycosidase gene. In some embodiments, at least one or more expression vectors encoding targeted RNA and a polynucleotide sequence encoding a CRISPR-related nuclease such as Cas9 are used to modify cells not to express the glycosidase gene.

[0165] In further embodiments, cells modified to express the binding polypeptide may be contacted with inhibitors of one or more glycosidases selected from the group consisting of α-glucosidase I, α-glucosidase II, and α-mannosidase I. In some embodiments, these enzyme inhibitors may be, for example, small molecules or small interfering RNAs (siRNAs). siRNAs are short (20-25 nt) double-stranded RNAs that inhibit the glycosidase of interest via post-transcriptional gene silencing. Glycosidase-specific siRNAs may be prepared and used as described in U.S. Patent No. 6,506,559 and / or by other preferred methods. See Appasani, RNA Interference Technology From Basic Science to Drug Development, Cambridge University Press, 1st ed., 2005; and Uei-Ti K et al. (2004), Nucleic Acids Res., vol.32(3):936-948. Examples of low-molecular-weight α-glucosidase I inhibitors include castanospermine (see Pan YT et al. (1983), Biochemistry, vol.22(16):3975-3984), deoxynojirimycin (known as "DNJ"; see Hettkamp H et al. (1984), Eur.J.Biochem., vol.142:85-90) and its N-alkyl and N-alkenyl derivatives (e.g., N-butyl-DNJ); 2,5-dihydromethyl-3,4-dihydroxypyrrolidine (known as "DMDP"; see Elbein AD et al. (1984), J.Biol.Chem., vol.259(2):12409-12413); and australine (Molyneux RJ et al.) (See al. (1988), J.Nat.Prod., vol.51:1198-1206). Examples of small molecule α-glucosidase II inhibitors include DNJ and its N-alkyl and N-alkenyl derivatives, as well as MDL25637.See Hettkamp H et al. (1984), Eur. J. Biochem., vol. 142: 85-90 and Kaushal GP, et al. (1988), J. Biol. Chem., vol. 263 (33): 17278-17283. Examples of low-molecular-weight α-mannosidase I inhibitors include deoxymannojirimycin (DMJ) (see Legler G and Julick E (1984), Carbohydr. Res., vol. 128(1): 61-72) and its derivatives (e.g., N-methyl derivatives as described in Bosch Jv et al. (1985), Virology, vol. 143(1): 342-346), 1,4-dideoxy-1,4-imino-D-mannitol (DIM) (see Fleet et al. (1984), J. Chem. Soc. Chem. Commun., vol. 1240-1241 and Palmarzyk G et al. (1985), Arch. Biochem. Biophys., vol. 243: 35-45), and kifunensin (Elbein AD et al.) See al. (1990), J. Biol. Chem., vol.265:15599-15605.

[0166] In one exemplary embodiment, cells modified to express a binding polypeptide are cultured in the presence of the α-mannosidase I inhibitor kifunensin. In certain embodiments, kifunensin may be used for periods of at least 12, 24, 48, 72 hours or 4, 7, 10, 20 days or more, or continuously, at concentrations of 0.01–100 μg / ml, 0.01–75 μg / ml, 0.01–50 μg / ml, 0.01–40 μg / ml, 0.01–30 μg / ml, 0.01–20 μg / ml, 0.1–10 μg / ml, 0.1–2.0 μg / ml, or 1–0.5 μg / ml. In one exemplary embodiment, CHO or hybridoma cells are incubated with approximately 0.5–10 μg / ml of kifunensin for 10 days. In one exemplary embodiment, kifunensin is used at concentrations ranging from 60 ng / mL to approximately 2500 ng / mL. In a further exemplary embodiment, kifunensin is used at a concentration of 2000 ng / mL.

[0167] In further embodiments, the oligomannose-type N-glycan on the bound polypeptide disclosed herein comprises one or more oligomannose-type oligosaccharides selected from the group consisting of Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and Man5(GlcNAc)2.

[0168] In other exemplary embodiments, the composition produced by the method disclosed herein contains at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more (in molar ratio to total N-glycans) of oligomannose-type glycans Man 5-9 Contains (GlcNAc)2. In some embodiments, a composition is provided comprising a group of isolated glycosylated polypeptides, each containing an Fc domain containing an N-glycan, wherein the composition contains at least 50% Man in molar ratio relative to the total N-glycan. 5-9 Contains (GlcNAc)2N-glycan.

[0169] In another exemplary embodiment, the conjugated polypeptide disclosed herein comprises Man8 and Man9N-glycans. In yet another exemplary embodiment, a composition is provided comprising a group of isolated glycosylated conjugated polypeptides, each comprising an Fc domain containing an N-glycan, wherein the composition contains at least 50% Man in molar ratio relative to the total N-glycan. 5-9 (GlcNAc)2N-glycan is included, and Man8 and Man9-containing N-glycans together constitute the main species.

[0170] In one embodiment, the binding polypeptide disclosed herein mainly contains Man9(GlcNAc)2N-glycan. In some embodiments, a composition is provided comprising a group of isolated glycosylated binding polypeptides, each containing an Fc domain containing an N-glycan, wherein the composition contains at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, or 99% Man9(GlcNAc)2N-glycan in molar ratio relative to the total N-glycan. 5-9 (GlcNAc)2N-glycan is included. In an exemplary embodiment, a composition is provided comprising a group of isolated glycosylated polypeptides, each comprising an Fc domain containing an N-glycan, wherein the composition contains at least 97% Man in molar ratio relative to the total N-glycan. 5-9 Contains (GlcNAc)2N-glycan.

[0171] In some embodiments, the bound polypeptides disclosed herein contain reduced or undetectable amounts of oligomannose-type N-glycans Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and Man5(GlcNAc)2, while also containing trace amounts (e.g., less than 10% of total N-glycans) or undetectable amounts of complex-type N-glycans (e.g., G0, C1, G2, G0F, G1F, G2F, and G0F-Gn).

[0172] In some embodiments, Man in the composition of the present disclosure 5-9(GlcNAc)2 is substantially defucosylated (i.e., defucosylated or nonfucosylated), meaning they contain 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or less than 30% (in molar ratio compared to total N-glycans) or less fucose. In some embodiments, the composition contains less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% (in molar ratio compared to total N-glycans) of Man5(GlcNAc)2 and / or Man6(GlcNAc)2N-glycans. In some embodiments, the composition contains trace amounts (i.e., less than 10% in molar ratio compared to total N-glycans) or undetectable amounts of Man4(GlcNAc)2. In some embodiments, the composition contains 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less than 1% (in molar ratio compared to total N-glycans) or less of complex glycans.

[0173] Glycan compositions can be evaluated using methods such as lectin blotting, HPLC, and / or mass spectrometry such as MALDI-TOF (e.g., Townsend et al. (1997), Techniques in Glybiology, CRC Press).

[0174] In some embodiments, binding polypeptides containing oligomannose-type glycans exhibit enhanced ADCC activity compared to the same binding polypeptide prepared without treatment with a mannosidase inhibitor (e.g., kifunensin). In other embodiments, binding polypeptides containing oligomannose-type glycans showed enhanced binding to Fc receptors. In exemplary embodiments, binding polypeptides with oligomannose-type glycans showed enhanced binding to Fcγ receptors. In further exemplary embodiments, binding polypeptides with oligomannose-type glycans showed enhanced binding to FcγRIIIa.

[0175] In further embodiments, the binding polypeptide containing the oligomannose-type glycan exhibits substantially the same or better binding specificity to the target. In some embodiments, the binding polypeptide containing the oligomannose-type glycan exhibits substantially the same or higher binding affinity to the target. In some embodiments, the binding polypeptide containing the oligomannose-type glycan exhibits substantially the same or lower binding affinity to the mannose receptor.

[0176] Determination of polypeptide binding and specificity The binding affinity of antibodies or Fc fusion proteins to the target and to the Fc receptor and mannose receptor can be evaluated using surface plasmon resonance, ELISA, or other suitable methods (see Shields RL et al. (2001), J. Biol. Chem., vol. 276: 6591-6604). In some embodiments, the binding constant K of the binding polypeptide to the Fc receptor is used. D The binding constant K of the binding polypeptide to its target (e.g., antigen) may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times higher than that of the wild-type control. D This may be substantially the same as (i.e., ±50%) as the wild-type control or may be better. In some embodiments, the binding constant K of the antibody or Fc fusion protein of the present invention to the mannose receptor DThis may be substantially the same as (i.e., ±50%) as the wild-type control, or it may be lower.

[0177] The binding specificity of the antibody or Fc fusion protein can be determined, for example, by flow cytometry, Western blotting, or another preferred method. In some embodiments, the binding polypeptide is directed toward a human target protein (e.g., a human antigen) expressed on the surface of a target cell. In some embodiments, this may be toward a soluble antigen. In some other embodiments, the binding polypeptide is toward a pathogenic target (e.g., a viral or bacterial protein). The binding polypeptide may be specific to a human target or may cross-react with corresponding targets from other species.

[0178] In some embodiments, certain pharmacokinetic parameters of the conjugated polypeptide of the present invention are the same as or better than those of the wild-type control. For example, in some embodiments, the excluded half-life (t 1 / 2 The concentration and / or area under the concentration curve (AUC) may be substantially the same as (i.e., ±50%) as the wild-type control, or may be higher. Pharmacokinetic parameters may be measured in humans or using appropriate animal models. See, for example, Shargel L and Yu A (1995), Applied Biopharmaceutics and Pharmacokinetics, 4th ed., McGraw-Hill / Appleton.

[0179] FC domain and FC modification In certain aspects of this disclosure, Fc domains, such as Fc domain variants, are provided. As used herein, the term “Fc region” or “Fc domain” refers to the portion of the heavy chain constant region that begins in the hinge region immediately upstream of the papain cleavage site (i.e., residue 216 in IgG, with the first residue of the heavy chain constant region being 114) and ends at the C-terminus of the antibody. Thus, a complete Fc region includes at least the hinge domain, the CH2 domain, and the CH3 domain.

[0180] The Fc region of an antibody is involved in non-antigen binding and can mediate effector function by binding to Fc receptors. There are several different types of Fc receptors, which are classified based on the type of antibody they recognize. For example, the Fc-gamma receptor (FcγR) binds to IgG class antibodies, the Fc-alpha receptor (FcαR) binds to IgA class antibodies, and the Fc-epsilon receptor (FcεR) binds to IgE class antibodies. The neonatal Fc receptor (FcRn) interacts with the Fc region of an antibody to promote antibody recycling through the rescue of normal lysosomal degradation. FcγR belongs to a family that includes several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb.

[0181] As used herein, the terms “natural Fc” or “wild-type Fc” refer to molecules corresponding to sequences of non-antigen-binding fragments produced by other means, whether resulting from antibody digestion or in monomeric or multimeric form, and which may contain hinge regions. The original immunoglobulin source of natural Fc is typically of human origin and can be any immunoglobulin, e.g., IgG1 and IgG2. Natural Fc molecules consist of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of a natural Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, and IgGA2). An example of natural Fc is a disulfide-bonded dimer resulting from papain digestion of IgG. As used herein, the term “natural Fc” can be used to refer to monomeric, dimeric, or polymeric forms.

[0182] As used herein, the terms “Fc domain variant,” “Fc variant,” or “modified Fc” refer to molecules or sequences that have been modified from natural / wild-type Fc but still contain a binding site to FcR. Therefore, the term “Fc variant” may include molecules or sequences that are humanized from non-human natural Fc. Furthermore, natural Fc may include regions that provide structural features or biological activity not required for the antibody-like binding polypeptides described herein and may therefore be removed. Accordingly, the term “Fc variant” includes molecules or sequences that lack one or more natural Fc sites or residues, or in which one or more Fc sites or residues are modified, and which affect or are involved in: (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) N-terminal heterogeneity upon expression in selected host cells, (4) glycosylation, (5) interaction with complements, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cell-mediated cytotoxicity (ADCC).

[0183] In certain exemplary embodiments, the Fc variants characterized herein have one or more of the following properties compared to wild-type Fc: extended serum half-life, enhanced FcRN-binding affinity, enhanced FcRN-binding affinity at acidic pH, enhanced FcγRIIIa-binding affinity, and / or similar thermal stability.

[0184] FcγRIIIa V158, or human CD16a-V receptor, or CD16aV, refers to a polypeptide construct containing a fragment of the CD16 human receptor that binds to the Fc region of native antibodies, mediates antibody-dependent cytotoxicity, and possesses valine (V) at position 158. This has also been reported in the literature as allotype CD16a V158. FcγRIIIa F158, or human CD16a-F receptor, or CD16aF, refers to a polypeptide construct containing a fragment of the CD16 human receptor that binds to the Fc region of native antibodies, mediates antibody-dependent cytotoxicity, and possesses phenylalanine (F) at position 158. This has also been reported in the literature as allotype CD16a F158.

[0185] As used herein, the term “Fc domain” encompasses natural / wild-type Fc and Fc variants and sequences as defined herein. Similar to Fc variants and natural Fc molecules, the term “Fc domain” includes molecules in monomeric or polymeric form, whether digested from the whole antibody or produced by other means. In certain exemplary embodiments, the Fc domains described herein are thermally stabilized.

[0186] In certain exemplary embodiments, the Fc domain described herein is glycosylated (e.g., via N-linked glycosylation). In some embodiments, the Fc domain includes N-linked glycosylation, for example, with an N-linked glycosylation motif containing the amino acid sequence NXT or NXS (where X is any amino acid residue other than proline). In some embodiments, the Fc domain is glycosylated at a native glycosylation site corresponding to amino acid position 297 of the Fc region, according to EU numbering.

[0187] In certain exemplary embodiments, the Fc domain is glycosylated with an oligomannose-type N-glycan. In other exemplary embodiments, the glycosylated Fc domain contains an oligomannose-type N-glycan selected from the group consisting of Man9(GlcNAc)2, Man8(GlcNAc)2, Man7(GlcNAc)2, Man6(GlcNAc)2, and Man5(GlcNAc)2. In yet another exemplary embodiment, the Fc domain contains an oligomannose-type N-glycan that is mainly Man9(GlcNAc)2 and Man8(GlcNAc)2. In some exemplary embodiments, the Fc domain contains an oligomannose-type N-glycan that is mainly Man9(GlcNAc)2. In certain exemplary embodiments, the Fc domain is glycosylated with an oligomannose-type N-glycan at the native Fc glycosylation site corresponding to EU position 297.

[0188] In other exemplary embodiments, the Fc domain is glycosylated by an oligomannose-type N-glycan at a modified (non-natural) Fc glycosylation site. The exemplary non-natural Fc glycosylation site includes an asparagine residue at EU position 298, a serine or threonine residue at amino acid position 300, and optionally, an alanine residue at EU position 299 and / or a glutamine residue at EU position 297. The exemplary non-natural Fc glycosylation site includes the “NNAS” glycosylation motif described in U.S. Patent No. 9,790,268, which is incorporated herein by reference.

[0189] In certain exemplary embodiments, the Fc domains described herein are any combination of thermal stabilization and contain oligomannose-type N-glycans and Fc variants.

[0190] In one embodiment, the present disclosure provides an Fc domain variant comprising effector-enhancing amino acid substitutions.

[0191] In one embodiment, an Fc domain variant having a modified FcγRIIIa bond, comprising one or more amino acid substitutions as disclosed herein. In one embodiment, an Fc domain variant with enhanced FcγRIIIa binding affinity, comprising one or more amino acid substitutions as disclosed herein. In one embodiment, the Fc domain variant with enhanced FcγRIIIa binding affinity comprises two or more amino acid substitutions as disclosed herein. In one embodiment, the Fc domain variant with enhanced FcγRIIIa binding affinity comprises three or more amino acid substitutions as disclosed herein. In one embodiment, the Fc domain variant with enhanced FcγRIIIa binding affinity comprises four or more amino acid substitutions as disclosed herein.

[0192] In one exemplary embodiment, the binding polypeptide or Fc domain variant disclosed herein has an increased affinity for binding to human FcγRIIIa, which is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher compared to the WT binding polypeptide.

[0193] In one embodiment, an Fc domain variant having a modified FcRN-binding includes an Fc domain having one or more amino acid substitutions as disclosed herein. In one embodiment, an Fc domain variant with enhanced FcRN-binding affinity includes an Fc domain having one or more amino acid substitutions as disclosed herein. In one embodiment, an Fc domain variant with enhanced FcRN-binding affinity includes an Fc domain having two or more amino acid substitutions as disclosed herein. In one embodiment, an Fc domain variant with enhanced FcRN-binding affinity includes an Fc domain having three or more amino acid substitutions as disclosed herein.

[0194] In some embodiments, the Fc domain variant may exhibit species-specific FcRN-binding affinity. In one embodiment, the Fc domain variant may exhibit human FcRN-binding affinity. In one embodiment, the Fc domain variant may exhibit cynomolgus monkey FcRN-binding affinity. In some embodiments, the Fc domain variant may exhibit interspecies FcRN-binding affinity. Such Fc domain variants are said to be cross-reactive across one or more different species. In one embodiment, the Fc domain variant may exhibit FcRN-binding affinity for both humans and cynomolgus monkeys.

[0195] The neonatal Fc receptor (FcRn) interacts with the Fc region of antibodies to promote recycling through rescue of normal lysosomal degradation. This process occurs in endosomes at acidic pH (e.g., pH <6.5) but is a pH-dependent process that does not occur under physiological pH conditions in blood flow (e.g., non-acidic pH). In some embodiments, Fc domain variants exhibit enhanced FcRN-binding affinity at acidic pH compared to the wild-type Fc domain. In some embodiments, Fc domain variants exhibit enhanced FcRN-binding affinity at pH <7, e.g., approximately pH 6.5, approximately pH 6.0, approximately pH 5.5, and approximately pH 5.0, compared to the wild-type Fc domain. In some embodiments, Fc domain variants exhibit enhanced FcRN-binding affinity at pH <7, e.g., approximately pH 6.5, approximately pH 6.0, approximately pH 5.5, and approximately pH 5.0, compared to the FcRN-binding affinity of the wild-type Fc domain at elevated non-acidic pH. The elevated non-acidic pH could be, for example, above pH 7, around pH 7, around pH 7.4, around pH 7.6, around pH 7.8, around pH 8.0, around pH 8.5, or around pH 9.0.

[0196] In certain embodiments, it may be desirable for the Fc domain variant to exhibit substantially the same FcRN-binding affinity as the wild-type Fc domain at non-acidic pH. In some embodiments, it may be desirable for the Fc domain variant to exhibit lower FcRN-binding affinity at non-acidic pH than the binding polypeptide containing a modified Fc domain having the double amino acid substitution M428L / N434S according to EU numbering. See U.S. Patent No. 8,088,376. Accordingly, it may be desirable for the Fc domain variant to exhibit minimal perturbation to pH-dependent FcRN-binding.

[0197] In some embodiments, Fc domain variants with enhanced FcRN-binding affinity at acidic pH exhibit a reduced (i.e., slower) FcRn off-rate compared to the wild-type Fc domain. In some embodiments, Fc domain variants with enhanced FcRN-binding affinity at acidic pH compared to the FcRN-binding affinity of the bound polypeptide at high non-acidic pH exhibit a slower FcRn off-rate at acidic pH compared to the FcRn off-rate of the wild-type Fc domain at high non-acidic pH.

[0198] Certain embodiments include an Fc domain variant in which at least one amino acid in one or more constant region domains is deleted or otherwise modified to provide desired biochemical characteristics, such as decreased or enhanced effector function, non-covalent dimerization ability, improved ability to localize to tumor sites, shortened or prolonged serum half-life, compared to a completely unchanged antibody of substantially the same immunogenicity.

[0199] In certain other embodiments, the Fc domain variant includes a constant region derived from a different antibody isotype (e.g., constant regions derived from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the Fc domain variant includes a chimeric hinge (i.e., a hinge including hinge domains from different antibody isotypes, e.g., an upper hinge domain derived from an IgG4 molecule and a hinge portion derived from an IgG1 intermediate hinge domain). In certain embodiments, the Fc domain may be mutated to increase or decrease effector function using techniques known in the art.

[0200] In some embodiments, Fc domain variants exhibit altered binding affinity to Fc receptors. There are several different types of Fc receptors, classified based on the type of antibody they recognize. For example, Fc-gamma receptors (FcγR) bind to IgG class antibodies, Fc-alpha receptors (FcαR) bind to IgA class antibodies, and Fc-epsilon receptors (FcεR) bind to IgE class antibodies. FcγR belongs to a family that includes several members, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb. In some embodiments, Fc domain variants exhibit altered FcγRIIIa binding affinity compared to the wild-type Fc domain. In some embodiments, Fc domain variants exhibit decreased FcγRIIIa binding affinity compared to the wild-type Fc domain. In some embodiments, Fc domain variants exhibit enhanced FcγRIIIa binding affinity compared to the wild-type Fc domain. In some embodiments, the Fc domain variant-modified Fc domain has nearly the same FcγRIIIa binding affinity as the wild-type Fc domain.

[0201] In some embodiments, the Fc domain variant exhibits altered binding affinity to the Fc receptor (e.g., improved affinity to the FcyRIIIa receptor) while maintaining thermal stability similar to the binding polypeptide having the wild-type Fc domain. In certain embodiments, the Fc variant has a Tm of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C or less than the binding polypeptide having the wild-type Fc domain. In one exemplary embodiment, the Fc variant has a melting temperature (Tm) of 10°C or less than that of the binding polypeptide having the wild-type Fc domain.

[0202] In some embodiments, the Fc domain variant has altered binding affinity to the Fc receptor (e.g., increased affinity to the FcyRIIIa receptor), and its thermal stability is similar to that of the binding polypeptide with the wild-type Fc domain. The binding polypeptide with the variant Fc domain is produced by culturing cells expressing the binding polypeptide in the presence of kifunensin, while the binding polypeptide with the wild-type Fc domain is produced by culturing cells in the presence of kifunensin. In certain embodiments, the Fc variant has a Tm of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10°C or less than that of the binding polypeptide with the wild-type Fc domain. The binding polypeptide with the wild-type Fc domain is produced by culturing cells expressing the binding polypeptide in the presence of kifunensin, while the binding polypeptide with the wild-type Fc domain is produced by culturing cells in the absence of kifunensin. In one exemplary embodiment, the Fc variant has a Tm of 10°C or less of the binding polypeptide with the wild-type Fc domain, the binding polypeptide with the variant Fc domain is produced by culturing cells expressing the binding polypeptide in the presence of kifunensin, and the binding polypeptide with the wild-type Fc domain is produced by culturing cells in the absence of kifunensin.

[0203] In some embodiments, the Fc domain variant has altered binding affinity to the Fc receptor (e.g., improved affinity to the FcyRIIIa receptor), exhibits thermal stability similar to the binding polypeptide with the wild-type Fc domain, and both the variant Fc domain-containing and wild-type Fc domain-containing binding polypeptides are produced by culturing cells expressing the binding polypeptide in the presence of kifunensin. In specific embodiments, the Fc variant has a Tm of 1, 2, 3, 4, or 5°C or less than that of the wild-type Fc domain-containing binding polypeptide, and both the variant Fc domain-containing and wild-type Fc domain-containing binding polypeptides are produced by culturing cells expressing the binding polypeptide in the presence of kifunensin. In one exemplary embodiment, the Fc variant has a Tm of 5°C or less than that of the wild-type Fc domain-containing binding polypeptide, and both the variant Fc domain-containing and wild-type Fc domain-containing binding polypeptides are produced by culturing cells expressing the binding polypeptide in the presence of kifunensin.

[0204] In certain embodiments, the binding polypeptide may include an antibody constant region (e.g., an IgG constant region, e.g., a human IgG constant region, e.g., a human IgG1 or IgG4 constant region) that mediates one or more effector functions. For example, binding of the C1 complex to an antibody constant region may activate the complement system. Complement system activation is important in the opsonization and lysis of cellular pathogens. Complement system activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies bind to various cell receptors via their Fc region (the Fc receptor binding site on the antibody Fc region binds to the cell's Fc receptor (FcR)). Several Fc receptors exist that are specific to different classes of antibodies, including IgG (gamma receptor), IgE (epsilon receptor), IgA (alpha receptor), and IgM (mu receptor). Antibody binding to Fc receptors on the cell surface triggers several important and diverse biological responses, including phagocytosis and destruction of antibody-coated particles, clearance of immune complexes, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. In some embodiments, the binding polypeptide (e.g., an antibody or its antigen-binding fragment) binds to the Fc-gamma receptor. In alternative embodiments, the binding polypeptide may include a constant region lacking one or more effector functions (e.g., ADCC activity) and / or unable to bind to the Fcγ receptor.

[0205] Binding polypeptides with enhanced ADCC activity are disclosed herein. As used herein, “ADCC activity” refers to the ability of a binding polypeptide to induce an ADCC response. ADCC is a cell-mediated response in which antigen-nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize a binding polypeptide bound to the surface of a target cell, and subsequently cause lysis (i.e., “death”) of the target cell. The primary mediator cell is the natural killer (NK) cell. NK cells express only FcγRIII, with FcγRIIIa being the activating receptor and FcγRIIIb being the inhibitory receptor; monocytes express FcγRI, FcγRII, and FcγRIII (Ravetch et al. (1991), Annu. Rev. Immunol., vol. 9: 457-92). ADCC activity can be directly evaluated using in vitro assays, such as release assays using peripheral blood mononuclear cells (PBMCs) and / or NK effector cells, or bioluminescent reporter bioassays as described in the examples (see also Shields RL et al. (2001) J. Biol. Chem., vol. 276(9): 6591-6604). ADCC activity can be expressed as the concentration of the conjugated polypeptide at which target cell lysis is at most half. Thus, in some embodiments, the concentration of the conjugated polypeptide of the present invention at which the lysis level is the same as the half-maximal lysis level by the wild-type control is at least 2, 3, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times lower than the concentration of the wild-type control itself. In addition, in some embodiments, the conjugated polypeptide of the present invention may exhibit higher maximum target cell lysis compared to the wild-type control. For example, the maximum target cell lysis of the antibody or Fc fusion protein of the present invention may be 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, or more higher than that of the wild-type control.

[0206] In one exemplary embodiment, the binding polypeptide or Fc domain variant disclosed herein exhibits enhanced antibody-dependent cytotoxicity (ADCC) activity compared to the WT-binding polypeptide. In further exemplary embodiments, the ADCC activity of the binding polypeptide or Fc domain variant is at least 1, 2, 3, 4, or 5 times higher compared to the WT-binding polypeptide.

[0207] Certain embodiments include antibodies in which at least one amino acid in one or more constant region domains is deleted or otherwise modified to result in desired biochemical characteristics, such as decreased or enhanced effector function, non-covalent dimerization ability, improved ability to localize to tumor sites, shortened or prolonged serum half-life, compared to a completely unaltered antibody of substantially the same immunogenicity. For example, certain antibodies for use in the diagnostic and treatment methods described herein are domain deletion antibodies that contain a polypeptide chain similar to an immunoglobulin heavy chain but lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the constant region of the modified antibody is deleted, for example, all or part of the CH2 domain is deleted.

[0208] In certain other embodiments, the conjugated polypeptide includes constant regions derived from different antibody isotypes (e.g., constant regions derived from two or more of human IgG1, IgG2, IgG3, or IgG4). In other embodiments, the conjugated polypeptide includes a chimeric hinge (i.e., a hinge comprising hinge portions derived from hinge domains of different antibody isotypes, e.g., an upper hinge domain from an IgG4 molecule and an IgG1 intermediate hinge domain). In one embodiment, the conjugated polypeptide includes an Fc region or a portion thereof derived from a human IgG4 molecule and a Ser228Pro mutation (EU numbering) in the core hinge region of the molecule.

[0209] The amino acid substitutions in the Fc variant can be located at any position within the Fc domain (i.e., any amino acid position according to the EU Code). In one embodiment, the Fc variant includes substitutions at an amino acid position located in or part of the hinge domain. In another embodiment, the Fc variant includes substitutions at an amino acid position located in or part of the CH2 domain. In yet another embodiment, the Fc variant includes substitutions at an amino acid position located in or part of the CH3 domain. In yet another embodiment, the Fc variant includes substitutions at an amino acid position located in or part of the CH4 domain.

[0210] The binding polypeptide may be any technically recognized Fc variant known to result in effector function and / or improvement (e.g., reduction or enhancement) of FcR binding. Such Fc variants are, for example, those found in the International PCT Publications, International Publications 88 / 07089A1, 96 / 14339A1, 98 / 05787A1, 98 / 23289A1, 99 / 51642A1, 99 / 58572A1, 00 / 09560A2, 00 / 32767A1, 00 / 42072A2, and 02 / 4421. Pamphlet No. 5A2, Pamphlet No. 02 / 060919A2, Pamphlet No. 03 / 074569A2, Pamphlet No. 04 / 016750A2, Pamphlet No. 04 / 029207A2, Pamphlet No. 04 / 035752A2, Pamphlet No. 04 / 063351A2, Pamphlet No. 04 / 074455A2, Pamphlet No. 04 / 099249A2, Pamphlet No. 05 / 040217A2, Pamphlet No. 05 / 070963A1 , and the same brochures No. 05 / 077981A2, No. 05 / 092925A2, No. 05 / 123780A2, No. 06 / 019447A1, No. 06 / 047350A2 and No. 06 / 085967A2 or U.S. Patent No. 5,648,260; No. 5,739,277; No. 5,834,250; No. 5,869,046; No. 6,096,871; No. 6 This may include any one of the amino acid substitutions disclosed in Specification No. 121,022; Specification No. 6,194,551; Specification No. 6,242,195; Specification No. 6,277,375; Specification No. 6,528,624; Specification No. 6,538,124; Specification No. 6,737,056; Specification No. 6,821,505; Specification No. 6,998,253; and Specification No. 7,083,784 (each of which is incorporated herein in whole by reference).In one exemplary embodiment, the conjugated polypeptide may include an Fc variant containing an amino acid substitution at EU position 268 (e.g., H268D or H268E). In another exemplary embodiment, the conjugated polypeptide may include an amino acid substitution at EU position 239 (e.g., S239D or S239E) and / or an amino acid substitution at EU position 332 (e.g., I332D or I332Q).

[0211] In certain embodiments, the binding polypeptide may include Fc variants containing amino acid substitutions that alter the antigen-independent effector function of the antibody, particularly the circulating half-life of the binding polypeptide. Such binding polypeptides exhibit either increased or decreased binding to FcRn compared to binding polypeptides lacking these substitutions, thus resulting in prolonged or shortened serum half-lives, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered antibody is desirable, for example, in treating chronic diseases or chronic disorders. In contrast, Fc variants with reduced FcRN-binding affinity are expected to have shorter half-lives, and such molecules are also useful in administration to mammals where a shorter circulating time may be advantageous, for example, in in vivo diagnostic imaging or in situations where prolonged circulating presence of the initiating antibody would result in toxic side effects. Fc variants with reduced FcRN-binding affinity are less likely to cross the placenta and are therefore useful for treating diseases or disorders in pregnant women. In addition, other applications where reduced FcRN-binding affinity may be desirable include applications localizing to the brain, kidneys, and / or liver. In one exemplary embodiment, the modified binding polypeptide (e.g., an antibody or its antigen-binding fragment) exhibits reduced transport from blood vessels to the epithelium of the renal glomeruli. In another embodiment, the altered binding polypeptide (e.g., an antibody or its antigen-binding fragment) exhibits reduced transport across the blood-brain barrier (BBB) ​​from the brain to the intervascular space. In one embodiment, the antibody with altered FcRN-binding contains an Fc domain having one or more amino acid substitutions within the “FcRN-binding loop” of the Fc domain. The FcRN-binding loop consists of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions that alter FcRN-binding activity are disclosed in International Publication No. 05 / 047327, which is incorporated herein by reference in its entirety.In certain exemplary embodiments, the conjugated polypeptide (e.g., an antibody or its antigen-binding fragment) comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering). In other exemplary embodiments, the conjugated molecule comprises a human Fc domain having the double mutation H433K / N434F (see, for example, U.S. Patent No. 8,163,881).

[0212] In other embodiments, the conjugated polypeptide for use in the diagnostic and treatment methods described herein has a constant region, e.g., an IgG1 or IgG4 heavy chain constant region, which is modified to reduce or eliminate glycosylation. For example, the conjugated polypeptide (e.g., an antibody or its antigen-binding fragment) may also include Fc variants that include amino acid substitutions that alter the glycosylation of the antibody Fc. For example, the Fc variant may have reduced glycosylation (e.g., N- or O-linked glycosylation). In exemplary embodiments, the Fc variant includes a reduction in glycosylation of an N-linked glycan typically found at amino acid position 297 (EU numbering). In another embodiment, the antibody has amino acid substitutions near or within an N-linked glycosylation motif containing a glycosylation motif, e.g., an amino acid sequence NXT or NXS. In specific embodiments, the antibody includes an Fc variant having an amino acid substitution at amino acid position 228 or 299 (EU numbering). In more specific embodiments, the antibody comprises an IgG1 or IgG4 constant region containing S228P and T299A mutations (EU numbering).

[0213] Exemplary amino acid substitutions conferring reduction or modified glycosylation are disclosed in International Publication No. 05 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, the conjugating polypeptide is modified to eliminate glycosylation. Such conjugating polypeptides may be called "agly" conjugating polypeptides (e.g., "agly" antibodies). While not bound by theory, "agly" conjugating polypeptides are thought to have improved in vivo safety and stability profiles. Agly conjugating polypeptides may be any isotype or subclass thereof, e.g., IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the agly conjugating polypeptide comprises a deglycosylated Fc region of an IgG4 antibody lacking Fc effector function, thereby eliminating the possibility of Fc-mediated toxicity to normal vital organs expressing IL-6. In yet other embodiments, the conjugating polypeptide comprises a modified glycan. For example, an antibody may be defucosylated by reducing the number of fucose residues on the N-glycan at Asn297 in the Fc region. Defucosylation increases FcγRII binding on NK cells, thereby potently enhancing ADCC. Diabodies containing anti-IL-6 scFv and anti-CD3 scFv have been shown to induce ADCC-mediated death of IL-6-expressing cells. Therefore, in one embodiment, a defucosylated anti-IL-6 antibody is used to target and kill IL-6-expressing cells. In another embodiment, the binding polypeptide may have a modified number of sialic acid residues on the N-glycan at Asn297 in the Fc region. Numerous technically recognized methods are available for producing "agly" antibodies with modified glycans. For example, such antibodies can be produced using genetically modified host cells (e.g., modified yeast, e.g., Picchia or CHO cells) in which the glycosylation pathway is modified (e.g., glycosyltransferase deletion).

[0214] In certain exemplary embodiments, the effector-enhancing Fc domain variant has one or more amino acid substitutions selected from the group consisting of: aspartic acid at amino acid position 221 (D); cysteine ​​at amino acid position 222 (C); tyrosine at amino acid position 234 (Y); alanine at amino acid position 236 (A); tryptophan at amino acid position 236 (W); aspartic acid at amino acid position 239 (D); leucine at amino acid position 243 (L); tyrosine at amino acid position 252 (Y); threonine at amino acid position 254 (T); aspartic acid at amino acid position 256 (D); glutamic acid at amino acid position 256 (E); glutamic acid at amino acid position 267 (E); phenylalanine at amino acid position 268 (F); proline at amino acid position 292 (P); amino Alanine (A) at amino acid position 298; leucine (L) at amino acid position 300; isoleucine (I) at amino acid position 305; tryptophan (W) at amino acid position 307; glutamine (Q) at amino acid position 307; threonine (T) at amino acid position 324; tryptophan (W) at amino acid position 326; alanine (A) at amino acid position 326; leucine (L) at amino acid position 330; glutamic acid (E) at amino acid position 332; alanine (A) at amino acid position 333; serine (S) at amino acid position 333; alanine (A) at amino acid position 334; alanine (A) at amino acid position 336; arginine (R) at amino acid position 345; leucine (L) at amino acid position 396; leucine (L) at amino acid position 428; and serine (S) at amino acid position 434 (according to EU numbering). See Saunders KO (2009), Front.Immunol., vol.10(1296):1-20; Mackness et al. (2019), MAbs, vol.11:1276-88; and International Publication No. 2019147973A1.

[0215] In some embodiments, the Fc domain variant may include amino acid substitutions at positions selected from amino acid positions 239, 267, 268, 298, 314, 330, 332, 339, and 373 according to EU numbering. In some embodiments, the Fc domain variant may include aspartic acid (D) at amino acid position 239. In other embodiments, the Fc domain variant may include glutamic acid (E) at amino acid position 332. In yet another embodiment, the Fc domain variant may include alanine (A) at amino acid position 298. In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332. In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

[0216] In some exemplary embodiments, the Fc domain variant may contain aspartic acid (D) at amino acid position 267. In other embodiments, the Fc domain variant may contain aspartic acid (D) at amino acid position 268. In yet another embodiment, the Fc domain variant may contain glutamic acid (E) at amino acid position 268. In some embodiments, the Fc domain variant may contain cysteine ​​(C) at amino acid position 298. In one exemplary embodiment, the Fc domain variant contains isoleucine (I) at amino acid position 314. In some embodiments, the Fc domain variant may contain methionine (M) at amino acid position 314. In other embodiments, the Fc domain variant may contain glutamine (Q) at amino acid position 314. In yet another embodiment, the Fc domain variant may contain tryptophan (W) at amino acid position 314. In one exemplary embodiment, the Fc domain variant contains phenylalanine (F) at amino acid position 330. In another exemplary embodiment, the Fc domain variant contains methionine (M) at amino acid position 330. In yet another exemplary embodiment, the Fc domain variant contains aspartic acid (D) at amino acid position 339. In yet another exemplary embodiment, the Fc domain variant contains isoleucine (I) at amino acid position 339. In yet another exemplary embodiment, the Fc domain variant contains proline (P) at amino acid position 339. In yet another exemplary embodiment, the Fc domain variant contains threonine (T) at amino acid position 339. In yet another exemplary embodiment, the Fc domain variant contains phenylalanine (F) at amino acid position 373. In yet another exemplary embodiment, the Fc domain variant contains tryptophan (W) at amino acid position 373.

[0217] In some embodiments, the Fc domain variant may include amino acid substitutions at positions selected from amino acid positions 252, 254, 256, 307, 428, and 434 according to EU numbering. In some embodiments, the Fc domain variant may include leucine (L) at amino acid position 428 and serine (S) at amino acid position 434. In other embodiments, the Fc domain variant may include tyrosine (Y) at amino acid position 252 and aspartic acid (D) at amino acid position 256. In yet another embodiment, the Fc domain variant may include aspartic acid (D) at amino acid position 256 and tryptophan (W) at amino acid position 307. In some embodiments, the Fc domain variant may include tyrosine (Y) at amino acid position 252, threonine (T) at amino acid position 254, and glutamic acid (E) at amino acid position 256.

[0218] In some embodiments, the Fc domain variant may further include amino acid substitutions at amino acid positions 256 and / or 307 according to EU numbering. In some embodiments, the Fc domain variant may include a combination of amino acid substitutions, with aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307. In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and aspartic acid (D) at amino acid position 239. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and alanine (A) at amino acid position 298. In yet another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and glutamic acid (E) at amino acid position 332. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, aspartic acid (D) at amino acid position 239, and glutamic acid (E) at amino acid position 332. In yet another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, aspartic acid (D) at amino acid position 239, and alanine (A) at amino acid position 298.

[0219] In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and aspartic acid (D) at amino acid position 267. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and aspartic acid (D) at amino acid position 268. In yet another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and glutamic acid (E) at amino acid position 268. In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and cysteine ​​(C) at amino acid position 298. In one exemplary embodiment, the Fc domain variant contains aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and isoleucine (I) at amino acid position 314. In another exemplary embodiment, the Fc domain variant contains aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and methionine (M) at amino acid position 314. In one exemplary embodiment, the Fc domain variant contains aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and glutamine (Q) at amino acid position 314. In another exemplary embodiment, the Fc domain variant contains aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tryptophan (W) at amino acid position 314. In yet another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 330. In yet another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and methionine (M) at amino acid position 330. In yet another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and aspartic acid (D) at amino acid position 339.In another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and isoleucine (I) at amino acid position 339. In another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and proline (P) at amino acid position 339. In another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and threonine (T) at amino acid position 339. In yet another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and phenylalanine (F) at amino acid position 373. In another exemplary embodiment, the Fc domain variant comprises aspartic acid (D) at amino acid position 256, glutamine (Q) at amino acid position 307, and tryptophan (W) at amino acid position 373.

[0220] Thermostabilized Fc domain variant The structure of the constant antibody domain is similar to that of the variable domain, which consists of β-strands connected by loops and short helices. The CH2 domain of the heavy chain constant region exhibits weak carbohydrate-mediated intermolecular protein interactions, in contrast to the extensive intermolecular interactions shown by other domains. The isolated mouse CH2 domain is relatively unstable at physiological temperature (Feige MJ et al. (2004), J. Mol. Biol., vol. 344(1):107-118), but previous studies have demonstrated that the thermal stability of the CH2 domain can be enhanced by the addition of intramolecular disulfide bonds, and that these can be used as scaffolds for conjugates (Gong R et al. (2009), J. Biol. Chem., vol. 284(21):14203-210).

[0221] Effector-enhanced Fc domain variants that exhibit an increase in thermal instability (i.e., a decrease in thermal stability) compared to the wild-type Fc domain are known. For example, the S239D / I332E and S239D / I332E / A330L variants result in a decrease in the stability of the CH2 domain, as shown by a decrease in the melting temperature (Tm) in differential scanning calorimetry (DSC) analysis. G236A / S239D / A330L / I332E shows a decrease in the thermal shift measurement of the protein and a significantly shortened half-life in hFcγR transgenic mice when compared to the wild-type. For reviews, see Liu Z et al. (2014), J. Biol. Chem., vol. 289(6):3571-90 and Liu R et al. (2020), Antibodies, vol. 9(4):64.

[0222] Effector-enhanced Fc domain variants with improved FcγR binding and not significantly decreased stability when compared to the wild-type are known. See, for example, European Patent No. 2940135B1 of Example 10.

[0223] It has further been discovered that thermostabilized Fc domain variants can be made by introducing one or more disulfide bonds into the Fc domain. Thus, in one aspect, the present disclosure provides Fc domain variants comprising one or more modified (e.g., non-natural) disulfide bonds, such as intra-chain disulfide bonds mediated by, for example, one or more cysteine pairs.

[0224] In certain exemplary embodiments, the disulfide bond is an intra-chain disulfide bond between two CH2 regions of the Fc domain. In certain exemplary embodiments, the disulfide bond is an intra-chain disulfide bond between two CH3 regions of the Fc domain. In certain exemplary embodiments, two or more intra-chain disulfide bonds are present between two CH2 regions of the Fc domain and / or between two CH2 regions of the Fc domain.

[0225] Thermal stability, or the tendency of an Fc domain (e.g., an Fc domain with or without a binding polypeptide) to unfold, can be determined using various methods known in the art. For example, the unfolding or denaturation temperature can be measured by nanoformat differential scanning calorimetry (nanoDSC) or nanoformat differential scanning fluorescence (nanoDSF) (Wen J et al. (2020), Anal. Biochem., vol. 593: 113581). The detectable temperature at which a protein begins to unfold is called the toneset.

[0226] In certain exemplary embodiments, the Tonset of a heat-stabilized Fc domain variant (e.g., having one or more modified disulfide bonds) is elevated compared to an unheat-stabilized Fc domain variant. In certain exemplary embodiments, the Tonset of a heat-stabilized Fc domain variant is approximately 1.0 compared to an unheat-stabilized Fc domain variant. o C, about 1.5 o C, about 2.0 o C, about 2.5 o C, about 3.0 o C, about 3.5 o C, about 4.0 o C, about 4.5 o C, about 5.0 o C, about 5.5 o C, about 6.0 o C, about 6.5 o C, about 7.0 o C, about 7.5 o C, about 8.0 o C, about 8.5 o C, about 9.0 o C, about 9.5 o C, about 10.0 o C, about 10.5 o C, about 11.0 o C, about 11.5 o C, about 12.0 o C, about 12.5 o C, about 13.0 o C, about 13.5 o C, about 14.0 o C, about 14.5o C, about 15.0 o C, about 15.5 o C, about 16.0 o C, about 16.5 o C, about 17.0 o C, about 17.5 o C, about 18.0 o C, about 18.5 o C, about 19.0 o C, about 19.5 o C, about 20.0 o C, about 20.5 o C, about 21.0 o C, about 21.5 o C, about 22.0 o C, about 22.5 o C, about 23.0 o C, about 23.5 o C, about 24.0 o C, about 24.5 o C or about 25.0 o C, increases.

[0227] In certain exemplary embodiments, the thermostabilized Fc domain variant has one or more amino acid substitution pairs selected from the group consisting of cysteine substitutions at the following positions: amino acid positions 242 and 334; amino acid positions 240 and 334; amino acid positions 287 and 306; amino acid positions 292 and 302; amino acid positions 323 and 332; amino acid positions 259 and 306; amino acid positions 350 and 441; amino acid positions 343 and 431; amino acid positions 375 and 404; amino acid positions 375 and 396; and amino acid positions 348 and 439 (according to EU numbering). For reviews, see Wozniak-Knopp G et al. (2012), PLoS One, vol. 7(1):e30083, Jacobsen FW et al. (2017), J. Biol. Chem. 292:1865-75 and WO 2014153063 pamphlet.

[0228] In certain exemplary embodiments, the heat-stabilized Fc domain variant includes a modified (e.g., unnatural) intrachain disulfide bond interposed by a pair of cysteines substituting (i) leucine (L) at amino acid position 242 and lysine (K) at amino acid position 334; (ii) alanine (A) at amino acid position 287 and leucine (L) at amino acid position 306; or (iii) arginine (R) at amino acid position 292 and valine (V) at amino acid position 302, according to EU numbering.

[0229] In some embodiments, the heat-stabilized Fc domain variant includes a modified (e.g., unnatural) intrachain disulfide bond interposed by a pair of cysteines substituting leucine (L) at amino acid position 242 and lysine (K) at amino acid position 334. In certain embodiments, the heat-stabilized Fc domain variant includes a modified (e.g., unnatural) intrachain disulfide bond interposed by a pair of cysteines substituting alanine (A) at amino acid position 287 and leucine (L) at amino acid position 306. In certain exemplary embodiments, the heat-stabilized Fc domain variant includes a modified (e.g., unnatural) intrachain disulfide bond interposed by a pair of cysteines substituting arginine (R) at amino acid position 292 and valine (V) at amino acid position 302. In certain exemplary embodiments, the heat-stabilized Fc domain variant may include at least one modified intrachain disulfide bond. In certain embodiments, the thermally stabilized Fc domain variant may include two or more modified intrachain disulfide bonds.

[0230] In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant may include glutamic acid (E) at amino acid position 332, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes alanine (A) at amino acid position 298, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239, glutamic acid (E) at amino acid position 332, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239, alanine (A) at amino acid position 298, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302.

[0231] In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 267, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 268, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes glutamic acid (E) at amino acid position 268, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes cysteine ​​(C) at amino acid position 298, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In one exemplary embodiment, the Fc domain variant includes isoleucine (I) at amino acid position 314, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes methionine (M) at amino acid position 314, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In one exemplary embodiment, the Fc domain variant includes glutamine (Q) at amino acid position 314, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes tryptophan (W) at amino acid position 314, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes phenylalanine (F) at amino acid position 330, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes methionine (M) at amino acid position 330, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In yet another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 339, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302.In another exemplary embodiment, the Fc domain variant includes isoleucine (I) at amino acid position 339, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes proline (P) at amino acid position 339, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes threonine (T) at amino acid position 339, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes phenylalanine (F) at amino acid position 373, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302. In another exemplary embodiment, the Fc domain variant includes tryptophan (W) at amino acid position 373, cysteine ​​(C) at amino acid position 292, and cysteine ​​(C) at amino acid position 302.

[0232] In other exemplary embodiments, the Fc domain variant includes cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307.

[0233] In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes alanine (A) at amino acid position 298, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239, alanine (A) at amino acid position 298, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes glutamic acid (E) at amino acid position 332, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 239, glutamic acid (E) at amino acid position 332, cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307. In one exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 267, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307. In one exemplary embodiment, the Fc domain variant includes cysteine ​​(C) at amino acid position 298, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307.In another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 268, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes glutamic acid (E) at amino acid position 268, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In one exemplary embodiment, the Fc domain variant includes isoleucine (I) at amino acid position 314, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In another exemplary embodiment, the Fc domain variant includes methionine (M) at amino acid position 314, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In one exemplary embodiment, the Fc domain variant includes glutamine (Q) at amino acid position 314, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In another exemplary embodiment, the Fc domain variant includes tryptophan (W) at amino acid position 314, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes phenylalanine (F) at amino acid position 330, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307.In another exemplary embodiment, the Fc domain variant includes methionine (M) at amino acid position 330, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes aspartic acid (D) at amino acid position 339, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes isoleucine (I) at amino acid position 339, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In another exemplary embodiment, the Fc domain variant includes proline (P) at amino acid position 339, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes threonine (T) at amino acid position 339, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In yet another exemplary embodiment, the Fc domain variant includes phenylalanine (F) at amino acid position 373, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307. In another exemplary embodiment, the Fc domain variant includes tryptophan (W) at amino acid position 373, cysteine ​​(C) at amino acid position 292, cysteine ​​(C) at amino acid position 302, aspartic acid (D) at amino acid position 256, and glutamine (Q) at amino acid position 307.

[0234] Fc-containing bound polypeptide In one embodiment, the disclosure provides conjugated polypeptides (e.g., antibodies, antibody fragments, antibody variants, and fusion proteins) containing an Fc domain (e.g., a variant Fc domain). In certain embodiments, the conjugated polypeptide is an antibody or a fragment or derivative thereof. Any antibody from any source or species may be used in the conjugated polypeptide disclosed herein. Suitable antibodies include, but are not limited to, human antibodies, humanized antibodies, or chimeric antibodies.

[0235] Any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and Fc domain from a species may be used in the binding polypeptides disclosed herein. Chimeric Fc domains, which include a portion of an Fc domain from a different species or Ig class, may also be used. In certain embodiments, the Fc domain is a human IgG1 Fc domain.

[0236] In other embodiments, the disclosure provides conjugated polypeptides (e.g., antibodies, antibody fragments, antibody variants, and fusion proteins) comprising at least one CH1 domain. CH1 domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA, and IgE) and species may be used in the conjugated polypeptides disclosed herein. Chimeric CH1 domains comprising a portion of a CH1 domain from a different species or Ig class may also be used. In certain embodiments, the CH1 domain is a human IgG1 CH1 domain.

[0237] In one embodiment, the disclosure provides a binding polypeptide comprising an isolated Fc domain variant that includes at least one binding domain (e.g., at least one binding polypeptide) or is complexed with it (e.g., fused). In certain embodiments, the binding domain comprises one or more antigen-binding domains. The antigen-binding domain does not need to originate from the same molecule as the parent Fc domain. In certain embodiments, the Fc domain variant is present in the antibody. In one embodiment, the Fc domain variant is present in the antibody or complexed with the antibody. Any antibody from any source or species may be used with the Fc domain variants disclosed herein. Suitable antibodies include, but are not limited to, chimeric antibodies, humanized antibodies, or human antibodies. Suitable antibodies include, but are not limited to, full-length antibodies, monoclonal antibodies, polyclonal antibodies, or immunoglobulin monovariable domain antibodies or VHHs.

[0238] The terms “multispecificity,” “multispecificity antibody,” or “multispecificity binding protein” may refer to a binding protein that specifically binds to two or more antigens. A multispecificity binding protein that binds to two antigens and / or two different epitopes of different antigens is also referred to herein as a “bispecificity” binding protein. A multispecificity binding protein that binds to three antigens and / or three different epitopes is also referred to herein as a “trispecificity” binding protein. Thus, multispecificity binding proteins can bind to two or more different targets simultaneously. Using genetic engineering, multispecificity binding proteins, or their binding fragments or derivatives, can be designed, modified, and produced having a desired set of binding properties and effector functions.

[0239] In one embodiment, the conjugated polypeptide composition described herein is an antibody. In some embodiments, the antibody is multispecific. In some embodiments, the multispecific antibody is selected from the group consisting of DVD-Ig, optionally CODV-Ig, CrossMab, CrossMab-Fab, and Tandem Fab, which are CODV-based formulations. In some embodiments, the multispecific antibody is T-cell engager multispecific. In some embodiments, the multispecific antibody is NK-cell engager.

[0240] In certain embodiments, the binding polypeptides of the Disclosure may include antigen-binding fragments of antibodies. The term “antigen-binding fragment” refers to a polypeptide fragment of an immunoglobulin or antibody that binds to an antigen or competes with an intact antibody (i.e., the intact antibody from which it is derived) for antigen binding (i.e., specific binding). Antigen-binding fragments may be prepared by recombinant or biochemical methods well known in the Art.

[0241] Examples of antigen-binding fragments include variable fragments (Fv), Fab, Fab', (Fab')2, minibody, diabody, triabody, tetrabody, tandemdi-scFv, tandemtri-scFv, immunoglobulin single variable domains (ISV), such as VHH (including humanized VHH), camelidized VHH, monodomain antibodies, domain antibodies, or dAb.

[0242] In certain exemplary embodiments, the conjugated polypeptide of the Disclosure comprises at least one antigen-binding fragment and an Fc domain variant. In certain exemplary embodiments, the conjugated polypeptide of the Disclosure comprises: (a) a variable fragment (Fv), Fab, Fab', (Fab')2, minibody, diabody, triabody, tetrabody, tandemdi-scFv, tandemtri-scFv, immunoglobulin single variable domain (ISV), e.g., VHH (including humanized VHH), camelidized VHH, monodomain antibody, domain antibody or dAb; and (b) an Fc domain variant.

[0243] In an exemplary embodiment, the binding polypeptide comprises a single-chain variable region array (ScFv). The single-chain variable region array comprises a single polypeptide having one or more antigen-binding sites, for example, a VL domain linked to a VH domain by a flexible linker. The ScFv molecule can be constructed in a VH-linker-VL orientation or a VL-linker-VH orientation. The flexible hinge connecting the VL and VH domains that constitute the antigen-binding site contains about 10 to about 50 amino acid residues. The linking peptide is known in the art. The binding polypeptide can comprise at least one scFv and / or at least one constant region. In one embodiment, the binding polypeptide of the present disclosure can comprise at least one scFv linked or fused to an Fc domain variant.

[0244] In certain specific exemplary embodiments, the binding polypeptide of the present disclosure is a multivalent (e.g., tetravalent) antibody produced by fusing a DNA sequence encoding an antibody with a ScFv molecule (e.g., a modified ScFv molecule). For example, in one embodiment, these sequences are combined such that the ScFv molecule (e.g., a modified ScFv molecule) is linked to an Fc domain variant via a flexible linker (e.g., a gly / ser linker) at its N-terminus or C-terminus. In another embodiment, the tetravalent antibody of the present disclosure can be produced by fusing a ScFv molecule to a linking peptide that is fused to an Fc domain variant to construct a ScFv-Fab tetravalent molecule.

[0245] In another embodiment, the binding polypeptide of the Disclosure is a modified minibody. The modified minibody of the Disclosure is a dimer molecule comprising two polypeptide chains, each containing an ScFv molecule fused to an Fc domain variant via a linking peptide. The minibody can be prepared by constructing the ScFv component and linking the peptide component using the methods described herein (see, for example, U.S. Patent No. 5,837,821 or International Publication No. 94 / 09817A1). In another embodiment, a tetravalent minibody can be constructed. The tetravalent minibody can be constructed in the same manner as the minibody, except that two ScFv molecules are linked using a mobile linker. The linked scFv-scFv construct is then linked to an Fc domain variant.

[0246] In another embodiment, the binding polypeptide of the present disclosure comprises a diabody. Each diabody has a polypeptide similar to an scFv molecule, but is typically a dimeric tetravalent molecule having a short (less than 10, e.g., about 1 to about 5) amino acid residue linker connecting both variable domains, so that the VL and VH domains on the same polypeptide chain cannot interact. Instead, the VL and VH domains of one polypeptide chain interact with the (respectively) VH and VL domains on a second polypeptide chain (see, for example, International Publication No. 02 / 02781). The diabody of the present disclosure comprises an scFv-like molecule fused to an Fc domain variant.

[0247] In another embodiment, the conjugated polypeptide of the present disclosure comprises any preferred fragment of any one of the following, which is fused to an immunoglobulin single variable domain (ISV), such as a domain antibody, "dAb", VHH (including humanized VHH), camelized VHH, another single variable domain, or an Fc domain variant.

[0248] The term "immunoglobulin single variable domain" (ISV or ISVD), which is used interchangeably with "single variable domain", defines an immunoglobulin molecule in which the antigen-binding site is present on a single immunoglobulin domain and is formed by a single immunoglobulin domain. This distinguishes immunoglobulin single variable domains from "conventional" immunoglobulins (such as monoclonal antibodies) or their fragments (Fab, Fab’, F(ab’)2, scFv, di-scFv, etc.), in which two immunoglobulin domains, particularly two variable domains, interact to form the antigen-binding site. Typically, in conventional immunoglobulins, the heavy-chain variable domain (V H ) and the light-chain variable domain (V L ) interact to form the antigen-binding site. In this case, both the complementarity-determining regions (CDRs) of V H and V L contribute to the antigen-binding site, i.e., a total of six CDRs are involved in the formation of the antigen-binding site. ISVs belonging to the so-called "V H 3 class" (i.e., ISVs having high sequence homology to V H 3 class human germline sequences such as DP-47, DP-51 or DP-29) or ISVs belonging to the so-called "VH4 class" (i.e., ISVs having high sequence homology to V H 4 class human germline sequences such as DP-78), as described in, for example, WO 2007 / 118670 A1 pamphlet, may be used herein.

[0249] The term "VHH" or "VHH antibody" refers to a type of single-domain antibody containing a variable heavy-chain domain lacking a light chain. Similar to conventional VH domains, VHH contains four FRs and three CDRs. VHH offers advantages over conventional antibodies. Because they are approximately 10 times smaller than IgG molecules, properly folded functional VHH can be produced by in vitro expression while achieving high yields. Furthermore, VHH is highly stable and resistant to protease action. The characterization and production of VHH are outlined by Harmsen and De Haard HJ (Appl. Microbiol. Biotechnol. 2007 November;77(1):13-22).

[0250] In certain exemplary embodiments, the conjugated polypeptide of the Disclosure comprises an Fc domain (e.g., an Fc domain variant) fused with one or more VHHs.

[0251] ISV (especially, V HH The sequence and partially humanized VHH) can be particularly characterized by the presence of one or more "Hallmark residues" (as described herein in Table 1 and in the subsequent paragraphs describing NANOBODY® immunoglobulin monovariable domains), so that the ISV is a NANOBODY® ISV.

[0252] Therefore, generally speaking, NANOBODY® ISV (especially (partially or completely) humanized V HH and Camelization V H V HH ) can be defined as an amino acid sequence having the (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (FR1-FR4 refer to framework regions 1-4, respectively, CDR1-CDR3 refer to complementarity-determining regions 1-3, respectively, and one or more Hallmark residues as further defined in Table 1). In particular, NANOBODY® ISV (in particular, (partially) humanized V HH and Camelization V H V HH) may be an amino acid sequence having the (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (FR1-FR4 each refer to framework regions 1-4, CDR1-CDR3 each refer to complementarity-determining regions 1-3, and the framework sequence is as further defined herein). In particular, ISV may be an amino acid sequence having the (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (FR1-FR4 each refer to framework regions 1-4, and CDR1-CDR3 each refer to complementarity-determining regions 1-3).

[0253] The term “Immunoglobulin Monovariable Domain (ISV)” encompasses NANOBODY® VHH as described in International Publication No. 08 / 020079 or International Publication No. 09 / 138519, and therefore in one aspect refers to VHH, humanized VHH or camelized VH (e.g., camelized human VHH) or generally optimized VHH (e.g., optimized for chemical stability and / or solubility, maximum overlap with known framework regions, and maximum expression).

[0254] Generally, NANOBODY® immunoglobulin single variable domain (ISV) (especially (partially) humanized V) HH Sequence and camelization V H V HH The sequence may be characterized by the presence of one or more "Hallmark residues" (as further described herein) in one or more framework sequences (as further described herein). Thus, generally, NANOBODY®ISV can be defined as an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (In the formula, FR1 to FR4 refer to framework regions 1 to 4, respectively, CDR1 to CDR3 refer to complementarity-determining regions 1 to 3, respectively, and one or more of the Hallmark residues are as further defined herein).

[0255] In particular, NANOBODY®ISV may be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (In the formula, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively, and the framework sequence is as further defined herein).

[0256] For more details, NANOBODY®ISV may be an immunoglobulin sequence having the following (general) structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 (In the formula, FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determination regions 1 to 3, respectively.) One or more amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104, and 108, according to Kabat numbering, are selected from the Hallmark residues listed in Table 1 below.

[0257] [Table 1]

[0258] [Table 2]

[0259] In other embodiments, the conjugated polypeptide includes a multispecific or multivalent antibody containing one or more variable domains in series on the same polypeptide chain, such as a tandem variable domain (TVD) polypeptide. An exemplary TVD polypeptide includes the “double-headed” or “double Fv” configuration described in U.S. Patent No. 5,989,830. In the double Fv configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate chains (one heavy chain and one light chain), with one polypeptide chain having two series-connected VH domains separated by a peptide linker (VH1-linker-VH2) and the other polypeptide chain consisting of complementary VL domains connected in series by a peptide linker (VL1-linker-VL2). In a cross-over double-head configuration, the variable domains of two different antibodies are expressed in tandem orientation on two separate polypeptide chains (one heavy chain and one light chain). One polypeptide chain has two VH domains in series separated by a peptide linker (VH1-linker-VH2), while the other polypeptide chain consists of complementary VL domains connected in series by a peptide linker (VL2-linker-VL1) in the opposite orientation. Further antibody variants based on the "dual Fv" scheme include dual variable domain IgG (DVD-IgG) bispecific antibodies (see U.S. Patent No. 7,612,181) and the TBTI scheme (see U.S. Patent Publication No. 2010 / 0226923A1). The addition of constant domains (CH1-Fc to the heavy chain and a kappa or lambda constant domain to the light chain) to each chain of the dual Fv results in a functionally bispecific antibody without requiring further modification (i.e., the obvious addition of constant domains to enhance stability). In some embodiments, the conjugated polypeptide comprises a multispecific or multivalent antibody containing one or more variable domains in series on the same polypeptide chain fused to the Fc domain variant.

[0260] In another exemplary embodiment, the binding polypeptide comprises a cross-double variable domain IgG (CODV-IgG) bispecific antibody based on a “double-head” configuration (see U.S. Patent Application Publication 20120251541A1, which is incorporated herein by reference in its entirety). The CODV-IgG antibody variant has one polypeptide chain (VL1-L1-VL2-L2-CL) in which the VL domain is linked in series to the CL domain, and a second polypeptide chain (VH2-L3-VH1-L4-CH1) in which the complementary VH domain is linked in series in the reverse direction to the CH1 domain, and these polypeptide chains form a cross-light-heavy pair. In certain embodiments, the second polypeptide may be further linked to an Fc domain (VH2-L3-VH1-L4-CH1-Fc). In certain embodiments, linker L3 is at least twice as long as linker L1, and / or linker L4 is at least twice as long as linker L2. For example, L1 and L2 may be amino acid residues of 1 to 3 lengths, L3 may be amino acid residues of 2 to 6 lengths, and L4 may be amino acid residues of 4 to 7 lengths. Examples of suitable linkers include a single glycine (Gly) residue; a diglycine peptide (Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with 4 glycine residues (Gly-Gly-Gly-Gly); a peptide with 5 glycine residues (Gly-Gly-Gly-Gly-Gly); a peptide with 6 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly); a peptide with 7 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly); and a peptide with 8 glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly). Other amino acid residue combinations that can be used include, for example, the peptides Gly-Gly-Gly-Gly-Ser and Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Ser.

[0261] In other embodiments, the conjugated polypeptide includes CrossMab or CrossMab-Fab multispecificity schemes. See International Publication No. 2009080253 and Schaefer, et al., PNAS (2011), 108:11187-1191. Antibody variants based on the CrossMab scheme have crossings of antibody domains within one arm of a bispecific IgG antibody, enabling precise chain association. In some embodiments, the conjugated polypeptide includes tandem Fab schemes. Tandem Fab is a class of bispecific antibody fragments based on Fab. Tandem Fab comprises two Fabs targeting different epitopes.

[0262] In certain embodiments, the binding polypeptide comprises an immunoadhesin molecule containing a non-antibody binding region (e.g., a receptor, ligand, or cell adhesion molecule) fused to an antibody constant region (see, for example, Ashkenazi et al. (1995), Methods, vol. 8(2), 104-115 (the whole of which is incorporated herein by reference)).

[0263] In certain embodiments, the bound polypeptide includes an immunoglobulin-like domain. Suitable immunoglobulin-like domains include fibronectin domains (e.g., Koide A and Koide S (2007), Methods Mol. Biol. 352:95-109 (the entire domain is incorporated herein by reference)), DARPin (e.g., see Stumpp MT et al. (2008), Drug Discov. Today, vol. 13 (15-16):695-701 (the entire domain is incorporated herein by reference)), protein A Z domain (e.g., see Nygren P et al. (2008), FEBS J., vol. 275 (11):2668-76 (the entire domain is incorporated herein by reference)), lipocalins (e.g., see Skerra A (2008), FEBS J., vol. 275 (11):2677-83 (the entire domain is incorporated herein by reference)), and affilins (e.g., see Ebersbach H et al. See al. (2007), J.Mol.Biol., vol.372(1):172-85 (the whole is incorporated herein by reference)), afitin (e.g., see Krehenbrink M et al. (2008), J.Mol.Biol., vol.383(5):1058-68 (the whole is incorporated herein by reference)), avimer (e.g., see Silverman J et al. (2005), Nat.Biotechnol., vol.23(12):1556-61 (the whole is incorporated herein by reference)), finomer (e.g., see Grabulovski D et al. (2007), J BiolChem, vol.282(5):3196-3204 (the whole is incorporated herein by reference)) and Kunitz domain peptide (e.g., Nixon et al. (2006), Curr Opin Drug Examples include, but are not limited to, DiscovDevel, vol.9(2):261-8 (the entire text of which is incorporated herein by reference).

[0264] In other embodiments, the binding polypeptide comprises a T-cell engager-type multispecific antibody. “T-cell engager” refers to a binding protein directed towards the host immune system, more specifically, the cytotoxic activity of T cells, as well as tumor target proteins. In some embodiments, the isolated effector-competent polypeptide comprises an NK-cell engager-type multispecific antibody. “NK-cell engager” refers to a binding protein comprising a monoclonal antibody fragment that targets the activated NK cell receptor, an antigen-specific targeting region, and an Fc region (Gauthier, et al. (2019) Cell, vol. 177(7): 1701-13).

[0265] The conjugated polypeptides of this disclosure, including the Fc domain variants described herein, may include the CDR sequence or variable domain sequence of a known “parent” antibody. In some embodiments, the parent antibody and the antibody of this disclosure may share similar or identical sequences, apart from modifications to the Fc domain as disclosed herein.

[0266] In another embodiment, the conjugated polypeptide comprises a therapeutic polypeptide. In some embodiments, the therapeutic polypeptide may be a receptor, ligand, or enzyme. In some embodiments, the therapeutic polypeptide may be a coagulation factor. In some embodiments, the coagulation factor is selected from the group consisting of FI, FII, FIII, FIV, FV, FVI, FVII, FVIII, FIX, FX, FXI, FXII, FXIII), VWF, prekallikrein, high molecular weight kininogen, fibronectin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitors (ZPIs), plasminogen, alpha-2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI-1), plasminogen activator inhibitor-2 (PAI2), any thymogen thereof, any active form thereof, and any combination thereof. In some embodiments, the therapeutic polypeptide may be a growth factor. The growth factor may be selected from any growth factor known in the art. In some embodiments, the growth factor is a hormone, and in other embodiments, the growth factor is a cytokine. In some embodiments, the growth factor is a chemokine. In some embodiments, the conjugated polypeptide comprises a therapeutic molecule or therapeutic polypeptide conjugated to the N-terminus and / or C-terminus of the Fc domain of the present invention. In some embodiments, the polypeptide is an Fc-fusion polypeptide.

[0267] In one embodiment, the binding polypeptide disclosed herein may be internalized by a cell. In another embodiment, the amount of binding polypeptide internalized by the cell is greater than the amount of reference binding polypeptide lacking a targeting moiety internalized by the cell.

[0268] In one embodiment, the targeting moiety binds to a receptor on the target cell. For example, the targeting moiety may include a mannose-6-phosphate moiety that binds to a mannose-6-phosphate receptor on the cell. In another exemplary embodiment, the targeting moiety binds to a Siglec on the target cell. Exemplary Siglecs include sialoadhesin (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), MAG (Siglec-4), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-14, or Siglec-15. In yet another embodiment, the targeting moiety includes an α2,3-, α2,6-, or α2,8-bound sialic acid residue. In a further embodiment, the targeting moiety includes an α2,3-sialyl lactose moiety or an α2,6-sialyl lactose moiety. Other exemplary receptors include, but are not limited to, C-type lectin receptors, galectin receptors, and L-type lectin receptors. Exemplary lectin receptors include TDEC-205, macrophage mannose receptor (MMR), Dectin-1, Dectin-2, macrophage-inducible type C lectin (Mincle), dendritic cell-specific ICAM3-binding non-intecglin (DC-SIGN, CD209), DC NK lectin receptor-1 (DNGR-1), Langerin (CD207), CD169, lectican, asialoglycoprotein receptor, DCIR, MGL, DC receptor, collectin, selectin, NK cell receptor, multi-CTLD endocytosis receptor, Reg-group (type VII) lectin, chondrolectin, tetranectin, polycystin, attractin (ATRN), eosinophil major basic protein (EMBP), DGCR2, thrombomodulin, Bimlec, SEEC, and CB CP / Frem 1 / QBRICK.

[0269] In certain embodiments, the binding polypeptide of the present disclosure comprises a modified reactive amino acid residue that is complexed with LYTAC via a reaction moiety. In further embodiments, a linker complexes the modified reactive amino acid residue with LYTAC.

[0270] In certain embodiments, the LYTAC region capable of binding to a cell surface lysosomal targeting receptor comprises mannose-6-phosphate (M6P) or its derivatives, GalNAc (e.g., trivalent GalNAc), and glycopeptides. In certain embodiments, the cell surface lysosomal targeting receptor comprises asialoglycoprotein receptor (ASGPR), mannose-6-phosphate receptor (M6PR) (including, but not limited to, cation-independent M6PR), and sialic acid-binding immunoglobulin lectin (Siglec).

[0271] Expression of binding polypeptide In one embodiment, polynucleotides encoding Fc domain variants and / or binding polypeptides disclosed herein are provided. Methods for producing Fc domain variants and / or binding polypeptides are also provided, comprising expressing these polynucleotides.

[0272] The polynucleotides encoding Fc domain variants and / or binding polypeptides disclosed herein are typically inserted into expression vectors for introduction into host cells that can be used to produce desired amounts of claimed antibodies, therapeutic polypeptides, and Fc fusion proteins. Accordingly, in certain embodiments, the present invention provides expression vectors comprising the polynucleotides disclosed herein, as well as host cells comprising these vectors and polynucleotides.

[0273] The terms “vector” or “expression vector” are used for the purposes of this specification and the claims to mean a vector used in accordance with the invention as a vehicle for introducing and expressing a desired gene in a cell. As is known to those skilled in the art, such vectors can be readily selected from the group consisting of plasmids, phages, viruses and retroviruses. Generally, a vector compatible with this disclosure includes a selection marker, a suitable restriction site for facilitating the cloning of the desired gene, and the ability to enter and / or replicate in eukaryotic or prokaryotic cells.

[0274] Numerous expression vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Furthermore, cells into which the DNA has been incorporated into their chromosomes can be selected by introducing one or more markers that enable selection of the host cells into which the gene has been transferred. Markers may provide prototrophicity to a trophic-dependent host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. Selectable marker genes can be directly ligated to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal mRNA synthesis. These elements may include signal sequences, splice signals, and transcription promoters, enhancers, and termination signals. In some embodiments, the cloned variable region gene is inserted into an expression vector along with the heavy chain constant region gene and light chain constant region gene (such as a human gene) synthesized as described above.

[0275] In other embodiments, the binding polypeptides featured in this disclosure may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as the heavy and light chains of antibodies, can be produced from a single polycistronic construct. These systems are beneficial because they utilize internal ribosome entry sites (IRESs) to yield relatively high levels of polypeptides in eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Patent No. 6,193,980 (incorporated herein by reference). Those skilled in the art will understand that such expression systems can be used to effectively produce the entire range of polypeptides disclosed herein.

[0276] More generally, once a vector or DNA sequence encoding an antibody or fragment thereof is prepared, the expression vector can be introduced into a suitable host cell. That is, the host cell can be transformed. Plasmid introduction into host cells can be carried out by various techniques well known to those skilled in the art. These techniques include, but are not limited to, gene transfer (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion using enveloped DNA, microinjection, and infection with intact viruses. See Ridgway, AAG "Mammalian Expression Vectors" Chapter 24.2, pp.470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). Transformed cells are grown under conditions suitable for light and heavy chain production, and assays are performed for the synthesis of heavy chain proteins and / or light chain proteins. Examples of assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activated cell sorter analysis (FACS), and immunohistochemistry.

[0277] As used herein, the term "transformation" broadly refers to the introduction of DNA into recipient host cells that alters their genotype and consequently alters the recipient cells.

[0278] Similarly, “host cells” refer to cells constructed using recombinant DNA technology and transformed with a vector encoding at least one heterologous gene. In describing the process for isolating polypeptides from recombinant hosts, the terms “cells” and “cell culture” are used interchangeably to indicate the source of antibodies unless otherwise clearly specified. In other words, the recovery of polypeptides from “cells” may mean recovery from either whole spin-down cells or from cell culture media containing both culture medium and suspended cells.

[0279] In one embodiment, the host cell line used for the expression of the Fc domain variant and / or binding polypeptide is of eukaryotic or prokaryotic origin. In one embodiment, the host cell line used for the expression of the Fc domain variant and / or binding polypeptide is of bacterial origin. In one embodiment, the host cell line used for the expression of the Fc domain variant and / or binding polypeptide is of mammalian origin. Those skilled in the art can determine the specific host cell line best suited to the desired gene product to be expressed therein. Examples of host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary cell line, DHFR-negative), HELA (human cervical cancer), CVI (monkey kidney cell line), COS (a derivative of CVI with SV40T antigen), R1610 (Chinese hamster fibroblast), BALBC / 3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cell), RAJI (human lymphocyte), and 293 (human kidney). In one embodiment, the cell line results in the modification of the antibody expressed therefrom, e.g., defucosylation (e.g., PER.C6.RTM. (Crucell) or FUT8 knockout CHO cell line (POTELLIGENT™ cells) (Biowa, Princeton, NJ)). In one embodiment, NS0 cells may be used. Host cell lines are typically available from the commercial service American Tissue Culture Collection or from publicly available literature.

[0280] In vitro production enables scale-up to obtain large quantities of the desired polypeptide. Techniques for mammalian cell culture under tissue culture conditions are known in the art, and these techniques include, for example, homogeneous suspension culture in an airlift reactor or a continuous stirring reactor, or culture of cells fixed or captured, for example, in hollow fibers, in microcapsules, on agarose microbeads or on ceramic cartridges. If necessary and / or desired, the polypeptide solution may be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography with DEAE-cellulose and / or (immuno)affinity chromatography.

[0281] One or more genes encoding a binding polypeptide can also be expressed in non-mammalian cells such as bacteria, yeast, or plant cells. In this regard, it will be found that various non-mammalian single-celled microorganisms (such as bacteria) can be transformed and grown in culture or by fermentation. Bacteria susceptible to transformation include strains of Enterobacteriaceae (e.g., Escherichia coli) or Salmonella, and members of Bacillaceae (e.g., Bacillus subtilis), Pneumococcus, Streptococcus, and Haemophilus influenzae. Furthermore, it will be found that when expressed in bacteria, the binding polypeptide can become part of an inclusion body. The binding polypeptide must be isolated, purified, and then assembled into a functional molecule.

[0282] In addition to prokaryotes, eukaryotic microorganisms can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used eukaryotic microorganism, but many other strains are also commonly available. For expression in the genus Saccharomyces, plasmid YRp7 is commonly used, for example (Stinchcomb DT et al. (1979), Nature, 282:39-43; Kingsman et al. (1979), Gene, vol.7:141-52; and Tschemper et al. (1980), Gene, vol.10:157-66). This plasmid already contains the TRP1 gene, which provides a selection marker for yeast mutants lacking the ability to grow on tryptophan, such as ATCC number 44076 or PEP4-1 (Jones EW (1977), Genetics, vol.85(1):23-33). The presence of trpl lesions as a characteristic of the yeast host cell genome then provides a suitable environment for detecting transformation due to proliferation in the absence of tryptophan.

[0283] Treatment method using binding polypeptides In one embodiment, the Disclosure provides a method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising administering an effective amount of the conjugated polypeptide disclosed herein to the subject. In a particular embodiment, the Disclosure provides a kit and method for treating a disease and disorder, such as cancer, in a mammalian subject requiring treatment of the disease and disorder, such as cancer.

[0284] The binding polypeptides of this disclosure are useful for many different applications. For example, in one embodiment, the subject binding polypeptide is useful for reducing or eliminating cells having an epitope recognized by the binding domain of the binding polypeptide. In another embodiment, the target binding polypeptide is effective for reducing or eliminating the concentration of a circulating soluble antigen. In one embodiment, the binding polypeptide may reduce tumor size, inhibit tumor growth, and / or extend the survival time of tumor-bearing animals. In another embodiment, the target binding polypeptide is effective as a T cell engager. Thus, this disclosure also relates to a method for treating tumors in humans or other animals, the method of administering an effective no-observed-adverse-effect level of a modified antibody to such humans or animals.

[0285] In another embodiment, the target binding polypeptide is useful for treating other disorders or diseases, including but not limited to infectious diseases, autoimmune disorders / diseases, inflammatory disorders / diseases, lung diseases, neurological or neurodegenerative diseases, liver diseases, spinal diseases, uterine diseases, and depressive disorders. Non-limited examples of infectious diseases include those caused by RNA viruses, e.g., Orthomyxoviridae (e.g., influenza), Paramyxoviridae (e.g., polynuclear respiratory viruses, parainfluenza viruses, metapneumoviruses), Rhabdoviridae (e.g., rabies virus), Coronaviridae (e.g., SARS-CoV), Togaviridae (e.g., chikungunya virus), Retroviridae (e.g., HIV), or DNA viruses. Examples of infectious diseases include, but are not limited to, bacterial infectious diseases caused by Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus, Streptococcus, and Escherichia coli, and other infectious diseases caused by fungi (e.g., Candida albicans) or parasites (e.g., malaria). Other infectious diseases include, but are not limited to, SARS, yellow fever, Lyme borreliosis, leishmaniasis, anthrax, and meningitis. Exemplary autoimmune disorders include, but are not limited to, psoriasis and lupus. Accordingly, this disclosure relates, for example, to methods for treating various pathological conditions that would benefit from using a subject effector-competent polypeptide with an extended half-life.

[0286] Those skilled in the art will be able to determine, through routine experiments, what the effective no-observed-adverse-effect level (NOAEL) of a conjugated polypeptide is for the purpose of treating malignant tumors. For example, the therapeutic activity of the conjugated polypeptide of this disclosure may vary depending on factors such as disease stage (e.g., stage I vs. stage IV), age, sex, medical complications (e.g., immunosuppressive state or disease), and body weight, as well as the ability of the modified antibody to induce the desired response in the subject. The administration regimen may be adjusted to provide the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation.

[0287] Generally, the compositions provided in this disclosure may be used to prophylactically or therapeutically treat any neoplasm, including antigen markers that enable the targeting of cancer cells by modified antibodies.

[0288] Pharmaceutical composition and administration thereof Methods for preparing and administering the conjugated polypeptides of this disclosure to a subject can be readily determined by those skilled in the art. The route of administration of the conjugated polypeptides of this disclosure may be oral, parenteral, inhaled, topical, or any other suitable method. As used herein, the term parenteral includes intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these dosage forms are obviously assumed to be within the scope of this disclosure, the dosage forms are solutions for injection, particularly intravenous or intra-arterial injection, or solutions for infusion. Typically, preferred pharmaceutical compositions for injection may include buffers (e.g., acetic acid, phosphoric acid, or citrate buffer), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). However, other methods conforming to the teachings herein may involve directly delivering modified antibodies to the site of a harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0289] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions containing saline and a buffer medium. In the compositions and methods of this disclosure, pharmaceutically acceptable carriers include, but are not limited to, 0.01 to 0.1 M, e.g., 0.05 M phosphate buffer or 0.8% physiological saline. Other common parenteral vehicles include sodium phosphate solution, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oil. Intravenous vehicles include fluids and nutritional supplements, and electrolyte solutions such as ringer's dextrose-based solutions. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present. More specifically, suitable pharmaceutical compositions for injection include sterile aqueous solutions (water-soluble) or dispersions, and sterile powders for immediate-use preparations of sterile injection solutions or dispersions. In such cases, the composition must be sterile and fluid enough to be easily injected. It must be stable under manufacturing and storage conditions and typically protected from microbial contamination, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol) and suitable mixtures thereof. Adequate fluidity may be maintained, for example, by the use of a coating material such as lecithin, by maintaining the required particle size in the case of dispersions, and by the use of a surfactant.

[0290] In many cases, the composition contains isotonic agents, such as sugars, polyhydric alcohols, such as mannitol, sorbitol, or sodium chloride. The inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition can lead to prolonged absorption of the injectable composition.

[0291] In any case, a sterile injection solution may be prepared by incorporating the required amount of the active compound (e.g., a modified conjugated polypeptide, either alone or in combination with other activators) into a suitable solvent, along with one or a combination thereof of the components listed herein, and subsequently by filtration sterilization as necessary. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other required components from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, exemplary preparation methods include vacuum drying and lyophilization, which yield a powder of the active component and any desired additional components from a pre-filtration sterilized solution thereof. The preparations for injection are processed and filled into containers such as ampoules, bags, bottles, syringes or vials and sealed under sterile conditions according to methods known in the art. Furthermore, the preparations may be packaged and sold in kit form, such as those described in U.S. Patent Application No. 2002-0102208 and U.S. Patent No. 6,994,840 (each incorporated herein by reference). Such products typically have labels or accompanying information indicating that the relevant composition is useful for treating subjects suffering from or predisposed to autoimmune disorders or neoplasms.

[0292] The effective dose of the compositions of this disclosure for treating the above-described conditions will vary depending on many different factors, including the means of administration, the target site, the patient's physiological state, whether the patient is human or animal, other pharmaceuticals administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, but non-human mammals, including transgenic mammals, may also be treated. To optimize safety and efficacy, the treatment dose may be titrated using conventional methods known to those skilled in the art.

[0293] The conjugated polypeptides of this disclosure may be administered on multiple occasions. The interval between single doses may be weekly, monthly, or yearly. The interval may also be irregular, as indicated by measuring the blood levels of the Fc domain variant or antigen in the patient. In some methods, the dosage is adjusted to achieve plasma-modified conjugated polypeptide concentrations of approximately 1–1000 μg / ml, and in other methods, approximately 25–300 μg / ml. Alternatively, the conjugated polypeptide may be administered as a sustained-release formulation, in which case the required frequency of administration is lower. In the case of antibodies, the dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-lives, followed by chimeric antibodies and non-human antibodies.

[0294] The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the polypeptides of the present invention or cocktails thereof are administered to patients who are not yet in a diseased state in order to increase patient tolerance. Such amounts are defined as “prophylactic effective doses.” In this use, the exact amount still depends on the patient’s health and overall immunity, but is generally in the range of about 0.1 to about 25 mg per dose, and particularly about 0.5 to about 2.5 mg per dose. Relatively low doses are administered at relatively infrequent intervals over a long period. Some patients continue to receive treatment for the remainder of their lives. In therapeutic applications, relatively high doses (e.g., about 1 to 400 mg / kg antibody / dose, with doses of about 5 to 25 mg more commonly used for radioimmune complexes and higher doses used for cytotoxic-drug-modified antibodies) may be required at relatively short intervals until the progression of the disease is mitigated or terminated, or until the patient shows partial or complete improvement of disease symptoms. A prophylactic regime may then be administered to the patient.

[0295] The conjugated polypeptides of this disclosure may be administered optionally in combination with other agents that are effective in treating disorders or conditions requiring treatment (e.g., prophylactic or therapeutic). The effective single-treatment dose (i.e., therapeutically effective dose) of the 90Y-labeled modified antibody of this disclosure is in the range of about 5 to about 75 mCi, for example, about 10 to about 40 mCi. The effective single-treatment non-myeloablative dose of the 131I-modified antibody is in the range of about 5 to about 70 mCi, or about 5 to about 40 mCi. The effective single-treatment ablative dose (i.e., potentially requiring autologous bone marrow transplantation) of the 131I-labeled antibody is in the range of about 30 mCi to about 600 mCi, for example, about 50 mCi to less than about 500 mCi. For vis-a-vis mouse antibodies with longer circulating half-lives, in conjunction with chimeric antibodies, the effective single-dose dose of iodine-131-labeled chimeric antibody for non-myelolytic disruption ranges from approximately 5 mCi to approximately 40 mCi, for example, less than approximately 30 mCi. For example, the imaging reference for 111In labeling is typically less than approximately 5 mCi.

[0296] While the conjugated polypeptide may be administered as described above, it should be emphasized that in other embodiments, the polypeptide may be administered as first-line therapy to otherwise healthy patients. In such embodiments, the conjugated polypeptide may be administered to patients with normal or average red bone marrow reserve and / or patients who have never received and will not receive treatment. As used herein, administering the polypeptide in combination with or in conjunction with adjuvant therapy means administering or applying the treatment and the disclosed antibody in succession, simultaneously, with the same extent, concurrently, synchronously, or in a simultaneous presence. Those skilled in the art will understand that the timing of administration or application of the various components of a combined therapeutic regimen may be adjusted to enhance the overall effect of the treatment.

[0297] As stated above, the conjugated polypeptides, antibodies, therapeutic polypeptides, or their Fc domain variant-fusion polypeptides of the present disclosure may be administered in pharmaceutically effective amounts for in vivo treatment of mammalian disorders. In this regard, it will be understood that the disclosed Fc domain variants may be formulated to facilitate administration and enhance the stability of the active agent.

[0298] The pharmaceutical compositions of this disclosure may include pharmaceutically acceptable non-toxic sterile carriers such as physiological saline, non-toxic buffers, and preservatives. For the purposes of this application, the pharmaceutically effective amount of the conjugated polypeptide, whether conjugated or not, in the therapeutic agent shall be maintained such that it is sufficient to achieve effective binding to an antigen and achieve a benefit, for example, improving the symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the polypeptide may interact with a selected antigen on neoplastic or immunoreactive cells, increasing the death of those cells. Naturally, the pharmaceutical compositions of this disclosure may be administered in single or multiple doses to provide a pharmaceutically effective amount of the modified conjugated polypeptide.

[0299] In accordance with the scope of this disclosure, the conjugated polypeptides of this disclosure may be administered to humans or other animals in an amount sufficient to produce a therapeutic or prophylactic effect, in accordance with the treatment methods described above. The conjugated proteins of this disclosure may be administered to such humans or other animals in conventional dosage forms prepared by combining the antibodies of this disclosure with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques. It will be recognized by those skilled in the art that the form and characteristics of the pharmaceutically acceptable carrier or diluent are determined by the amount of the active ingredient to be combined with it, the route of administration, and other well-known variable factors. Those skilled in the art will further understand that a cocktail containing one or more species of conjugated polypeptides described in this disclosure may prove particularly effective.

[0300] The contents of articles, patents and patent applications, and all other documents and electronically available information referred to or cited herein are incorporated herein by reference in whole, to the same extent that each individual publication is shown to be incorporated by specific and individual reference. The applicant reserves the right to physically incorporate into this application any and all material and information from such articles, patents, patent applications, or other physical and electronic documents. Embodiments described herein as exemplary may be well carried out in the absence of any elements or elements, limitations or restrictions that are specifically disclosed herein or not. Accordingly, for example, in each case herein, any of the terms “including,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms, while retaining their ordinary meanings. Any single term, single element, single phrase, group of terms, group of phrases, or group of elements described herein may each be specifically excluded from a claim.

[0301] While the present invention has been described with reference to its specific embodiments, it should be understood by those skilled in the art that various modifications may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein can be made by using suitable equivalents without departing from the scope of the embodiments disclosed herein. Furthermore, many modifications may be made to adapt specific circumstances, materials, substance compositions, processes, process stages or steps to the object, spirit and scope of the invention. Any such modifications are intended to fall within the scope of the appended claims. While specific embodiments have been described in detail here, this will be better understood by referring to the following examples, which are included for illustrative purposes only and are not intended to limit the scope. [Examples]

[0302] This disclosure is further illustrated by the following examples, which should not be construed as further limitations. The sequence lists, drawings, tables, and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

[0303] Example 1. Effect of glycosylation on the effector function of monoclonal antibodies containing Fc mutations. introduction Numerous efforts have been made to enhance ADCC to improve the effectiveness of treatments for the disease. Protein engineering of the Fc-CH2 domain using site-directed mutagenesis can significantly improve FcγRIIIa binding and ADCC activity (Lazar Ga et al. (2006), Proc. Natl. Acad. USA, vol. 103(11): 4005-10 and Liu Z et al. (2014), J Biol Chem, vol. 289(6): 3571-90). Similarly, ADCC has also been significantly enhanced through Fc-glycotechnology, particularly via core fucose removal (Shields RL et al. (2002), J Biol Chem, vol. 277(30): 26733-40 and Shinkawa T et al. (2003), J Biol Chem., vol. 278(5): 3466-73). Both Fc modification strategies resulted in a 10- to 100-fold increase in potency (Masuda K et al. (2007), Mol Immunol, vol. 44(12): 3122-31). This study investigated whether the combination of the double mutant S239D / I332E (DE mutation) with different Fc-glycosylation, including defucosylation, mannosylation, and hypergalactosylation, further affects both FcγRIIIa binding and ADCC. The results presented herein demonstrate the synergistic effect on the effector function of recombinant human monoclonal IgG1 antibody, mAb A, when combined with Fc mutations and glycan modifications.

[0304] Materials and methods Cell culture, antibody expression / modification, and SDS-PAGE: Human monoclonal antibodies (IgG1) were produced from Chinese hamster ovary (CHO) cells containing wild-type (WT) Fc sequences and DE mutations (S239D / I332E) (Lazar Ga et al. (2006), Proc. Natl. Acad. USA, vol. 103(11): 4005-10). CHO cells expressing the antibodies were cultured in suspension in CD-CHO containing 4 mM glutamine (ThermoFisher Scientific®). Defucosylated WT and DE antibodies were produced by gene transfer into antibody-expressing CHO cells using genes encoding the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexose reductase (RMD) and green fluorescent protein (GFP). Gene transfer was performed using the Gene Pulser Xcell electroporation system (Bio-Rad®), and then the cells that had received the gene transfer were selected in G418. Subsequently, RMD-expressing cells were sorted by flow cytometry using GFP fluorescence (von Horsten HH, et al. Production of non-fucosylated antibody by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase Glycobiology 2010;20(12):1607-18). Defucosylated antibodies were also prepared by treating antibody-expressing cells with 1,3,4-tri-O-acetyl-2-deoxy-2-fluoro-L-fucose, peracetylated 2-fluoro-2-deoxy-L-fucose (2FF, 0-200 μM) (Okeley NM et al. (2013), Proc. Natl. Acad. USA, vol. 110(14): 5404-9). Antibodies containing oligomannose were prepared by treating cells with kifunensin, a potent α-mannosidase I inhibitor (Zhou Q et al. (2008), Biotechnol Bioeng, vol. 99(3): 652-65).On day 0, kifunensin (2 μg / mL) was added to the antibody-expressing cell culture, and the cells were grown for 11 days before being harvested for antibody purification. Highly galactosylated antibodies were prepared in vitro using β-galactosyltransferase (Houde D et al. (2010), Mol Cell Proteomics, vol.9(8):1716-28).

[0305] The antibody IgG1 was purified using protein A chromatography. Briefly, the protein A affinity medium, MAbSelect (GE Healthcare®), was equilibrated with PBS (pH 7.2). The sample was added to the column, washed with 10 column volumes of equilibration buffer, then eluted with 12.5 mM citrate, and the pH was immediately adjusted to approximately 7.0 with 0.5 M HEPES buffer (pH 7.2). The antibody underwent five buffer changes with PBS (pH 7.2). The antibodies were analyzed using SDS-PAGE under reducing and non-reducing conditions using 4-12% NuPAGE (ThermoFisher Scientific®).

[0306] N-linked glycan analysis: N-linked glycans were released from the antibody using PNGase F and purified using solid-phase extraction. Aliquots of the sample were mixed with a 2,5-dihydroxybenzoic acid matrix and applied to the target. MALDI-TOF mass spectra were obtained using a Brukar Autoflex® Speed ​​MALDI-TOF (Bruker Daltonics, Billerica, MA) in cation reflectance mode. A portion of the released N-linked glycans were also labeled with 2-aminobenzamide (2-AB) and analyzed using hydrophilic interaction liquid chromatography-ultrahigh performance liquid chromatography (HILIC-UPLC) (Reusch D et al. (2015), MAbs, vol.7(1):167-79). Glycans were separated using glycanBEHamide along with a glycanBEHamide column (2.1 x 150 mM) on an Acquity UPLC® H-class Bio System (Waters®). The column was equilibrated in 25% solvent A (50 mM ammonium formate, pH 4.4) and 75% solvent B (100% acetonitrile). 2-AB labeled glycans were eluted at 60°C for 36.5 minutes using a linear gradient from 75% to 0% solvent B at a flow rate of 0.4–0.2 mL / min.

[0307] FcγRIIIa binding analysis: The binding of antibodies to recombinant human FcγRIIIa-V158 was analyzed using surface plasmon resonance (SPR) with a Biacore™ T200 instrument. The antibody was diluted to 5 μg / ml in HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) and injected onto a sensor chip immobilized with protein A (Cytiva) at a flow rate of 10 μL / min for 30 seconds at 25°C to obtain intermediate capture levels. Recombinant human FcγRIIIa-V158 was successively diluted 3-fold in running buffer from 900 to 3.7 nM (100 nM shown in Figures 4A-B), injected in pairs onto the captured antibody for 2 minutes, and then dissociated in buffer at a flow rate of 30 μL / min for 3 minutes. The surface was regenerated with 10 mM glycine at pH 1.5 for 30 seconds. Sensorgrams were processed using BiaEvaluation software (GE® Healthcare), fitted to a 1:1 binding model, and the binding affinity (KD) was calculated.

[0308] ADCC Activity Assay: ADCC potency was measured using a bioluminescent reporter bioassay (Promega®). This method utilizes target cells and effector cells, as well as Jurkat cells, that express a cell surface target antigen, modified to express FcγRIIIa (V158) and a luciferase reporter. In the presence of target cells, antibodies, and modified effector cells, pathway activation leads to the induction of the luciferase reporter in the effector cells. Luciferase production is proportional to the level of ADCC activity present. Similar assays have been reported as alternative methods for measuring ADCC without isolating peripheral blood mononuclear cells (PBMCs) from fresh blood (Parekh BS et al. (2012), MAbs, vol.4(3):310-8 and Kurogochi M et al. (2015), PloS One, vol.10(7):e0132848). This has been shown to correlate well with standard approaches using PBMCs with good accuracy, precision, and robustness (Parekh BS et al. (2012), MAbs, vol.4(3):310-8).

[0309] Multiple doses of the antibody were added to target cells in pairs. Effector cells were then added to assay plates containing the target cells and antibody. After set incubation, a luciferase substrate was added, and the response for luciferase production was measured. The luciferase response to each dose of the sample and reference material fit a four-parameter model. The dose-response curve for the sample was compared to that of the reference material. Due to the fact that various molecules were compared for these studies, not all curves were parallel to or relative to the reference material. Therefore, a multiplier increase in the estimated ADCC activity was determined for molecular comparison.

[0310] result Antibody Fc Engineering for Enhanced Effector Function: N-linked glycans in protein 1-specific wild-type (WT) and DE mutant antibodies were modified enzymatically, metabolically, or recombinantly to achieve high galactosylation, mannosylation, or defucosylation. As shown in Figure 2, analysis of these antibodies using SDS-PAGE revealed profiles with expected antibody size and minimal impurities or aggregates. Their N-linked glycans were released with PNGase F and analyzed using MALDI-TOF MS. As shown in Figures 3A and 3B, the results demonstrate that both WT and DE antibodies primarily contain G0F(GlcNAc2Man3GlcNAc2Fuc1) and G1F(Gal1GlcNAc2Man3GlcNAc2Fuc1) glycans, which are the major species found in most recombinant human antibodies. As shown in Figures 3C and 3D, when antibodies were modified in vitro with galactosyltransferase, the N-linked glycans on WT and DE mutants became the highly galactosylated species, G2F (Gal2GlcNAc2Man3GlcNAc2Fuc1). As shown in Figures 3E and 3F, when WT and DE antibodies were purified from kifunensin-treated cell cultures, they mainly contained oligomannose-type glycans, Man9 (Man9GlcNAc2) and Man8 (Man8GlcNAc2). In WT and DE antibodies purified from cells transfected with the gene encoding the bacterial oxidoreductase GDP-6-deoxy-D-lyxo-4-hexose reductase (RMD), the main components were defucosylated glycans, G0 (GlcNAc2Man3GlcNAc2) and G1 (Gal1GlcNAc2Man3GlcNAc2) (Figures 3G and 3H).

[0311] FcγRIIIa binding of Fc-modified antibodies: Surface plasmon resonance (SPR) was used to investigate FcγRIIIa binding of WT and DE antibodies specific to protein 1 with different N-linked glycans. As a result of DE mutations, the interaction of the antibody with FcγRIIIa was strongest compared to glycan-modified WT antibodies (Tables 1 and 2 below). The order of binding can be ranked as follows: DE > defucosylated WT > mannosylated WT > high galactosylated WT ≥ WT. Combining DE mutations with glycan modifications for high galactosylation or mannosylation had a synergistic effect on increasing FcγRIIIa binding (Figure 4A). Antibodies with high galactose and oligomannose mainly had a dissociation rate constant (k d Due to a decrease in ), they showed 1.8 times and 2.3 times higher affinity compared to unmodified DE antibodies, respectively (Table 1). When DE mutations were combined with defucosylation, receptor binding of defucosylated DE improved 3.5 times compared to DE antibodies (Figure 4B). This enhanced interaction is mainly a result of a decrease in the dissociation rate (Table 2).

[0312] [Table 3]

[0313] [Table 4]

[0314] Synergistic effects of modified antibodies against ADCC: The ADCC activity of DE antibodies specific to glycan-modified protein 1 was evaluated using an ADCC reporter gene assay. DE mutations, in combination with mannosylation, show potential synergistic enhancement (Figure 5A). Modified DE antibodies containing oligomannose showed slightly higher ADCC activity (1.4-fold) compared to unmodified DE antibodies. ADCC activity was found to be similar for DE antibodies with or without high galactosylation.

[0315] Interestingly, the FcγRIIIa affinity of DE mutants was higher than that of defucosylated WT antibodies (Table 2), but their ADCC activity was similar (Figure 5B). Furthermore, the DE mutation plus defucosylation resulted in a synergistic enhancement of ADCC activity (Figure 5B). Compared to unmodified DE antibodies, ADCC activity increased by approximately 3.2 times.

[0316] Correlation between effector function and defucosylation levels in antibodies with DE mutations: To gain a more detailed understanding of the effect of defucosylation due to Fc mutations on effector function, different amounts of defucosylation were induced by treating DE antibody-expressing cells with fucose analogs, specifically peracetylated 2-fluorofucose, for fucosyltransferase inhibition. For more accurate quantification, N-linked glycans released from the antibodies were labeled with 2-AB and analyzed using UPLC. As shown in Table 3 below, there was a decrease in α1,6-linked fucose in the glycans after antibody purification from cells treated with increasing amounts of fucose analogs. Effector function of the antibodies was determined using FcγRIIIa binding and ADCC reporter gene assays. As shown in Figure 6A, there was a linear correlation between the amount of defucosylation and receptor binding (r 2 (=0.99). Furthermore, there was a positive correlation between enhanced ADCC activity and an increase in the amount of defucosylation in the same antibody (r 2 =0.84) (Figure 6B). The results suggest that, in association with DE mutations, lower fucose content leads to higher FcγRIIIa affinity and ADCC for the antibody.

[0317] [Table 5]

[0318] conclusion To gain a more detailed understanding of the effects of glycosylation on Fc mutations, we investigated the effects of complex protein mutagenesis involving glycan modification. We used glycan modifications from double mutations and DE mutations. Human recombinant monoclonal antibody IgG1, containing a double mutation (DE), was modified for defucosylation, mannosylation, and hypergalactosylation. Synergistic effects on FcγRIIIa binding and ADCC activity were demonstrated with antibodies containing both DE mutations and defucosylation. Synergistic effects were also observed in FcγRIIIa binding with mannosylation and hypergalactosylation combined with DE mutations. Improved interaction between FcγRIIIa and DE antibodies containing three different types of glycans was primarily due to a decrease in dissociation rate. Different mechanisms are thought to contribute to the synergistic enhancement of FcγRIIIa interactions. Furthermore, enhanced effector function correlated with increased levels of defucosylation in the antibody. These results suggest that different glycosylations in antibodies with Fc mutations can have various effects on receptor binding and ADCC activity.

[0319] While there was a slight effect on FcγRIIIa binding and no further enhancement of ADCC by DE+ hypergalactosylation, the DE antibody containing oligomannose showed greater receptor binding and slightly higher ADCC activity than unmodified DE.

[0320] In conclusion, these results demonstrate the synergistic effects of glycosylation, including defucosylation, mannosylation, and hypergalactosylation, on antibodies with effector-enhancing Fc mutations. Therefore, these results provide the first evidence of a synergistic effect between n-effector-enhancing mutations and Fc glycosylation in the Fc domain.

[0321] Example 2. Human FcγRIIIa affinity of antibody variants specific to protein 2. introduction To test the synergistic effect on enhanced effector function, Fc mutants of protein 2-specific antibodies were generated with and without kifunensin treatment. After purification, human FcγRIIIa binding affinity was measured.

[0322] Materials and methods FcγRIIIa binding analysis: The following modifications were made, and FcγRIIIa binding analysis was performed using surface plasmon resonance (SPR) on a Biacore® T200 instrument as described above in Example 1. The antibody was diluted to 0.5 μg / ml, and recombinant human FcγRIIIa-V158 was sequentially diluted 3-fold in HBS-EP + pH 7.4 running buffer at 3000 nM to 0.457 nM and injected at 50 μL / min for 2 minutes. Sensorgrams were processed and fitted using a 1:1 dynamic coupling model.

[0323] Table 4 below shows the expression and purification of antibody variants specific to protein 2 with and without kifunensin. Expression was performed using Expi293® expression medium (20 mL scale) with 2 μg / ml kifunensin. Purification was performed using a 1 mL MabSelect Sure® column with single-step elution of 5 column volumes (CV), followed by buffer exchange on an Amicon® unit. Figures 7A and 7B show SDS-PAGE of Fc variants of antibody specific to protein 2 with and without kifunensin treatment. Figure 7A shows the non-reduced state. Figure 7B shows the reduced conditions. 4-12% BT SDS-PAGE was used with MES buffer and 4 μg protein / lane.

[0324] [Table 6]

[0325] result Glycan Analysis: The glycan structures of various Fc variant antibodies were analyzed using MALDI-TOF with and without kifunensin, as shown in Figures 8A-F. Figure 8A shows the wild-type antibody, which mainly contains G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. Figure 8B shows the S298A antibody, which mainly contains G0F and G1F glycans without kifunensin treatment, and Man9 glycans with kifunensin treatment. Figure 8C shows the S239D antibody, which mainly contains G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. S239D / S298A antibody, as shown in Figure 8D, containing mainly G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man9 glycan with kifunensin treatment. I332E antibody, as shown in Figure 8E, containing mainly G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment. S239D / I332E antibody, as shown in Figure 8F, containing mainly G0F-Gn, G0F, and G1F glycans without kifunensin treatment, and Man8 and Man9 glycans with kifunensin treatment.

[0326] Results of human FcγRIIIa binding affinity: The binding affinity to human FcγRIIIa was measured for various Fc variants with and without kifunensin treatment, and the sensorgrams are shown in Figures 9B-G. Figure 9A shows the measured binding affinity for the following antibodies: WT, S239D, S239D / S298A, S298A, I332E, and S239D / I332E. These results show a synergistic effect, with kifunensin treatment increasing the affinity to hFcγRIIIa by 2.4 to 7 times for all Fc variants tested. For example, without kifunensin treatment, S239D / S298A has lower affinity than S239D / I332E, but with kifunensin treatment, it shows higher affinity than S239D / I332E without kifunensin treatment.

[0327] Example 3. Rescue of thermal stability loss caused by Fc mutation and kifunensin treatment. introduction In this example, the thermal stability and Fcγ receptor binding of mAbs containing the Fc variant were tested with and without kifunensin treatment. Since kifunensin treatment reduced the thermal stability of the antibody, the ability of the R292C / V302C (disulfide) mutation to rescue the loss of thermal stability was tested.

[0328] Materials and methods NanoDSF Thermal Denaturation: mAbs containing the Fc variant (specific to protein 2 or protein 4) were diluted with buffer A (1 mg / mL at 10 mM histidine, pH 6.0), and then buffer-exchanged with buffer A (4x) to remove salt traces from the purification process. The final concentration of all mAbs was then normalized to 0.5 mg / mL. Thermal stability was determined by a nano-format of differential scanning fluorescence (nanoDSF) using a Prometheus NT48 with a "high-sensitivity" capillary, applying a linear temperature gradient from 20°C to 95°C at a heating rate of 1°C / min. NanoDSF monitors protein unfolding with increasing temperature using changes in the protein's intrinsic fluorescence. The protein solution was excited using a 266 nm wavelength light source, and fluorescence emission from tyrosine and tryptophan residues at 330 nm and 350 nm was detected. The maximum emission values ​​and intensities of tyrosine and tryptophan residues are highly dependent on their direct environment and can change as the protein unfolds during thermal denaturation. By monitoring the change in the ratio of fluorescence intensities at 330 nm and 350 nm as a function of temperature, a sigmoid curve representing the transition of protein unfolding can be obtained. The midpoint of the sigmoid curve represents the melting temperature (Tm). Tonset is the detectable temperature at which the protein begins to unfold. There are three inflection points (IPs), two of which correspond to CH2 and CH3 in the Fc domain, and the third inflection point corresponds to the Fab domain. Tagg is the temperature at which the protein tends to aggregate.

[0329] Fcγ receptor binding analysis: Using protein A capture, antibody binding to recombinant human FcγRIIIa-V158 was analyzed using surface plasmon resonance (SPR) on a Carterra LSA instrument. Protein 4-specific antibodies were diluted to 0.5, 0.2, and 0.05 μg / ml in HBS-EP+ (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20) in pairs of wells of three 96-well plates. For each plate, the antibodies were printed for 10 minutes using a capture array method with separate quadrants on sensor chips immobilized with protein A / G (Carterra PAGHC30M chip). His-tagged recombinant human FcγRIIIa-V158 was sequentially diluted 2-fold in HBS-EP+ pH 7.4 and injected onto the capture antibody for 2 minutes of association followed by 3 minutes of dissociation, and affinity was measured. Each infusion series contained a total of 12 different concentrations of FcγRIIIa ranging from 1.95 nM to 4000 nM. Buffer infusions were evenly distributed between receptor infusions for appropriate blank subtraction. Sensorgrams were processed using Carterra Kinetics analysis software and fitted to a 1:1 binding model to obtain rate constants. For reported binding affinities, the average was calculated from the paired levels on the plate and from each antibody capture level.

[0330] Results and Conclusions Table 5 below shows the Tm of the CH2 domain when determined by thermal denaturation for antibodies specific to protein 2 with different Fc variants, in or out of the presence of kifunensin treatment. Kifunensin treatment reduces the Tm of the CH2 domain by 3-6°C. In the case of the S239D / I332E Fc variant and kifunensin treatment, the Tm is 42.8°C, compared to 68.9°C for the wild-type (WT) antibody without kifunensin treatment. Figure 10 shows the selected antibodies from Table 5 (WT without kifunensin, WT with kifunensin, S239D / I332E without kifunensin, and S239D / S298A with kifunensin).

[0331] [Table 7]

[0332] Table 6 below shows the Tm of the CH2 domain, as determined by thermal denaturation, for antibodies specific to protein 4 having different Fc variants with and without kifunensin treatment. The disulfide bond (DSB) between R292C and V302C significantly rescues the loss of thermal stability caused by the Fc variant and kifunensin treatment.

[0333] [Table 8]

[0334] SPR results for binding to rhFcγRIIIa-V158 show WT-like binding affinity for R295C / V305C, T256D / T307Q, and T256D / T307Q+R295C / V305C. Kifunesin treatment increases affinity by 1.6 to 7.7 times for all Fc variants, as shown in Table 7 and Figure 16 below.

[0335] [Table 9]

[0336] Example 4. Effect of kifunensin treatment on characterization of FcRn-enhanced variants introduction Experiments were conducted to characterize a series of antibody variants specific to protein 3, expressed with and without kifunensin. Previous experiments had shown that kifunensin modifies the antibody's glycan structure to increase oligomannose and enhance Fcγ receptor functionality, so experiments were conducted to determine its effect on FcRN binding.

[0337] Materials and methods N-linked glycan analysis: Glycan analysis was completed using mass spectrometry (as described in Example 1 above).

[0338] Fcγ receptor binding analysis: FcγRIIIa binding was determined using SPR and measured with a Biacore® T200 instrument. Recombinant human HPC4-tagged FcγRIIIa-V158 was diluted to 1.25 μg / mL in HBS-P+ w / CaCl2 (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% surfactant P20, 2 mM CaCl2) running buffer and injected at a flow rate of 5 μL / min for 30 seconds into a CM5 chip immobilized with anti-HPC4-tagged antibody (Roche). The antibody was diluted to 500 nM in running buffer and injected into the captured receptor in triplicates for 3 minutes. Dissociation was measured for 3 minutes, and the chip was regenerated with HBS-EP+ buffer with 7 mM EDTA for 3 minutes at a flow rate of 20 μL / min. The steady state of the 300 nM antibody was determined in triplicates and averaged. The magnification change in the response to WT (response magnification change) was determined for comparison between variants in each skeleton.

[0339] Human FcRN-binding analysis: FcRN-binding was determined using SPR and measured on a Biacore™ T200 instrument using a biotin CAPture kit (Cytiva). The running buffer was PBS containing 0.05% surfactant P-20 (PBSP+, GE Healthcare) buffered with HCl, at pH 6.0 for kinetics or pH 7.4 for binding. The CAPture reagent was captured on the CAP tip to a surface density of >2000 RU, followed by the capture of 0.2 μg / ml biotinylated recombinant human FcRn at 30 μL / min for 24 seconds to a final surface density of approximately 20 RU. Antibodies were sequentially diluted 3-fold from 1000 nM to a total of 5 concentrations in running buffer at pH 6.0, injected in pairs for 3 minutes, and then dissociated in buffer for 5 minutes. The surface was regenerated for 2 minutes at 50 μL / min with 6 M guanidine hydrochloride and 250 mM NaOH. Using the same conditions as above, except for a tenfold increase in the FcRn capture level, steady-state RU measurements at pH 7.4 were obtained in triplicate at 1000 nM. The kinetic parameters at pH 6.0 were fitted to a bivalent model using Biacore T200 Evaluation Software. Each concentration series was fitted independently to obtain average rate and affinity. Apparent binding affinity was calculated for the first on and off rates from the bivalent model. For response comparison, residual binding at pH 7.4 was measured using 1000 nM of each antibody simultaneously.

[0340] NanoDSF thermal denaturation: The thermal stability was determined using DSF in a triple dose of 0.2 mg / mL (as described above in Example 3).

[0341] FcRn affinity chromatography: pH dependence was determined using FcRn affinity chromatography with immobilized human FcRn and an MES-BTP pH gradient. FcRn affinity columns were adapted to biotinylated recombinant human FcRn on a 1 mL streptavidin HP HiTrap column (GE Healthcare) as described by Schlothauer et al. 300 μg antibody in a low pH buffer (20 mM 2-(N-morpholino)ethanesulfonic acid (MES,Sigma) pH 5.5; 150 mM NaCl) was injected into the column on an AKTA Pure System (AKTA). Antibodies were eluted using pH gradients prepared at 0.5 mL / min over more than 30 column volumes (CV) with low and high pH buffers (20 mM 1,3-bis(tris(hydroxymethyl)methylamino)propane (bistrispropane, Sigma) pH 9.5; 150 mM NaCl), and absorbance and pH were monitored. The generation of a linear pH gradient (linear regression, R² > 0.99) was achieved through the following stepwise method: 0% to 30% high pH buffer over 9 CV, 30% to 70% over 16.5 CV, and 70% to 100% over 4.5 CV. The columns were re-equilibriumated with low pH buffer for subsequent runs. All variants were performed in triplicates. FcRn affinity column elution profiles were fitted to a single Gaussian distribution in Sigmaplot 11 (Systat Software, Inc.) to determine elution volume, full width at half maximum (FWHM), and pH at UV280 maxima.

[0342] result N-linked glycan analysis: Figures 11A-F show mass spectrometry glycan analysis of WT, LS, YTE, YD, DQ, and DW antibodies specific to protein 3, with and without kifunensin treatment. All kifunensin-treated antibodies have an oligomannose content of >97% Man9(GlcNAc)2. All untreated antibodies are >80% defucosylated.

[0343] FcγRIIIa binding: Figure 12 shows the triplicate FcγRIIIa binding affinity response to WT, DQ, DW, LS, YD, and YTE antibodies specific to protein 3, with and without kifunensin treatment. Table 8 below shows the multiplicative change in FcγRIIIa binding affinity to protein 3-specific antibodies with and without kifunensin. Table 9 shows the multiplicative change in FcγRIIIa binding affinity to protein 3-specific antibodies compared to the wild type with kifunensin treatment. Enhanced FcγRIIIa binding was observed for all variants expressed with kifunensin, as well as for the WT antibody.

[0344] [Table 10]

[0345] [Table 11]

[0346] Human FcRN binding: Human FcRN binding at pH 6.0 and pH 7.4 is shown in Figures 13A and 13B, respectively. A scatter plot of the results at pH 6.0 is shown in Figure 13C. FcRN binding assays were completed for WT, LS, YTE, DQ, DW, and YD antibodies specific to protein 3, both with and without kifunensin treatment. At pH 6.0, no significant changes in on or off binding rates were observed. DQ, DW, and YD all exhibited faster on and off binding rates compared to LS. At pH 7.0, a slight decrease in the human FcRN binding response was observed in the kifunensin-treated samples. Table 10 below shows the results at pH 6.0 and pH 7.4. D Alternatively, binding data for antibody variants in resonance units (RUs) and standard deviation (SD) are shown. Binding affinity and steady-state RU for treated and untreated antibodies are within the margin of error. No improvement in FcRN binding was observed.

[0347] [Table 12]

[0348] Thermal Stability: As shown in Figure 14, the thermal stability of protein 3-specific WT, LS, YTE, DQ, DW, and YD antibodies, as determined by DSF, was analyzed with and without kifunensin treatment. The solid black curve represents the case without kifunensin treatment, and the dotted curve represents the case with kifunensin treatment. Table 11 shows the change in Tm compared to WT. Kifunensin treatment makes all antibody variants even more unstable by 4–8°C (compared to Fc mutation alone). DW with kifunensin treatment shows a 16°C decrease in thermal stability compared to WT.

[0349] [Table 13]

[0350] FcRn Affinity Chromatography: As shown in Figure 15, FcRn affinity was determined by chromatography against protein 3-specific WT, LS, YTE, DQ, DW, and YD antibodies with and without kifunensin treatment. The solid black curve represents the untreated sample, and the dotted curve represents the treated sample. Table 12 provides the pH of elution for the antibody variants. The kifunensin-treated sample shows a similar pH elution profile to the untreated sample. This data supports the FcRN-binding results, indicating that kifunensin treatment has little effect on the overall binding affinity.

[0351] [Table 14]

[0352] conclusion Regarding the analysis of the N-glycan structure, kifunensin treatment resulted in the acquisition of an antibody specific to protein 3 with an oligomannose content of >97% Man9(GlcNAc)2. A significant improvement in FcγRIIIa binding was also observed, at approximately 1.8 compared to the wild type (WT). There was little effect on FcRN-binding affinity at pH 6.0. At pH 7.4, the binding response to FcRN-binding decreased slightly. The thermal stability of the antibody decreased by 4-8°C, and some Fc mutations decreased by >12°C compared to the wild type. Finally, no effect on the pH elution profile was observed.

[0353] Example 5. FcγRIIIa affinity of stability variants of kifunensin-containing and kifunensin-absorbed antibodies specific to protein 4. introduction Using SPR, experiments were conducted to compare the human FcγRIIIa binding affinity of human IgG1 variants of stability-enhancing mutants and kifunensin-treated protein 4-specific antibodies. The variants tested were S239D(D), S239D / S298A(DA), S239D / I332E(DE), R292C / V302C, T256D / T307Q(DQ), and combinations. M428L / N434S(LS) was included for comparison.

[0354] Materials and methods Fcγ receptor binding analysis: As described above in Example 3, antibody binding to recombinant human FcγRIIIa-V158 was analyzed using surface plasmon resonance (SPR) on a Carterra LSA instrument.

[0355] Results and Conclusions As shown in Figures 16A-B, the human FcγRIIIa binding affinity of various human IgG1 antibodies specific to protein 4, with and without kifunensin treatment, was tested. The antibodies tested were as follows: WT, S239D (D), D+R292C / V302C (SEFL2.2), S239D / S298A (DA), DA+SEFL2.2, S239D / I332E (DE), DE+SEFL2.2, T256D / T307Q (DQ), DQ+D, DQ+D+SEFL2.2, DQ+DA, DQ+DA+SEFL2.2, DQ+DE, DQ+DE+SEFL2.2, DQ+SEFL2.2, LS, and SEFL2.2. Figure 16B shows the selective binding affinity results, including the following antibodies: WT, D, DA, and DE. Figures 17A-E show the results for selected sensorgrams, WT (Figure 17A), DE (Figure 17B), DA treated with kifunensin (Figure 17C), DQ+D+R292C / V302C treated with kifunensin (Figure 17D), and DQ+DA+R292C / V302C treated with kifunensin (Figure 17E). These results indicate that kifunensin treatment increases the affinity for hFcγRIIIa by 1.6 to 7.7 times for all Fc variants tested. DE showed the highest affinity with kifunensin treatment. R292C / V302C, DQ, DQ+R292C / V302C, and LS maintained similar binding affinity to the WT antibody.

[0356] Example 6. Human FcγRIIIa affinity of antibody variants specific to protein 5. introduction To test the enhancement of effector function, the human FcγRIIIa binding affinity of Fc-stable mutants of protein 5-specific antibodies treated with or without kifunensin was measured.

[0357] Materials and methods A defucosylated antibody specific to protein 5 was obtained from Creative Biolabs. To measure Fcγ receptor IIIa binding, the antibody was diluted to 1 μg / ml and captured on a Protein A chip for 30 seconds at 10 μL / min using HBS-EP+ pH 7.4 running buffer (Biacore® T200 instrument). Recombinant human FcγRIIIa-V158 was sequentially diluted 3-fold in HBS-EP+ to concentrations from 111.111 nM to 1.372 nM. Five different concentrations of rhFcγRIIIa were injected onto the captured antibody for 2 minutes, followed by dissociation at a flow rate of 30 μL / min for 3 minutes. After 1 minute of stabilization, the surface was regenerated for 30 seconds using 10 mM glycine-HCl pH 1.5 at 20 μL / min. Sensorgrams were processed and fitted to a 1:1 dynamic coupling model. SDS-PAGE was performed under reducing and non-reducing conditions as described in Example 1 (Figure 18). Glycan analysis was performed using MALDI-TOF mass spectrometry as described in Example 1. Binding affinity to protein 5 was determined using Octet (Forte Bio) with a HIS2 biosensor. Thermal stability was determined by NanoDSF as described in Example 3 above.

[0358] Results and Conclusions Glycan Analysis: As shown in Figures 19A-D, MALDI-TOF glycan analysis of antibodies specific to protein 5 was completed. For the WT antibody, the major glycans were determined to be G0F and G1F (Figure 19A). The WT antibody was also determined to be 95.1% fucosylated and 4.9% defucosylated. For the DE+R292C / V302C antibody, the major glycans were determined to be G0F and G1F (Figure 19B). For the DA+kyfunensin antibody, the major glycans were determined to be Man9(GlcNAc)2 and Man8(GlcNAc)2 (Figure 19C). For the defucosylated antibody, the major glycan was determined to be G0 (Figure 19D).

[0359] Binding affinity to protein 5: Figures 20A-D show sensorgrams of binding analysis of various antibodies to protein 5, including WT (Figure 20A), DA+kyfunensin (Figure 20B), DE+R292C / V302C (disulfide) (Figure 20C), and defucosylated (Figure 20D).

[0360] Human FcγRIIIa affinity of protein 5-specific antibodies: As shown in Figures 21A-D, the binding affinity of various protein 5-specific antibodies to human FcγRIIIa was tested, including WT (Figure 21A), DA+kifunensin (Figure 21B), DE+R292C / V302C (disulfide) (Figure 21C), and defucosylated (Figure 21D). The binding affinity measurements for all variants are provided in Table 13 below. All variants showed higher binding affinity to human FcγRIIIa than WT.

[0361] [Table 15]

[0362] Thermal Stability: Table 14 below shows the thermal stability of various antibodies specific to protein 5 as determined by NanoDSF. Fc mutants DA and DE reduce the thermal stability of the CH2 domain, but not the Fab domain.

[0363] [Table 16]

[0364] Example 7. Modification of a thermally stable Fc domain with adjusted Fc function for effective biopharmaceuticals. introduction 1) CH2 loop residues without perturbing of hydrophobic core residues such as I332, and 2) CH2-CH3 interface residues, were used to generate 285 single-point Fc mutations using saturation mutagenesis. Saturated libraries of these residues were screened for thermal stability and hFcγRIIIa binding. Mutations with minimal impact on thermal stability and enhanced FcγR binding (especially hFcγRIIIa binding) were identified. The expressed variants were analyzed with and without kifunensin.

[0365] Materials and methods Fc fragments were captured on immobilized protein AG, and binding was measured using a flow with multiple concentrations of hFcγRIIIa-V158.

[0366] Sample preparation: PEPP samples were filtered to 0.22 μM in a 96-well filter plate. A280 was measured on a Stunner. Samples were normalized to 200 μg / mL in PBS at pH 7.2 on a Hamilton scale. Samples were diluted to 20 μg / mL in a 96-well plate, and then to 2.5 μg / mL in a 384-well plate using a Benchesmart.

[0367] Procedure at Carterra LSA: Protein AG was immobilized onto the HC30M sensor chip using amine chemistry. Loading antibodies were diluted to 0.25 μg / ml or 5 μg / ml per 10 minutes in multiple prints. Multiple concentrations of hFcγRIIIa-V158 were infused for 2 minutes, followed by 5 minutes of dissociation, which included 12 sequential 2-fold dilutions ranging from 4000 nM to 1.9 nM in HBS-EP+ at pH 7.4. The chip was regenerated for 3 x 30 seconds with 10 mM glycine at pH 1.5, followed by 1 minute of stabilization. Finally, the sensorgram was fitted using a 1:1 dynamic coupling model.

[0368] Thermal stability: The thermal stability of single-point Fc mutations was measured by nanoDSF with and without kifunensin. The top 46 mutants (with and without kifunensin) with higher affinity than the wild type were used to complete buffer exchange to 10 mM histidine pH 6.0.

[0369] Results and Conclusions Figures 22A-F show sensorgrams of human FcγRIIIa binding affinity for the following antibodies: WT (Figure 22A), WT treated with kifunensin (Figure 22B), S298A (Figure 22C), S298A treated with kifunensin (Figure 22D), H268D (Figure 22E), and H268D treated with kifunensin (Figure 22F). Both variants showed higher binding affinity to human FcγRIIIa than the WT variant.

[0370] Figures 23A and 23B show numerical values ​​for human FcγRIIIa binding affinity with and without kifunensin (Figure 23A) and with kifunensin (Figure 23B). Values ​​relative to WT in Figures 23A and 23B are shown in bold. These figures show that for several variants tested, the binding affinity to human FcγRIIIa is increased compared to WT. For example, Figure 23A shows that the KD(M) for WT not treated with kifunensin is 2.5E-07, and for S298C not treated with kifunensin, the KD(M) is 8.6E-08. Figure 23B shows that the binding affinity is further enhanced by kifunensin, with a KD(M) value of 2.4E-08 for S298C treated with kifunensin.

[0371] Figure 24 shows the numerical values ​​for human FcγRIIIa binding affinity with and without kifunensin, as well as the Tm values ​​for the tested Fc variants and WT. This figure shows that for several of the tested variants, the binding affinity to human FcγRIIIa increased compared to WT. For example, for A330A (WT) not treated with kifunensin, the KD(M) was 2.5E-07, and for A330F not treated with kifunensin, the KD(M) was 2.2E-07. The binding affinity was further enhanced by kifunensin, with a KD(M) value of 2.7E-08 for A330F treated with kifunensin, and the Tm (approximately 66.5°C) being within 5°C of WT (69.9°C) cultured in the absence of kifunensin.

[0372] Table 15 below shows the Fc variants selected for optimal hFcγRIIIa binding affinity and thermal stability. The Fc variants shown below (for which Tm data is available) had equivalent or enhanced binding affinity compared to the WT when both were cultured without kifunensin. When both were cultured with kifunensin, the Fc variants had higher binding affinity compared to the WT. In addition, the Tm of these variants was within 5°C of the WT. For example, Table 16 shows that the binding affinity of H268D is enhanced with kifunensin, with a KD(M) of 2.0E-08 compared to a KD(M) of 7.6E-08 without kifunensin. Furthermore, for H268D, the Tm with kifunensin is 64.6°C, which is about 1°C lower than the Tm without kifunensin, which is 65.7°C. Furthermore, the Tm of H268D with kifunensin is within 10°C of the Tm of the WT Fc domain-containing conjugated polypeptide cultured in the absence of kifunensin at 69.9°C.

[0373] [Table 17]

Claims

1. A composition comprising a group of isolated glycosylated polypeptides, each containing an N-glycan-containing Fc domain, wherein the Fc domain is a mutation (i) to (ix) according to EU numbering: (i) Aspartic acid (D) at amino acid position 239, (ii) Aspartic acid (D) at amino acid position 267, (iii) Aspartic acid (D) or glutamic acid (E) at amino acid position 268, (iv) Alanine (A) or cysteine ​​(C) at amino acid position 298, (v) At amino acid position 314, isoleucine (I), methionine (M), glutamine (Q), or tryptophan (W), (vi) Phenylalanine (F) or methionine (M) at amino acid position 330, (vii) Glutamic acid (E) at amino acid position 332, (viiii) At amino acid position 339, aspartic acid (D), isoleucine (I), proline (P), or threonine (T), (ix) Phenylalanine (F) or tryptophan (W) at amino acid position 373 It further includes at least one of the following: Man, in molar ratio, of at least 50% of total N-glycans ５－９ (GlcNAc) ２ A composition containing N-glycan.

2. Man ８ and Man ９ together, Man ５－９ (GlcNAc) ２ The composition according to claim 1, wherein the N-glycan is the main species.

3. Man, in molar ratio, 70%, 75%, 80%, 85%, 90%, or more than 95% of total N-glycans ９ (GlcNAc) ２ The composition according to claim 1, comprising an N-glycan.

4. Man at a molar ratio of at least 97% with respect to total N-glycan ９ (GlcNAc) ２ The composition according to claim 1, comprising N-glycan.

5. The composition according to any one of claims 1 to 4, wherein at least 80% of the N-glycans in the composition are defucosylated in molar ratio with respect to the total N-glycans.

6. The composition according to any one of claims 1 to 5, wherein the conjugated polypeptide is produced by culturing cells expressing the conjugated polypeptide in the presence of a mannosidase inhibitor.

7. The composition according to claim 6, wherein the mannosidase inhibitor is kifunensin.

8. The composition according to claim 7, wherein the concentration of kifunensin is about 60 ng / mL to about 2500 ng / mL.

9. The composition according to claim 8, wherein the concentration of kifunensin is approximately 2000 ng / mL.

10. Man ５－９ (GlcNAc) ２ A bound polypeptide containing an N-glycan is Man ５－９ (GlcNAc) ２ The composition according to any one of claims 1 to 9, having improved affinity for binding to the Fcγ receptor compared to a reference polypeptide that is otherwise identical but does not contain N-glycans.

11. The composition according to claim 10, wherein the Fcγ receptor is human FcγRIIIa.

12. Man ５－９ (GlcNAc) ２ The composition according to claim 11, wherein the bound polypeptide containing the N-glycan has an increased affinity for binding to human FcγRIIIIa that is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than that of the reference polypeptide.

13. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 1 to 12, wherein the antibody-dependent cytotoxic (ADCC) activity of the bound polypeptide containing N-glycan is improved compared to the reference polypeptide.

14. The composition according to claim 13, wherein the ADCC activity of the bound polypeptide is at least 1, 2, 3, 4, or 5 times higher than that of the reference polypeptide.

15. The composition according to any one of claims 10 to 14, wherein the reference polypeptide has a wild-type (WT) Fc domain.

16. The composition according to any one of claims 10 to 15, wherein the reference polypeptide is not prepared by culturing cells expressing the reference polypeptide in the presence of kifunensin.

17. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239.

18. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains glutamic acid (E) at amino acid position 332.

19. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

20. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 267.

21. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 268.

22. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains glutamic acid (E) at amino acid position 268.

23. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains alanine (A) at amino acid position 298.

24. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

25. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains cysteine ​​(C) at amino acid position 298.

26. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains isoleucine (I) at amino acid position 314.

27. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains methionine (M) at amino acid position 314.

28. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains glutamine (Q) at amino acid position 314.

29. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains tryptophan (W) at amino acid position 314.

30. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains phenylalanine (F) at amino acid position 330.

31. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains methionine (M) at amino acid position 330.

32. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 339.

33. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains isoleucine (I) at amino acid position 339.

34. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains proline (P) at amino acid position 339.

35. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains threonine (T) at amino acid position 339.

36. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains phenylalanine (F) at amino acid position 373.

37. The composition according to any one of claims 1 to 16, wherein the Fc domain of the conjugated polypeptide contains tryptophan (W) at amino acid position 373.

38. A composition comprising a group of isolated glycosylated polypeptides, each containing an Fc domain containing an N-glycan, The Fc domain further includes a mutation that increases binding to the Fc receptor, Man, in molar ratio, of at least 50% of total N-glycans ５－９ (GlcNAc) ２ Contains N-glycans, A composition wherein the Fc domain further comprises cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302, according to EU numbering.

39. Man ８ and Man ９ - together, Man ５－９ (GlcNAc) ２ The composition according to claim 38, wherein the main species of N-glycan is...

40. Man in molar ratios of 70%, 75%, 80%, 85%, 90%, or more than 95% relative to total N-glycans ９ (GlcNAc) ２ The composition according to claim 38, comprising an N-glycan.

41. Man, in molar ratio, of at least 97% of total N-glycans ９ (GlcNAc) ２ The composition according to claim 38, comprising an N-glycan.

42. A composition according to any one of claims 38 to 41, wherein at least 80% of the N-glycans in the composition are defucosylated in molar ratio to the total N-glycans.

43. The composition according to any one of claims 38 to 42, wherein the conjugated polypeptide is produced by culturing cells expressing the conjugated polypeptide in the presence of a mannosidase inhibitor.

44. The composition according to claim 43, wherein the mannosidase inhibitor is kifunensin.

45. The composition according to claim 44, wherein the concentration of kifunensin is about 60 ng / mL to about 2500 ng / mL.

46. The composition according to claim 45, wherein the concentration of kifunensin is approximately 2000 ng / mL.

47. Man ５－９ (GlcNAc) ２ The linked polypeptide containing N-glycan is Man ５－９ (GlcNAc) ２ The composition according to any one of claims 38 to 46, having improved affinity for binding to the Fcγ receptor compared to a reference polypeptide that is otherwise identical but does not contain N-glycans.

48. The composition according to claim 47, wherein the receptor is human FcγRIIIa.

49. Man ５－９ (GlcNAc) ２ The composition according to claim 47 or 48, wherein the bound polypeptide containing the N-glycan has an improved affinity for binding to human FcγRIIIIa that is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than that of the reference bound polypeptide.

50. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 47 to 49, wherein the ADCC activity of the bound polypeptide containing the N-glycan is improved compared to the reference polypeptide.

51. Man ５－９ (GlcNAc) ２ The composition according to claim 50, wherein the ADCC activity of the bound polypeptide containing the N-glycan is at least 1, 2, 3, 4, or 5 times higher than that of the reference bound polypeptide.

52. The composition according to any one of claims 47 to 51, wherein the reference-bound polypeptide has a wild-type (WT) Fc domain.

53. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239.

54. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains glutamic acid (E) at amino acid position 332.

55. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

56. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 267.

57. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 268.

58. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains glutamic acid (E) at amino acid position 268.

59. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains alanine (A) at amino acid position 298.

60. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

61. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains cysteine ​​(C) at amino acid position 298.

62. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains isoleucine (I) at amino acid position 314.

63. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains methionine (M) at amino acid position 314.

64. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains glutamine (Q) at amino acid position 314.

65. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains tryptophan (W) at amino acid position 314.

66. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains phenylalanine (F) at amino acid position 330.

67. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains methionine (M) at amino acid position 330.

68. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 339.

69. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains isoleucine (I) at amino acid position 339.

70. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains proline (P) at amino acid position 339.

71. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains threonine (T) at amino acid position 339.

72. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains phenylalanine (F) at amino acid position 373.

73. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide contains tryptophan (W) at amino acid position 373.

74. The composition according to any one of claims 38 to 52, wherein the Fc domain of the conjugated polypeptide further comprises aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307.

75. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 1 to 74, wherein the bound polypeptide containing the N-glycan has a melting temperature (Tm) of 10°C or less than that of a reference polypeptide having a WT Fc domain.

76. The reference polypeptide having the WT Fc domain is expressed by cells cultured in the absence of kifunensin, Man ５－９ (GlcNAc) ２ The composition according to claim 75, wherein the binding polypeptide containing N-glycan is expressed by cells cultured in the presence of kifunensin.

77. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 1 to 74, wherein the bound polypeptide containing the N-glycan has a Tm of 5°C or less of a reference polypeptide having a WT Fc domain.

78. The reference polypeptide having a WT Fc domain is expressed by cells cultured in the presence of kifunensin, and Man ５－９ (GlcNAc) ２ The composition according to claim 77, wherein the binding polypeptide containing N-glycan is expressed by cells cultured in the presence of kifunensin.

79. A composition comprising a group of isolated glycosylated polypeptides, each containing an Fc domain containing an N-glycan, The Fc domain further includes a mutation that increases binding to the Fc receptor, Man, in molar ratio, of at least 50% of total N-glycans ５－９ (GlcNAc) ２ Contains N-glycans, The Fc domain further contains aspartic acid (D) at amino acid position 256 and glutamine (Q) at amino acid position 307, according to EU numbering. composition.

80. Man ８ and Man ９ - together, Man ５－９ (GlcNAc) ２ The composition according to claim 79, wherein the main species of N-glycan is...

81. Man in molar ratios of 70%, 75%, 80%, 85%, 90%, or more than 95% relative to total N-glycans ９ (GlcNAc) ２ The composition according to claim 79, comprising an N-glycan.

82. Man, in molar ratio, of at least 97% of total N-glycans ９ (GlcNAc) ２ The composition according to claim 79, comprising an N-glycan.

83. The composition according to any one of claims 79 to 82, wherein at least 80% of the N-glycans in the composition are defucosylated in molar ratio with respect to the total N-glycans in the composition.

84. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 79 to 83, wherein the N-glycan-containing conjugated polypeptide is produced by culturing cells expressing the conjugated polypeptide in the presence of a mannosidase inhibitor.

85. Man ５－９ (GlcNAc) ２ The linked polypeptide containing N-glycan is Man ５－９ (GlcNAc) ２ The composition according to any one of claims 79 to 84, having improved affinity for binding to the Fcγ receptor compared to a reference binding polypeptide that does not contain an N-glycan but is otherwise identical.

86. The composition according to claim 85, wherein the Fc receptor is human FcγRIIIa.

87. Compared to the aforementioned reference-bonded polypeptide, Man ５－９ (GlcNAc) ２ The composition according to claim 86, wherein the affinity of the isolated bound polypeptide containing N-glycan to human FcγRIIIIa is at least 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher.

88. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 85 to 87, wherein the ADCC activity of the bound polypeptide containing the N-glycan is improved compared to the reference polypeptide.

89. Man ５－９ (GlcNAc) ２ The composition according to claim 88, wherein the ADCC activity of the bound polypeptide containing the N-glycan is at least 1, 2, 3, 4, or 5 times higher than that of the reference polypeptide.

90. The composition according to any one of claims 85 to 89, wherein the reference polypeptide has a wild-type (WT) Fc domain.

91. The composition according to any one of claims 84 to 90, wherein the mannosidase inhibitor is kifunensin.

92. The composition according to claim 91, wherein the concentration of kifunensin is about 60 ng / mL to about 2500 ng / mL.

93. The composition according to claim 92, wherein the concentration of kifunensin is approximately 2000 ng / mL.

94. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239.

95. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains glutamic acid (E) at amino acid position 332.

96. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239 and glutamic acid (E) at amino acid position 332.

97. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 267.

98. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 268.

99. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains glutamic acid (E) at amino acid position 268.

100. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains cysteine ​​(C) at amino acid position 298.

101. The composition according to any one of claims 79 to 93, wherein the Fc of the conjugated polypeptide domain contains alanine (A) at amino acid position 298.

102. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 239 and alanine (A) at amino acid position 298.

103. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains isoleucine (I) at amino acid position 314.

104. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains methionine (M) at amino acid position 314.

105. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains glutamine (Q) at amino acid position 314.

106. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains tryptophan (W) at amino acid position 314.

107. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains phenylalanine (F) at amino acid position 330.

108. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains methionine (M) at amino acid position 330.

109. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains aspartic acid (D) at amino acid position 339.

110. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains isoleucine (I) at amino acid position 339.

111. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains proline (P) at amino acid position 339.

112. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains threonine (T) at amino acid position 339.

113. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains phenylalanine (F) at amino acid position 373.

114. The composition according to any one of claims 79 to 93, wherein the Fc domain of the conjugated polypeptide contains tryptophan (W) at amino acid position 373.

115. Man ５－９ (GlcNAc) ２ The composition according to any one of claims 79 to 114, wherein the binding polypeptide containing N-glycan has a higher binding affinity to the neonatal Fc receptor (FcRn) compared to a binding polypeptide having a WT Fc domain.

116. The composition according to any one of claims 79 to 115, wherein the Fc domain of the conjugated polypeptide further comprises cysteine ​​(C) at amino acid position 292 and cysteine ​​(C) at amino acid position 302, according to EU numbering.

117. The composition according to any one of claims 1 to 116, wherein one or more of the conjugated polypeptides are antibodies.

118. The composition according to claim 117, wherein the antibody is a monoclonal antibody.

119. The composition according to claim 117 or 118, wherein the antibody is a chimeric, humanized, or human antibody.

120. The composition according to claim 117 or 118, wherein the antibody is a multispecific antibody.

121. The composition according to claim 120, wherein the multispecific antibody is selected from the group consisting of a DVD-Ig, CODV-based method, and is optionally CODV-Ig, CrossMab, CrossMab-Fab, and tandem Fab.

122. The composition according to claim 120 or 121, wherein the multispecific antibody is a T cell engager.

123. The composition according to claim 120 or 121, wherein the multispecific antibody is an NK cell engager.

124. The composition according to any one of claims 1 to 123, wherein one or more of the conjugated polypeptides comprises at least one antigen-binding fragment selected from the group consisting of variable fragment (Fv), Fab, Fab', (Fab')2, minibody, diabody, triabody, tetrabody, tandem-scFv, tandem-tri-scFv, and immunoglobulin single variable domain (ISV).

125. The composition according to any one of claims 1 to 123, wherein one or more of the conjugated polypeptides comprises an immunoglobulin monovariate domain (ISV).

126. The composition according to any one of claims 1 to 123, wherein one or more of the bound polypeptides contain VHH.

127. The composition according to any one of claims 1 to 126, wherein one or more of the conjugated polypeptides include a single-chain variable region (ScFv) sequence.

128. The composition according to any one of claims 1 to 127, wherein one or more of the conjugated polypeptides contain an IgG Fc domain.

129. The composition according to claim 128, wherein the Fc domain is an IgG1 domain.

130. The composition according to claim 128 or 129, wherein the Fc domain is a human Fc domain.

131. The composition according to any one of claims 1 to 130, wherein one or more of the conjugated polypeptides comprises a lysosome-targeted chimera (LYTAC).

132. The composition according to any one of claims 38 to 131, wherein the Fc receptor comprises a human FcγRIIIa receptor.

133. A pharmaceutical composition, as described in any one of claims 1 to 132.

134. A method for preparing the composition according to any one of claims 1 to 133, comprising culturing cells expressing the binding polypeptide in the presence of kifunensin.

135. The method according to claim 134, wherein the concentration of kifunensin is approximately 60 ng / mL to approximately 2500 ng / mL.

136. The method according to claim 135, wherein the concentration of kifunensin is approximately 2000 ng / mL.

137. An isolated nucleic acid molecule comprising a nucleic acid capable of expressing one or more conjugated polypeptides of the composition according to any one of claims 1 to 136.

138. A vector comprising the isolated nucleic acid molecule described in claim 137.

139. The vector according to claim 138, which is an expression vector.

140. A host cell comprising the vector according to claim 138 or 139.

141. A method for treating a disease or disorder in a subject requiring treatment of the disease or disorder, comprising administering an effective amount of the pharmaceutical composition described in claim 133 to the subject.

142. The method according to claim 141, wherein the disease or disorder is cancer.

143. The method according to claim 141, wherein the disease or disorder is an inflammatory disease.

144. The method according to claim 141, wherein the disease or disorder is an autoimmune disease.

Images