Polypeptides with controlled association

Abstract

The present disclosure relates to polypeptides for which association between polypeptides is controlled, and, in one embodiment, polypeptides for which association between Fc regions is controlled; a method for producing a polypeptide for which the association between the Fc region (in one embodiment, CH3 region) is controlled; a method for controlling association between Fc regions; compositions comprising such Fc regions; compositions comprising nucleic acids encoding such polypeptides, etc.

Description

Technical Field

[0001] The present disclosure relates to: polypeptides for which the association between polypeptides is controlled, for example, polypeptides for which the association between heavy chains or between Fc regions is controlled; methods for producing polypeptides for which the association between Fc regions (in one embodiment, CH3 regions) is controlled; methods for controlling the association between heavy chains or between Fc regions; compositions comprising polypeptides having an Fc region for which the association is controlled; and compositions containing nucleic acids encoding such polypeptides, etc. Background Art

[0002] Methods for co-expressing multiple antibodies in a single cell are important for efficiently producing drug mixtures or generating drug mixtures in living organisms using mRNA, DNA plasmids, gene therapy, etc. However, when antibody A composed of HA (heavy chain) and LA (light chain) and antibody B composed of HB (heavy chain) and LB (light chain) are co-expressed, in addition to the desired antibodies A and B, eight antibody species are formed in a random manner. This results in the production of a heterogeneous drug mixture or heterogeneous efficacy. Therefore, a technique is needed to suppress the expression of antibodies other than antibodies A and B and to allow the effective expression of antibodies A and B.

[0003] As a method for solving this problem, a method utilizing the difference in heavy chain association specificity between isotypes such as IgG and IgA, and a method utilizing the difference in heavy chain association specificity between subclasses IgG1 and IgG3 have been reported (PTL 1). However, since antibodies commonly used for therapeutic purposes are IgG, a technique applicable to all IgG subclasses and capable of even co-expressing antibodies of the same IgG subclass is needed. To solve this problem, methods for promoting the association of heavy chains of the same type have been reported, in which amino acid modifications are introduced into the heavy chain CH3 region to suppress the expression of IgG in which HA and HB associate (PTLs 2-4). These are all methods for controlling the association of heavy chains of the same type using the attraction and repulsion of charges, but in fact, only limited modifications and their combinations have been tested. Therefore, they cannot achieve the effective co-expression of three or more antibodies. In addition, these previous studies aimed to simultaneously produce multiple antibodies, rather than expressing antibodies in living organisms. In the case of expressing therapeutic antibodies in living organisms, the lack of association with endogenous IgG would reduce side effects. However, there has been no report of a technique for co-expressing multiple antibodies that do not associate with endogenous IgG.

[0004] [Citation List]

[0005] [Patent Document]

[0006] [PTL 1]WO 2004 / 009618

[0007] [PTL 2]WO 2013 / 157953

[0008] [PTL 3]WO 2014 / 015804

[0009] [PTL 4]WO 2017 / 205014 SUMMARY OF THE INVENTION

[0010] [TECHNICAL PROBLEM]

[0011] In view of the above, the present invention has been achieved. The object of the present invention is to provide polypeptides for which the association between polypeptides is controlled, methods for producing polypeptides for which the association is controlled, methods for controlling the association of polypeptides, nucleic acids encoding polypeptides for which the association is controlled, compositions containing such nucleic acids, and the like.

[0012] In one embodiment, a further object of the present invention is to provide polypeptides for which the association between Fc regions is controlled, methods for producing polypeptides for which the association between Fc regions is controlled, expression of polypeptides for which the association between Fc regions is controlled in a living organism, and methods for controlling the association between Fc regions. Further, in one embodiment, the object of the present disclosure is to provide antibodies for which the association in the interface between CH3 regions is controlled, and methods for co-expressing two or more antibodies in an effective manner.

[0013] [SOLUTION TO THE PROBLEM]

[0014] The present inventors selected the heavy chain constant region CH3 as the Fc intervening region that undergoes association control and conducted a special study on the control of the association between Fc regions of the same type. Thus, the inventors found that by using steric hindrance caused by simultaneously modifying amino acid residues in the CH3 interface with bulky and small amino acids, or using artificially introduced disulfide bonding, or by introducing a new combination of modifications to utilize the attractive and repulsive forces of charges to inhibit the association between different types of Fc regions, multimers of the same type of Fc region can be effectively formed.

[0015] Therefore, based on the findings of the present inventors, the association between heavy chains of the same type can be controlled. Further, the present disclosure is not only applicable to controlling the association between heavy chains of the same type, but also capable of controlling the association between any Fc-containing polypeptides.

[0016] The present inventors further confirmed that the antibodies for which the association between Fc regions is controlled according to the present disclosure retain the function of Fc.

[0017] As described above, the present inventors have successfully developed polypeptides for which the association between Fc regions is controlled, thus completing the present invention.

[0018] The present disclosure relates to polypeptides for which the association between polypeptides is controlled, methods for producing polypeptides for which the association is controlled, methods for controlling the association of polypeptides, nucleic acids encoding polypeptides for which the association is controlled, compositions containing such nucleic acids, and the like.

[0019] In one embodiment, the present disclosure relates to polypeptides for which the association between Fc regions is controlled, methods for producing polypeptides for which the association between Fc regions is controlled, the expression of polypeptides for which the association between Fc regions is controlled in a living organism, and methods for controlling the association between Fc regions, and more specifically relates to the following:

[0020] [1] A nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises an Fc region into which a modification has been introduced, and wherein due to the modification introduced into the Fc region, the first polypeptide associates with a first polypeptide having the modification via the Fc region more readily than with a first polypeptide comprising an Fc region into which the modification has not been introduced.

[0021] [1-1] The foregoing nucleic acid, wherein the first polypeptide associates less readily with a polypeptide into which the modification has not been introduced than with a polypeptide having the modification.

[0022] [1-1-1] The foregoing nucleic acid, wherein the first polypeptide is less likely to form a heterodimer with another polypeptide into which the modification has not been introduced.

[0023] [1-1-2] The foregoing nucleic acid, which is less likely to form a heterodimer than a homodimer.

[0024] [1-1-3] The foregoing nucleic acid, wherein the first polypeptide does not associate or associates less readily with endogenous IgG or an antibody fragment thereof containing an Fc region.

[0025] [1-2] The foregoing nucleic acid, wherein the first polypeptides having the modification associate with each other.

[0026] [1-2-1] The foregoing nucleic acid, wherein the first polypeptides having the modification form a homopolymer.

[0027] [1-2-2] The foregoing nucleic acid, which is more likely to form a homodimer than a heterodimer.

[0028] [1-2-3] The foregoing nucleic acid, wherein the first polypeptide exhibits a stronger homodimerization promoting ability than a control (e.g., wild-type IgG).

[0029] [1-2-4]The foregoing nucleic acid, wherein a homodimer of a polypeptide other than the first polypeptide (for example, a second polypeptide) is further formed.

[0030] [1-2-5]The foregoing nucleic acid, wherein a homodimer of one or two or more types of polypeptides is formed.

[0031] [1-2-6]The foregoing nucleic acid, wherein the modifications in the two or more types of polypeptides are different from each other. [1-2-7]The foregoing nucleic acid, wherein the combination of the different modifications is the combination shown in Table 7. [1-3]The foregoing nucleic acid, wherein the first polypeptide having the modification has an increased ability to form an associated multimer with a polypeptide having the modification as compared with the polypeptide into which the modification has not been introduced.

[0032] [1-4]The foregoing nucleic acid, wherein the nucleic acid is RNA, DNA, or a vector or plasmid carrying the nucleic acid.

[0033] [1-5]The foregoing nucleic acid, wherein the first polypeptide is a polypeptide whose association is controlled.

[0034] [1-5-1]The foregoing nucleic acid, wherein the association is the association between Fc regions.

[0035] [1-6]The foregoing nucleic acid, wherein the first polypeptide has a CH3.

[0036] [1-6-1]The foregoing nucleic acid, wherein the association is the association at the interface between CH3 regions.

[0037] [1-6-2]The foregoing nucleic acid, wherein the modification is a modification in CH3.

[0038] [2]The foregoing nucleic acid, wherein due to at least one of the following effects caused by the introduced modification, the association of the first polypeptide via the Fc region with the first polypeptide having the modification is easier than the association with the first polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity (also referred to as "steric hindrance"), (2) disulfide bonding (also referred to as "disulfide bond"), and (3) electrostatic charge (also referred to as "charge").

[0039] [2-1]The foregoing nucleic acid, wherein the steric complementarity is caused by the presence of a knob and a hole in the Fc region.

[0040] [2-1-1]The foregoing nucleic acid, wherein the modification is a modification for introducing a knob and a hole into the Fc region.

[0041] [2-1-2]The foregoing nucleic acid, wherein the pestle and the mortar in the Fc region induce the formation of a homodimer of the first polypeptide.

[0042] [2-1-3]The foregoing nucleic acid, wherein both the pestle and the mortar are introduced into the first polypeptide.

[0043] [2-1-4]The foregoing nucleic acid, which associates more readily with the polypeptide having the modification than with the polypeptide into which the modification has not been introduced, at least due to a combination of (1) steric complementarity and (3) the effect of electrostatic charge.

[0044] [2-2]The foregoing nucleic acid, wherein the disulfide bonding is formed by replacing one or more amino acids in the Fc region with cysteine (C).

[0045] [2-2-1]The foregoing nucleic acid, wherein the modification is a modification that introduces one or more cysteines into the Fc region.

[0046] [2-2-2]The foregoing nucleic acid, wherein the modification is replacing one or more amino acids in the Fc region with cysteine.

[0047] [2-2-3]The foregoing nucleic acid, wherein the disulfide bonding is a bond between the cysteines modified (introduced) in the Fc region.

[0048] [2-2-4]The foregoing nucleic acid, which associates more readily with the polypeptide having the modification than with the polypeptide into which the modification has not been introduced, at least due to a combination of (2) disulfide bonding and (3) the effect of electrostatic charge.

[0049] [2-3]The foregoing nucleic acid, wherein the effect of the electrostatic charge is generated by one or more charged amino acids modified (introduced) in the Fc region.

[0050] [2-3-1]The foregoing nucleic acid, wherein the introduced charged amino acids are positively charged amino acids and / or negatively charged amino acids.

[0051] [2-3-2]The foregoing nucleic acid, wherein at least two amino acids in the Fc region are replaced with a positively charged amino acid and a negatively charged amino acid.

[0052] [2-3-3]The foregoing nucleic acid, wherein the positively charged amino acids are selected from lysine (K), arginine (R), and histidine (H), and / or the negatively charged amino acids are selected from aspartic acid (D) and glutamic acid (E).

[0053] [2-4]The foregoing nucleic acid, wherein the effect is a combination of more than one effect (two or three effects) selected from (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0054] [3] A composition comprising:

[0055] The nucleic acid according to [1]; and

[0056] A nucleic acid encoding a second polypeptide,

[0057] wherein the second polypeptide comprises an Fc region into which a modification has been introduced, and wherein due to the introduction of the modification into the Fc region, the second polypeptide associates via the Fc region more readily with a second polypeptide having the modification than with a second polypeptide comprising an Fc region into which the modification has not been introduced.

[0058] [3-1] The aforementioned composition, wherein the modification in the second polypeptide is different from the modification in the first polypeptide.

[0059] [3-2] The aforementioned composition, wherein the function of the second polypeptide is different from the function of the first polypeptide.

[0060] [3-3] The aforementioned composition, wherein the amino acid sequence of the second polypeptide before modification is different from the amino acid sequence of the first polypeptide before modification.

[0061] [3-4] The aforementioned composition, which further comprises a nucleic acid encoding a polypeptide different from the first polypeptide or the second polypeptide (in one embodiment, a third and / or fourth polypeptide).

[0062] [4] A composition comprising:

[0063] The nucleic acid according to [1];

[0064] A nucleic acid encoding a second polypeptide; and

[0065] A nucleic acid encoding a third polypeptide,

[0066] wherein the second polypeptide associates more readily with the third polypeptide than with the second polypeptide.

[0067] [5] A composition comprising:

[0068] The nucleic acid according to [2]; and

[0069] A nucleic acid encoding a second polypeptide, wherein the second polypeptide does not have the modification that the first polypeptide has.

[0070] [6] The aforementioned composition, wherein the modification introduced into the first polypeptide is different from the modification introduced into the second polypeptide.

[0071] [7] The foregoing composition, wherein due to at least one of the following effects resulting from the introduced modification, the second polypeptide associates more readily via the Fc region with the second polypeptide having the modification than with the second polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0072] [7-1] The foregoing composition, wherein the modification in the second polypeptide is different from the modification in the first polypeptide.

[0073] [7-2] The foregoing composition, wherein the effect in the second polypeptide is different from the effect in the first polypeptide.

[0074] [8] The foregoing nucleic acid or composition, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 2.

[0075] [9] The foregoing nucleic acid or composition, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 4.

[0076]

[10] The foregoing nucleic acid or composition, wherein the modification introduced into the first polypeptide and the modification introduced into the second polypeptide are at least one of the combinations of the modifications listed in Table 6.

[0077]

[11] The foregoing nucleic acid or composition, wherein the polypeptide is an antibody.

[0078]

[12] The foregoing nucleic acid or composition, wherein the Fc region is the Fc region of IgG.

[0079]

[13] The foregoing nucleic acid or composition, wherein the polypeptide into which the modification has been introduced maintains the function of the polypeptide before the modification.

[0080]

[14] The foregoing nucleic acid or composition, wherein the Fc region into which the modification has been introduced maintains the function of the Fc of IgG.

[0081]

[15] The foregoing nucleic acid or composition, wherein the Fc region is derived from any one of IgG1, 2, 3, and 4.

[0082]

[16] A host cell into which the foregoing nucleic acid or composition has been introduced.

[0083]

[17] A polypeptide expressed by the foregoing nucleic acid.

[0084]

[18] A composition comprising a nucleic acid encoding a polypeptide,

[0085] wherein the polypeptide comprises an Fc region into which a modification has been introduced,

[0086] Wherein the modification is at least one of the modifications listed in Table 2.

[0087]

[19] A polypeptide comprising an Fc region into which a modification has been introduced, wherein due to at least one of the following effects resulting from the introduced modification, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0088] [19-1] The aforementioned polypeptide, wherein the modification is a modification in CH3.

[0089]

[20] A method for obtaining a polypeptide with controlled association, the method comprising:

[0090] The step of obtaining a nucleic acid encoding the polypeptide; and

[0091] The step of expressing the nucleic acid,

[0092] wherein the polypeptide comprises an Fc region, and

[0093] wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced.

[0094] [20-1] The aforementioned method, wherein the expression is in vivo, ex vivo or in vitro expression.

[0095]

[21] A method for controlling the association of a homomer of a polypeptide, the method comprising:

[0096] The step of obtaining a nucleic acid encoding the polypeptide; and

[0097] The step of expressing the nucleic acid,

[0098] wherein the polypeptide comprises an Fc region, and

[0099] wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced.

[0100]

[22] A method for promoting the expression of a homomer of a polypeptide, the method comprising:

[0101] The step of obtaining a nucleic acid encoding the polypeptide; and

[0102] The step of expressing the nucleic acid,

[0103] wherein the polypeptide comprises an Fc region, and

[0104] Among them, due to the introduction of the modification in the Fc region, the association of the polypeptide via the Fc region with a polypeptide having the modification is easier than the association with a polypeptide comprising an Fc region into which the modification has not been introduced.

[0105] In addition, the present disclosure relates to the following. The technical features listed in the above [1]-

[22] are also incorporated into the following inventions as appropriate.

[0106]

[101] A polypeptide for which the association is controlled, wherein the polypeptide comprises an Fc region into which a modification has been introduced, and wherein due to the introduction of the modification in the Fc region, the association of the polypeptide via the Fc region with a polypeptide having the modification is easier than the association with a polypeptide into which the modification has not been introduced.

[0107] [101-1] The aforementioned polypeptide, wherein the association of the polypeptide with a polypeptide into which the modification has not been introduced is less easy than the association with a polypeptide having the modification.

[0108] [101-1-1] The aforementioned polypeptide, wherein the polypeptide is less likely to form a heterodimer with another polypeptide that does not have the modification.

[0109] [101-1-2] The aforementioned polypeptide, which is less likely to form a heterodimer compared to a homodimer.

[0110] [101-1-3] The aforementioned polypeptide, wherein the polypeptide does not associate or is less likely to associate with endogenous IgG or an antibody fragment containing an Fc region thereof.

[0111] [101-2] The aforementioned polypeptide, wherein the polypeptides having the modification associate with each other.

[0112] [101-2-1] The aforementioned polypeptide, wherein the polypeptides having the modification form a homopolymer.

[0113] [101-2-2] The aforementioned polypeptide, which is more likely to form a homodimer compared to a heterodimer.

[0114] [101-2-3] The aforementioned polypeptide, which exhibits a stronger ability to promote homodimerization than a control (e.g., wild-type IgG).

[0115] [101-3] The aforementioned polypeptide, wherein the polypeptide having the modification has an increased ability to form an associated multimer with a polypeptide having the modification compared to a polypeptide that does not have the modification.

[0116] [101-3-1] The aforementioned polypeptide, wherein the association is an association between Fc regions.

[0117] [101-4]The foregoing polypeptide, wherein the polypeptide has a CH3.

[0118] [101-4-1]The foregoing polypeptide, wherein the association is an association at the interface between CH3 regions.

[0119] [101-4-2]The foregoing polypeptide, wherein the modification is a modification in CH3.

[0120]

[102] The foregoing polypeptide, wherein due to at least one of the following effects resulting from the introduced modification, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide in which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0121] [102-1]The foregoing polypeptide, wherein the steric complementarity is generated by the presence of a knob and a hole in the Fc region.

[0122] [102-1-1]The foregoing polypeptide, wherein the modification is a modification that introduces a knob and a hole into the Fc region.

[0123] [102-1-2]The foregoing polypeptide, wherein the knob and the hole in the Fc region induce the formation of a homodimer of the first polypeptide.

[0124] [102-1-3]The foregoing polypeptide, wherein both the knob and the hole are introduced into the first polypeptide.

[0125] [102-2]The foregoing polypeptide, wherein the disulfide bonding is formed by replacing one or more amino acids in the Fc region with cysteine (C).

[0126] [102-2-1]The foregoing polypeptide, wherein the modification is a modification that introduces one or more cysteines into the Fc region.

[0127] [102-2-2]The foregoing polypeptide, wherein the modification is replacing one or more amino acids in the Fc region with cysteine.

[0128] [102-2-3]The foregoing polypeptide, wherein the disulfide bonding is a bond between cysteines modified (introduced) in the Fc region.

[0129] [102-2-4]The foregoing polypeptide associates more readily with a polypeptide having the modification than with a polypeptide in which the modification has not been introduced, at least due to the combined effects of (2) disulfide bonding and (3) electrostatic charge.

[0130] [102 - 3]The foregoing polypeptide, wherein the effect of the electrostatic charge is generated by one or more charged amino acids modified (introduced) in the Fc region.

[0131] [102 - 3 - 1]The foregoing polypeptide, wherein the introduced charged amino acids are positively charged amino acids and / or negatively charged amino acids.

[0132] [102 - 3 - 2]The foregoing polypeptide, wherein at least two amino acids in the Fc region are replaced by a positively charged amino acid and a negatively charged amino acid.

[0133] [102 - 3 - 3]The foregoing polypeptide, wherein the positively charged amino acids are selected from lysine (K), arginine (R), and histidine (H), and / or the negatively charged amino acids are selected from aspartic acid (D) and glutamic acid (E).

[0134] [102 - 4]The foregoing polypeptide, wherein the effect is more than one effect (a combination of two or three effects) selected from (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0135]

[103] The foregoing polypeptide, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 2.

[0136]

[104] The foregoing polypeptide, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 4.

[0137]

[105] The foregoing polypeptide, wherein the steric complementarity includes a mortise and tenon.

[0138]

[106] The foregoing polypeptide, wherein the polypeptide is an antibody.

[0139]

[107] The foregoing polypeptide, wherein the Fc region is the Fc region of IgG.

[0140]

[108] The foregoing polypeptide, wherein the Fc region is derived from any one of IgG1, 2, 3, and 4.

[0141]

[201] A method for producing a polypeptide with controlled association between Fc regions, the method comprising:

[0142] (a) Obtaining a nucleic acid encoding a polypeptide in which a modification has been introduced into the Fc region, wherein due to at least one of the following effects, the association of the polypeptide with a polypeptide having the modification is easier than the association with a polypeptide in which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge;

[0143] (b) Introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and

[0144] (c) Recovering the polypeptide from a culture of the host cell.

[0145] [201-1] The foregoing method, further comprising introducing a modification into the Fc region such that (1) steric complementarity, (2) disulfide bonding, or (3) the effect of electrostatic charge occurs in the polypeptide.

[0146] [202-2] The foregoing method, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 2 or Table 4.

[0147]

[202] A method for producing a polypeptide with controlled association between Fc regions, the method comprising:

[0148] (a) Modifying a nucleic acid encoding a polypeptide comprising an Fc region such that the polypeptide associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge;

[0149] (b) Introducing the modified nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and

[0150] (c) Recovering the polypeptide from a culture of the host cell.

[0151]

[203] A method for controlling the association between polypeptides comprising an Fc region, the method comprising modifying the polypeptide such that the polypeptide associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0152]

[204] A method for generating a polypeptide with controlled association between polypeptides comprising an Fc region in a living organism, the method comprising:

[0153] (a) Preparing a nucleic acid encoding a polypeptide comprising an Fc region such that the polypeptide comprising the Fc region is modified into a polypeptide that associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge; and

[0154] (b) Introducing the nucleic acid of step (a) into a living organism.

[0155] [204-1]The foregoing method, which includes introducing a modification into the Fc region such that (1) steric complementarity, (2) disulfide bonding, or (3) the action of electrostatic charge occurs in the polypeptide.

[0156] [204-2]The foregoing method, wherein the nucleic acid is included in a vesicle.

[0157] [204-3]The foregoing method, which includes contacting the nucleic acid with a vesicle.

[0158] [204-4]The foregoing method, wherein the vesicle is a lipid nanoparticle (LNP), a virus, an extracellular vesicle (EV), or a liposome.

[0159] [204-5]The foregoing method, wherein the nucleic acid is RNA, DNA, or a vector or plasmid carrying the nucleic acid.

[0160] [204-6]The foregoing method, wherein the RNA is mRNA, genomic RNA, or circular RNA.

[0161] [204-7]The foregoing method, wherein the polypeptide comprising the Fc region is an antibody.

[0162] [204-8]The foregoing method, wherein the polypeptide comprising the Fc region is a single-domain antibody.

[0163] [204-9]The foregoing method, wherein the polypeptide comprising the Fc region is IgG.

[0164] [204-10]The foregoing method, wherein the polypeptide comprising the Fc region does not associate with endogenous IgG.

[0165] [204-11]The foregoing method, wherein the polypeptide comprising the Fc region retains the function of the Fc of wild-type IgG.

[0166] [204-12]The foregoing method, which includes administering the nucleic acid to a subject.

[0167] [204-13]The foregoing method, wherein the subject is a human.

[0168] [204-14]The foregoing method, wherein the polypeptide comprising the Fc region is a humanized antibody or a human antibody.

[0169]

[205] A method for expressing in a cell a polypeptide in which the association between polypeptides comprising an Fc region is controlled, the method comprising:

[0170] (a) Prepare a nucleic acid encoding a polypeptide comprising an Fc region such that the polypeptide comprising the Fc region is modified into a polypeptide that associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge; and

[0171] (b) Introduce the nucleic acid of step (a) into a cell.

[0172] [205-1] The foregoing method, which comprises introducing a modification into the Fc region such that an effect of (1) steric complementarity, (2) disulfide bonding, or (3) electrostatic charge occurs in the polypeptide.

[0173] [205-2] The foregoing method, wherein the nucleic acid is RNA, DNA, or a vector or plasmid carrying the nucleic acid.

[0174] [205-3] The foregoing method, wherein the RNA is mRNA, genomic RNA, or circular RNA.

[0175] [205-4] The foregoing method, wherein the polypeptide comprising the Fc region is an antibody.

[0176] [205-5] The foregoing method, wherein the polypeptide comprising the Fc region is a single-domain antibody.

[0177] [205-6] The foregoing method, wherein the polypeptide comprising the Fc region is IgG.

[0178] [205-7] The foregoing method, wherein the cell is a human cell.

[0179] [205-8] The foregoing method, which is an in vivo, ex vivo, or in vitro method.

[0180]

[206] A method for expressing two or more types of polypeptides comprising an Fc region in a cell, wherein the association between polypeptides of the same type is controlled:

[0181] (a) Prepare a nucleic acid encoding a polypeptide comprising an Fc region such that a first polypeptide comprising the Fc region is modified into a polypeptide that associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge; and

[0182] (b) Prepare a nucleic acid encoding a polypeptide comprising an Fc region such that a second polypeptide comprising the Fc region is modified into a polypeptide that associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) spatial complementarity, (2) disulfide bonding, and (3) electrostatic charge; wherein the modification in the second polypeptide is different from the modification in the first polypeptide.

[0183] [206-1] The foregoing method, wherein two or more types of polypeptides comprising an Fc region are expressed and homopolymers are allowed to form among polypeptides of the same type.

[0184] [206-2] The foregoing method, wherein the modifications of the two or more types of polypeptides to be expressed are different from each other.

[0185] [206-3] The foregoing method, wherein the combination of the modifications that are different from each other is the combination shown in Table 7.

[0186]

[207] A method for controlling the association of two or more types of polypeptides comprising an Fc region, wherein the association between polypeptides of the same type is controlled, and wherein the two or more types of polypeptides are modified such that they associate more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) spatial complementarity, (2) disulfide bonding, and (3) electrostatic charge, and further wherein the modifications in the two or more types of polypeptides are different from each other.

[0187] [207-1] The foregoing method, wherein the combination of the modifications that are different from each other is the combination shown in Table 7.

[0188]

[208] A method for producing a pharmaceutical composition comprising vesicles, the vesicles comprising a nucleic acid encoding a polypeptide in which the association between Fc regions is controlled, the method comprising:

[0189] (a) Obtain a nucleic acid encoding a polypeptide into which a modification has been introduced in the Fc region, wherein the polypeptide associates more readily with a polypeptide having the modification than with a polypeptide into which the modification has not been introduced due to at least one of the following effects: (1) spatial complementarity, (2) disulfide bonding, and (3) electrostatic charge; and

[0190] (b) Contact the nucleic acid of step (a) with the vesicles.

[0191] [208-1] The foregoing method, wherein the vesicles are lipid nanoparticles (LNP), viruses, extracellular vesicles (EV), or liposomes.

[0192] [208-2]The foregoing method, wherein there is further contact with a pharmaceutical carrier.

[0193] [208-3]The foregoing method, wherein the nucleic acid is RNA, DNA, or a vector or plasmid carrying the nucleic acid.

[0194]

[301] A pharmaceutical composition comprising the foregoing nucleic acid or the foregoing polypeptide and a pharmaceutical carrier.

[0195] [301-1]A pharmaceutical composition comprising a nucleic acid encoding a polypeptide comprising an Fc region with controlled association.

[0196] [301-2]The foregoing pharmaceutical composition, which comprises the nucleic acid encapsulated in a vesicle.

[0197] [301-3]A pharmaceutical composition comprising a nucleic acid encoding a polypeptide comprising an Fc region with controlled association and a vesicle.

[0198] [301-4]The foregoing pharmaceutical composition, wherein the vesicle is a lipid nanoparticle (LNP), a virus, an extracellular vesicle (EV), or a liposome.

[0199] [301-5]The foregoing pharmaceutical composition, wherein the nucleic acid is RNA, DNA, or a vector or plasmid carrying the nucleic acid.

[0200] [301-6]The foregoing pharmaceutical composition for generating a polypeptide with controlled association between Fc regions in a living organism.

[0201] [301-7]The foregoing pharmaceutical composition for expressing a polypeptide with controlled association between Fc regions in a cell.

[0202] In one embodiment, the present disclosure relates to the following:

[0203] [Example 1]

[0204] A nucleic acid encoding a first polypeptide,

[0205] wherein the first polypeptide comprises an Fc region into which a modification has been introduced,

[0206] wherein due to the modification introduced into the Fc region, the first polypeptide associates more readily via the Fc region with a first polypeptide having the modification than with a first polypeptide comprising an Fc region into which the modification has not been introduced.

[0207] [Example 2]

[0208] The nucleic acid according to Example 1, wherein due to at least one of the following effects resulting from the introduced modification, the association of the first polypeptide via the Fc region with the first polypeptide having the modification is easier than the association with the first polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0209] [Example 3]

[0210] A composition comprising:

[0211] The nucleic acid according to Disclosure 1 of the present application; and

[0212] A nucleic acid encoding a second polypeptide,

[0213] wherein the second polypeptide comprises an Fc region into which a modification has been introduced,

[0214] wherein due to the modification introduced into the Fc region, the association of the second polypeptide via the Fc region with the second polypeptide having the modification is easier than the association with the second polypeptide comprising an Fc region into which the modification has not been introduced.

[0215] [Example 4]

[0216] A composition comprising:

[0217] The nucleic acid according to Example 1;

[0218] A nucleic acid encoding a second polypeptide; and

[0219] A nucleic acid encoding a third polypeptide,

[0220] wherein the association of the second polypeptide with the third polypeptide is easier than the association of the second polypeptide.

[0221] [Example 5]

[0222] A composition comprising:

[0223] The nucleic acid according to Example 2; and

[0224] A nucleic acid encoding a second polypeptide,

[0225] wherein the second polypeptide does not have the modification that the first polypeptide has.

[0226] [Example 6]

[0227] The composition according to Example 3, wherein the modification introduced into the first polypeptide is different from the modification introduced into the second polypeptide.

[0228] [Example 7]

[0229] The composition according to embodiment 3, wherein due to at least one of the following effects resulting from the introduced modification, the association of the second polypeptide via the Fc region with the second polypeptide having the modification is easier than the association with the second polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0230] [Example 8]

[0231] The nucleic acid according to embodiment 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 2.

[0232] [Example 9]

[0233] The nucleic acid according to embodiment 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 4.

[0234] [Example 10]

[0235] The nucleic acid according to embodiment 6, wherein the modification introduced into the first polypeptide and the modification introduced into the second polypeptide are at least one of the combinations of the modifications listed in Table 6.

[0236] [Example 11]

[0237] The nucleic acid according to embodiment 1, wherein the polypeptide is an antibody.

[0238] [Example 12]

[0239] The nucleic acid according to embodiment 1, wherein the Fc region is the Fc region of IgG.

[0240] [Example 13]

[0241] The nucleic acid according to embodiment 1, wherein the polypeptide into which the modification has been introduced retains the function of the polypeptide before the modification.

[0242] [Example 14]

[0243] The nucleic acid according to embodiment 12, wherein the Fc region into which the modification has been introduced retains the function of the Fc of IgG.

[0244] [Example 15]

[0245] The nucleic acid according to embodiment 1, wherein the Fc region is derived from any one of IgG1, 2, 3, and 4.

[0246] [Example 16]

[0247] A host cell into which a nucleic acid according to any one of Examples 1, 2, and 8 to 14 or a composition according to any one of Examples 3 to 7 has been introduced.

[0248] [Example 17]

[0249] A polypeptide expressed from the nucleic acid according to Example 1.

[0250] [Example 18]

[0251] A composition comprising a nucleic acid encoding a polypeptide,

[0252] wherein the polypeptide comprises an Fc region into which a modification has been introduced,

[0253] wherein the modification is at least one of the modifications listed in Table 2.

[0254] [Example 19]

[0255] A polypeptide comprising an Fc region into which a modification has been introduced, wherein due to at least one of the following effects resulting from the introduced modification, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0256] [Example 20]

[0257] A method for obtaining a polypeptide with controlled association, the method comprising:

[0258] obtaining a nucleic acid encoding the polypeptide; and

[0259] expressing the nucleic acid,

[0260] wherein the polypeptide comprises an Fc region, and

[0261] wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced.

[0262] [Example 21]

[0263] A method for controlling the association of a homopolymer of a polypeptide, the method comprising:

[0264] obtaining a nucleic acid encoding the polypeptide; and

[0265] expressing the nucleic acid,

[0266] wherein the polypeptide comprises an Fc region, and

[0267] Among them, due to the introduction of the modification in the Fc region, the association of the polypeptide via the Fc region with the polypeptide having the modification is easier than the association with the polypeptide containing the Fc region in which the modification has not been introduced.

[0268] [Example 22]

[0269] A method for promoting the expression of a homopolymer of a polypeptide, the method comprising:

[0270] obtaining a nucleic acid encoding the polypeptide; and

[0271] expressing the nucleic acid,

[0272] wherein the polypeptide comprises an Fc region, and

[0273] Among them, due to the introduction of the modification in the Fc region, the association of the polypeptide via the Fc region with the polypeptide having the modification is easier than the association with the polypeptide containing the Fc region in which the modification has not been introduced.

[0274] [Example 23]

[0275] The composition according to Example 4, wherein the second polypeptide comprises an Fc region into which a modification has been introduced, and wherein due to the introduction of the modification in the Fc region, the association of the second polypeptide via the Fc region with the third polypeptide is easier than the association with the second polypeptide.

[0276] [Example 24]

[0277] The composition according to Example 1, wherein the Fc region into which the modification has been introduced comprises an amino acid modification at one position combination or two or more position combinations selected from the following position combinations shown in (a) to (d):

[0278] (a) positions 394 and 405;

[0279] (b) positions 366, 368 and 407;

[0280] (c) positions 347, 360, 399, 405 and 409; and

[0281] (d) positions 356, 392, 399 and 439, according to EU numbering.

[0282] [Example 25]

[0283] The composition according to Example 24, wherein the Fc region into which the modification has been introduced comprises at least one amino acid selected from the group consisting of:

[0284] (a) W, F, or Y at position 394, and

[0285] A, S, T, C, G, or V at position 405;

[0286] (b) W, Y, or F at position 366,

[0287] A, S, T, C, V, or G at position 368, and

[0288] V, L, I, M, A, S, T, C, N, or Q at position 407;

[0289] (c) R, K, Y, or H at position 347,

[0290] E or D at position 360,

[0291] V, L, I, M, S, T, C, H, A, N, Q, or G at position 399,

[0292] T, A, V, S, C, N, D, or G at position 405, and

[0293] W, F, Y, or H at position 409; and

[0294] (d) K or R at position 356,

[0295] D or E at position 392,

[0296] K or R at position 399, and

[0297] E or D at position 439,

[0298] According to EU numbering.

[0299] [Example 26]

[0300] The composition according to Example 24, wherein the Fc region into which the modification has been introduced comprises at least one amino acid selected from the group consisting of:

[0301] (a) W or F at position 394, and

[0302] A, S, T, or G at position 405;

[0303] (b) W, Y, or F at position 366,

[0304] A, T, C, V, or G at position 368, and

[0305] V, L, I, M, A, or C at position 407;

[0306] (c) R, K, Y, or H at position 347,

[0307] E or D at position 360,

[0308] V, L, I, M, S, T, C, H, A, N, or G at position 399,

[0309] T, A, or V at position 405, and

[0310] W, F, or Y at position 409; and

[0311] (d) K at position 356,

[0312] D at position 392,

[0313] K at position 399, and

[0314] E at position 439,

[0315] According to EU numbering.

[0316] [Example 27]

[0317] The nucleic acid according to Example 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 15.

[0318] [Example 28]

[0319] The nucleic acid according to Example 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 17.

[0320] In one embodiment, the present disclosure relates to the following:

[0321] [Example 29]

[0322] The composition according to Example 1, wherein the Fc region into which the modification has been introduced contains amino acid modifications at one position combination or two or more position combinations selected from the position combinations shown in (a) to (d) below:

[0323] (a) Positions 345, 347, 360, 366, 399, 407, and 409;

[0324] (b) Positions 356, 399, 409, and 439;

[0325] (c) Positions 356, 392, 399, and 409; and

[0326] (d) Positions 392, 399, and 409, according to EU numbering.

[0327] [Example 30]

[0328] The composition according to Example 29, wherein the Fc region into which the modification has been introduced comprises at least one amino acid selected from the group consisting of:

[0329] (a) R or E at position 345,

[0330] R or K at position 347,

[0331] D or E at position 360,

[0332] V at position 366,

[0333] M, Q, N, H, I, F, Y, T, S, V or L at position 399,

[0334] A at position 407, and

[0335] V, Q, N, H, L, I, F, Y, T or S at position 409;

[0336] (b) K, R or H at position 356,

[0337] K, R or H at position 399,

[0338] E or D at position 409, and

[0339] E or D at position 439,

[0340] (c) K, R or H at position 356,

[0341] D or E at position 392,

[0342] K, R or H at position 399, and

[0343] D or E at position 409; and

[0344] (d) D or E at position 392,

[0345] K or R at position 399, and

[0346] D or E at position 409,

[0347] According to EU numbering. Brief Description of the Drawings

[0348] Figure 1Shows the concept of CH3 interface association control, which promotes the formation of homodimers of heavy chains. (1) Amino acid modifications with charges that create an attraction between the modified CH3 regions but a repulsion from the wild-type CH3. (2) Amino acid modifications for generating modified CH3s with protrusions and cavities, where the modified CH3 regions can bind to each other but not to the wild-type CH3 due to steric hindrance. (3) Amino acid modifications that allow the formation of multiple artificial disulfide bonds between the modified CH3 regions. Association with the wild-type CH3 is inhibited.

[0349] Figure 2 Shows a method for screening heavy chain homodimerization-promoting modifications by SEC analysis.

[0350] Figure 3( Figures 3-1 to 3-47 ) shows the evaluation of antibody heavy chain association by CIEX analysis. In Figure 3-1 , an antibody without CH3 modification was analyzed.

[0351] In Figure 3-2 , an antibody with CH3 modification was analyzed.

[0352] In Figure 3-3 , an antibody without CH3 modification was analyzed.

[0353] In Figure 3-4 , an antibody with CH3 modification was analyzed.

[0354] In Figure 3-5 , an antibody without CH3 modification was analyzed.

[0355] In Figure 3-6 , an antibody with CH3 modification was analyzed.

[0356] In Figure 3-7 , an antibody without CH3 modification was analyzed.

[0357] In Figure 3-8 , an antibody with CH3 modification was analyzed.

[0358] Figure 3-9 Shows the analysis results of co-expression of two antibodies without CH3 modification. Each peak position was identified and assigned based on the elution positions observed when expressing each antibody.

[0359] Figure 3-10 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position was identified and assigned based on the elution positions observed when expressing each antibody.

[0360] Figure 3-11The analysis results of the co-expression of two antibodies without CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0361] Figure 3-12 The analysis results of the co-expression of two antibodies with CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0362] Figure 3-13 The analysis results of the co-expression of two antibodies without CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0363] Figure 3-14 The analysis results of the co-expression of two antibodies with CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0364] Figure 3-15 The analysis results of the co-expression of two antibodies without CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0365] Figure 3-16 The analysis results of the co-expression of two antibodies with CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0366] Figure 3-17 The analysis results of the co-expression of two antibodies without CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0367] Figure 3-18 The analysis results of the co-expression of two antibodies with CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0368] Figure 3-19 The analysis results of the co-expression of two antibodies without CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0369] Figure 3-20 The analysis results of the co-expression of two antibodies with CH3 modification are shown. Each peak position is identified and assigned based on the elution positions observed when each antibody is expressed.

[0370] Figure 3-21 The analysis results of the co-expression of three antibodies without CH3 modification are shown. Each peak is assigned by referring to the peak positions observed when one antibody is expressed or two antibodies are co-expressed.

[0371] Figure 3-22 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0372] Figure 3-23 Shows the analysis results of co-expression of three antibodies without CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0373] Figure 3-24 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0374] Figure 3-25 Shows the analysis results of co-expression of three antibodies without CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0375] Figure 3-26 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0376] Figure 3-27 Shows the analysis results of co-expression of four antibodies without CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0377] Figure 3-28 Shows the analysis results of co-expression of four antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0378] In Figure 3-29 one antibody with CH3 modification was analyzed.

[0379] Figure 3-30 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position is identified and assigned based on the elution positions observed when expressing each antibody.

[0380] Figure 3-31 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position is identified and assigned based on the elution positions observed when expressing each antibody.

[0381] Figure 3-32 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position is identified and assigned based on the elution positions observed when expressing each antibody.

[0382] Figure 3-33 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0383] Figure 3-34 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0384] Figure 3-35 Shows the analysis results of co-expression of four antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0385] In Figure 3-36 one antibody with CH3 modification was analyzed.

[0386] Figure 3-37 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position is identified and assigned based on the elution positions observed when expressing each antibody.

[0387] Figure 3-38 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position is identified and assigned based on the elution positions observed when expressing each antibody.

[0388] Figure 3-39 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position is identified and assigned based on the elution positions observed when expressing each antibody.

[0389] Figure 3-40 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0390] Figure 3-41 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0391] Figure 3-42 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0392] Figure 3-43 Shows the analysis results of co-expression of four antibodies with CH3 modification. Each peak is assigned by referring to the peak positions observed when expressing one antibody or co-expressing two antibodies.

[0393] Figure 3-44 Shows the analysis results of co-expression of two antibodies with CH3 modification. Each peak position was identified and assigned based on the elution positions observed when each antibody was expressed.

[0394] Figure 3-45 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak was assigned by referring to the peak positions observed when one antibody was expressed or when two antibodies were co-expressed.

[0395] Figure 3-46 Shows the analysis results of co-expression of three antibodies with CH3 modification. Each peak was assigned by referring to the peak positions observed when one antibody was expressed or when two antibodies were co-expressed.

[0396] Figure 3-47 Shows the analysis results of co-expression of four antibodies with CH3 modification. Each peak was assigned by referring to the peak positions observed when one antibody was expressed or when two antibodies were co-expressed.

[0397] Figure 4 Shows the evaluation of ECM binding of CH3-modified antibodies. The non-specific binding of each antibody into which a modification for promoting heavy-chain homodimerization was introduced was evaluated.

[0398] Figure 5( Figures 5-1 to 5-14 ) shows the evaluation of heavy-chain association of two antibodies by SEC analysis. Due to the presence of multimeric components generated by amino acid modification or antibody format, the focus was on comparing the heavy-chain heteromultimeric components indicated by the arrows in the figure. The elution positions of the heteromultimeric components in the chromatogram were identified by comparing with the elution positions of each heavy-chain homodimer reference sample. The analysis results of the reference samples are also shown as in the figure. In Figure 5-1 , the elution positions of the reference antibodies without CH3 modification were analyzed, as well as the ability of heavy-chain homodimerization under co-expression of the full-length heavy chain and the Fc fragment.

[0399] In Figure 5-2 , the elution positions of the reference antibodies with CH3 modification were analyzed, as well as the ability of heavy-chain homodimerization under co-expression of the full-length heavy chain and the Fc fragment.

[0400] In Figure 5-3 , the elution positions of the reference antibodies with CH3 modification were analyzed, as well as the ability of heavy-chain homodimerization under co-expression of the full-length heavy chain and the Fc fragment.

[0401] In Figure 5-4 , the elution positions of the reference antibodies with CH3 modification were analyzed, as well as the ability of heavy-chain homodimerization under co-expression of the full-length heavy chain and the Fc fragment.

[0402] In Figure 5-5Among them, the elution position of the reference antibody without CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0403] In Figure 5-6 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0404] In Figure 5-7 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0405] In Figure 5-8 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0406] In Figure 5-9 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0407] In Figure 5-10 Among them, the elution position of the reference antibody without CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0408] In Figure 5-11 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0409] In Figure 5-12 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0410] In Figure 5-13 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0411] In Figure 5-14 Among them, the elution position of the reference antibody with CH3 modification was analyzed, as well as the ability of heavy chain homodimerization under the co-expression of full-length heavy chain and Fc fragment.

[0412] Figure 6( Figures 6-1 to 6-10 ) shows the evaluation of the independent heavy chain heteromeric association ability and heavy chain homomeric association ability of the antibody by CIEX analysis. Figure 6-1Shows the analysis results when co-expressing a homodimeric antibody and a heterodimeric antibody whose heteromeric association is promoted by knobs-into-holes modification. In each case where the homodimer has no CH3 modification (sample number 1157) and has CH3 modification (sample numbers 1161, 1165, and 1169), the area ratios of the homodimer, heterodimer, and unintended heavy-chain polymers are calculated. In all cases, compared with the case without CH3 modification, the pair containing the modification for promoting homodimerization inhibited the unintended heavy-chain association.

[0413] Figure 6-2 Shows the analysis results when co-expressing a homodimeric antibody and a heterodimeric antibody whose heteromeric association is promoted by knobs-into-holes modification (including disulfide bonds). In each case where the homodimer has no CH3 modification (sample number 1176) and has CH3 modification (sample numbers 1180, 1184, and 1188), the area ratios of the homodimer, heterodimer, and unintended heavy-chain polymers are calculated. In all cases, compared with the case without CH3 modification, the pair containing the modification for promoting homodimerization inhibited the unintended heavy-chain association.

[0414] Figure 6-3 Shows the peak assignment results in the CIEX analysis of sample number 1157. The plasmids for expressing sample number 1157 were expressed in various combinations, and each resulting reference antibody was analyzed. The peaks of the target heavy-chain homodimer and heavy-chain heterodimer and other unintended peaks were assigned. The assigned heavy-chain associations are indicated at each peak in the figure.

[0415] Figure 6-4 Shows the peak assignment results in the CIEX analysis of sample number 1161. The plasmids for expressing sample number 1161 were expressed in various combinations, and each resulting reference antibody was analyzed. The peaks of the target heavy-chain homodimer and heavy-chain heterodimer and other unintended peaks were assigned. The assigned heavy-chain associations are indicated at each peak in the figure.

[0416] Figure 6-5 Shows the peak assignment results in the CIEX analysis of sample number 1165. The plasmids for expressing sample number 1165 were expressed in various combinations, and each resulting reference antibody was analyzed. The peaks of the target heavy-chain homodimer and heavy-chain heterodimer and other unintended peaks were assigned. The assigned heavy-chain associations are indicated at each peak in the figure.

[0417] Figure 6-6Shows the peak assignment results in the CIEX analysis of sample number 1169. The plasmids used to express sample number 1169 were expressed in various combinations, and each resulting reference antibody was analyzed. Peaks for the target heavy-chain homodimers and heavy-chain heterodimers were assigned, as well as other unexpected peaks. The assigned heavy-chain associations are indicated at each peak in the figure.

[0418] Figure 6-7 Shows the peak assignment results in the CIEX analysis of sample number 1176. The plasmids used to express sample number 1176 were expressed in various combinations, and each resulting reference antibody was analyzed. Peaks for the target heavy-chain homodimers and heavy-chain heterodimers were assigned, as well as other unexpected peaks. The assigned heavy-chain associations are indicated at each peak in the figure.

[0419] Figure 6-8 Shows the peak assignment results in the CIEX analysis of sample number 1180. The plasmids used to express sample number 1180 were expressed in various combinations, and each resulting reference antibody was analyzed. Peaks for the target heavy-chain homodimers and heavy-chain heterodimers were assigned, as well as other unexpected peaks. The assigned heavy-chain associations are indicated at each peak in the figure.

[0420] Figure 6-9 Shows the peak assignment results in the CIEX analysis of sample number 1184. The plasmids used to express sample number 1184 were expressed in various combinations, and each resulting reference antibody was analyzed. Peaks for the target heavy-chain homodimers and heavy-chain heterodimers were assigned, as well as other unexpected peaks. The assigned heavy-chain associations are indicated at each peak in the figure.

[0421] Figure 6-10 Shows the peak assignment results in the CIEX analysis of sample number 1188. The plasmids used to express sample number 1188 were expressed in various combinations, and each resulting reference antibody was analyzed. Peaks for the target heavy-chain homodimers and heavy-chain heterodimers were assigned, as well as other unexpected peaks. The assigned heavy-chain associations are indicated at each peak in the figure.

[0422] Figure 7-1 Shows the plasma concentrations of anti-CD3 homodimer, anti-GPC3 homodimer, and anti-CD3 / GPC3 heterodimer on day 3 after administration.

[0423] Figure 7-2 Shows the plasma concentrations of anti-FIXa homodimer, anti-FX homodimer, and anti-FIXa / FX heterodimer on day 3 after administration.

[0424] Figure 7-3 Shows the plasma concentrations of anti-CD3 homodimer, anti-GPC3 homodimer, and anti-CD3 / GPC3 heterodimer on day 7 after administration.

[0425] Figure 7-4 Shows the plasma concentrations of anti-FIXa homodimer, anti-FX homodimer and anti-FIXa / FX heterodimer on the 7th day after administration. Detailed implementation manner

[0426] Those skilled in the art generally easily understand and typically use conventional methods to utilize the techniques and procedures described or referenced herein, such as, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current Protocols in Molecular Biology (edited by F.M. Ausubel et al., (2003)); Methods in Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (edited by M.J. MacPherson, B.D. Hames and G.R. Taylor (1995)), Antibodies, A Laboratory Manual, edited by Harlow and Lane (1988), and Animal Cell Culture (edited by R.I. Freshney (1987)); Oligonucleotide Synthesis (edited by M.J. Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by J.E. Cellis, 1998) Academic Press; Animal Cell Culture (edited by R.I. Freshney, 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, J.B. Griffiths, and D.G. Newell, 1993 - 8) J.Wiley and Sons; Handbook of Experimental Immunology (edited by D.M. Weir and C.C. Blackwell); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P.as described in the widely used methods in Calos, Ed., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., Ed., 1994); Current Protocols in Immunology (J.E. Coligan et al., Ed., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (D. Catty, Ed., IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P. Shepherd and C. Dean, Ed., Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, Ed., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., Ed., J.B. Lippincott Company, 1993).

[0427] The following definitions and detailed descriptions are provided to facilitate understanding of the present disclosure as shown herein.

[0428] Definitions

[0429] Amino acid

[0430] In this document, amino acids are described by one-letter or three-letter codes or both, such as Ala / A, Leu / L, Arg / R, Lys / K, Asn / N, Met / M, Asp / D, Phe / F, Cys / C, Pro / P, Gln / Q, Ser / S, Glu / E, Thr / T, Gly / G, Trp / W, His / H, Tyr / Y, Ile / I or Val / V.

[0431] Amino acid modification

[0432] For amino acid modifications (also referred to herein as "amino acid substitutions" or "amino acid mutations") in the amino acid sequence of a polypeptide, known methods can be appropriately employed, such as site-directed mutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap extension PCR. In addition, several known methods can also be used as amino acid alteration methods to substitute unnatural amino acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and Proc. Natl. Acad. Sci. U.S.A. (2003) 100(11), 6353-6357). For example, a cell-free translation system containing tRNA (Clover Direct (Protein Express)) can be used, and the tRNA has an unnatural amino acid that binds to a complementary amber suppressor tRNA for one of the stop codons, the UAG codon (amber codon).

[0433] In this specification, when describing amino acid modification sites, the meaning of the term "and / or" includes all combinations in which "and" and "or" are appropriately combined. Specifically, for example, "the amino acid at position x, y and / or z is substituted" includes the following variants of amino acid alteration: the amino acid at (a) position x, (b) position y, (c) position z, (d) positions x and y, (e) positions x and z, (f) positions y and z, and (g) positions x, y and z.

[0434] In addition, herein, as an expression showing an amino acid modification, an expression that shows the single-letter or three-letter code of the amino acid before and after the modification, respectively, before and after the number indicating a specific position can be appropriately used. For example, the modification E345K used when substituting an amino acid contained in the variable region of an antibody indicates that Glu (E) is substituted with Lys (K) at position 345 (according to EU numbering). That is, the number shows the amino acid position according to EU numbering, the single-letter or three-letter amino acid code written before the number shows the amino acid before substitution, and the single-letter or three-letter amino acid code written after the number shows the amino acid after substitution.

[0435] Polypeptide

[0436] As used herein, the term "polypeptide" refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also referred to as peptide bonds). The term "polypeptide" does not refer to a product of a specific length. The term "polypeptide" also is intended to refer to post-expression modification products of polypeptides, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide may be derived from natural biological sources or produced by recombinant techniques and not necessarily translated from a designated nucleic acid sequence. It may be produced in any manner, including by chemical synthesis. In one embodiment, a polypeptide as described herein may be 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, or 1,000 or more amino acids in size.

[0437] In one embodiment, a polypeptide of the present disclosure is produced in a living organism. For example, a polypeptide of the present disclosure may be produced in a living organism by introducing a nucleic acid encoding the polypeptide of the present disclosure into the living organism. In one embodiment, a polypeptide of the present disclosure is a polypeptide expressed in a living organism.

[0438] Recombinant methods and compositions

[0439] Polypeptides that are antibodies or antigen-binding molecules may be produced using recombinant methods and compositions, e.g., as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid encoding a polypeptide as described herein is provided. In one embodiment, such nucleic acid may encode a polypeptide having a heavy chain Fc region. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one of such embodiments, the host cell comprises (e.g., has been transformed with) a vector comprising a nucleic acid encoding an amino acid sequence comprising a heavy chain Fc region. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell or a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cells). In one embodiment, a method for producing a polypeptide with controlled association according to the present disclosure is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding a polypeptide as provided herein under conditions suitable for expressing the polypeptide, and optionally recovering the polypeptide from the host cell (or the host cell culture medium).

[0440] For the recombinant production of the polypeptides (in one embodiment, antibodies) described herein, the nucleic acids encoding the polypeptides are isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acids can be readily isolated and sequenced using conventional procedures, e.g., by using oligonucleotide probes that specifically bind to the genes encoding the heavy chain Fc region.

[0441] Suitable host cells for vectors encoding polypeptides (in one embodiment, antibodies) for cloning or expression include the prokaryotic or eukaryotic cells described herein. For example, polypeptides (in one embodiment, antibodies) can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For the expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (ed. B.K.C. Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of polypeptide (antibody) fragments in E. coli.) The polypeptides can be isolated from the bacterial cell paste in the soluble fraction after expression and can be further purified.

[0442] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding polypeptides (antibodies), including such fungal and yeast strains in which the glycosylation pathways have been "humanized" such that polypeptides (in one embodiment, antibodies) with a partially or fully human glycosylation pattern are produced. (See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).)

[0443] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. A number of baculovirus strains have been identified that can be used with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0444] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe the PLANTIBODIES TM technology for the production of polypeptides (in one embodiment, antibodies) in transgenic plants).

[0445] Vertebrate cells can also be used as hosts. For example, mammalian cell lines suitable for growth in suspension may be useful. Other examples of available mammalian host cell lines are: the monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney lines (such as the 293 or 293 cells described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse testicular Sertoli cells (such as the TM4 cells described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); dog kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as those described in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as Y0, NS0, and Sp2 / 0. A review of certain mammalian host cell lines suitable for the production of polypeptides (antibodies) can be found, for example, in Yazaki and Wu, Methods in Molecular Biology, Volume 248 (edited by B.K.C. Lo, Humana Press, Totowa, NJ), pages 255-268 (2003).

[0446] The recombinant production of the polypeptides (in one embodiment, antibodies) described herein can be accomplished by using host cells containing (e.g., transformed with) one or more vectors containing nucleic acids encoding amino acid sequences that comprise the entire polypeptide (antibody) or a portion of the polypeptide (antibody), using methods similar to those described above.

[0447] The polypeptides (in one embodiment, antibodies) of the present disclosure include polypeptides that have undergone post-translational modifications. Examples of the polypeptides of the present disclosure that have undergone post-translational modifications include, in the case where the polypeptide is an antibody, an antibody that has undergone pyroglutamination at the N-terminus of the heavy-chain variable region and / or lysine deletion at the C-terminus of the heavy chain. As is known in the art, such post-translational modifications caused by pyroglutamination at the N-terminus and lysine deletion at the C-terminus have no effect on the activity of the antibody (Analytical Biochemistry, 2006, Vol. 348, pp. 24-39).

[0448] As used herein, the terms "first", "second", etc. with respect to polypeptides, etc. are used for convenience of distinction. Unless expressly stated, the use of these terms is not intended to impart a particular order or orientation.

[0449] The polypeptide can be a TCR (T cell receptor) or a part of a TCR.

[0450] Expression in living organism cells

[0451] In one embodiment, the polypeptides of the present disclosure can be expressed in cells of a living organism.

[0452] In one embodiment, the expression of the polypeptides of the present disclosure in cells is achieved by introducing a nucleic acid encoding the polypeptides of the present disclosure into target cells. Any standard method for introducing nucleic acids into cells can be used. Such methods include, for example, microinjection, biolistic injection, electroporation, calcium phosphate precipitation, liposomes, and transfection with retroviral, adenoviral, adeno-associated viral, and vaccinia vectors carrying the target nucleic acid.

[0453] In one embodiment, nucleic acids encoding the polypeptides of the present disclosure can be introduced into cells in a subject (in one embodiment, a human) by in vivo and ex vivo methods. In one example of in vivo delivery, the nucleic acid is directly injected into the subject (e.g., the site in need of treatment). In a further example of in vivo delivery, viral vectors (such as adenovirus, herpes simplex type I virus, or adeno-associated virus) and lipid-based systems (e.g., useful lipids for lipid-mediated gene transfer are DOTMA, DOPE, and DC-Chol) are used to transfect the nucleic acid into cells (for a review of certain gene marking and gene therapy protocols, see Anderson et al., Science 256:808-813 (1992) and WO 93 / 25673 and the references cited therein.). In an example of ex vivo treatment, cells of the subject are removed, the nucleic acid is introduced into those isolated cells, and the modified cells are directly administered to the subject or, for example, encapsulated within a porous membrane implanted in the subject (see, for example, U.S. Patent Nos. 4,892,538 and 5,283,187). In one embodiment, a commonly used vector for ex vivo delivery of nucleic acids is a retroviral vector.

[0454] Intracellular antibody

[0455] In one embodiment, the polypeptides of the present disclosure can be expressed intracellularly as intracellular antibodies (antibodies expressed intracellularly).

[0456] As used herein, the term "intracellular antibody" refers to an antibody or an antigen-binding portion thereof that is expressed intracellularly and is capable of selectively binding to a target molecule, as described, for example, in Marasco, Gene Therapy 4:11-15 (1997); Kontermann, Methods 34:163-170 (2004); U.S. Patent Nos. 6,004,940 and 6,329,173; U.S. Patent Application Publication Nos. 2003 / 0104402 and PCT Publication No. WO2003 / 077945 (see also, for example, WO96 / 007321, published March 14, 1996, regarding the generation of intracellular antibodies using gene therapy).

[0457] In one embodiment, intracellular expression of an intracellular antibody can be effected by introducing nucleic acids encoding the antibody or an antigen-binding portion thereof into a target cell. One or more nucleic acids encoding all or part of an antibody comprising the polypeptides of the present disclosure can be delivered to the target cell such that one or more intracellular antibodies are expressed.

[0458] In one embodiment, the polypeptides of the present disclosure in which the association between polypeptides is controlled can be used to generate intracellular antibodies intracellularly.

[0459] Single domain antibody

[0460] In one embodiment, the polypeptide of the present disclosure may be a polypeptide having a single-domain antibody.

[0461] As used herein, the term "single-domain antibody" is not limited by its structure as long as the domain can exhibit antigen-binding activity on its own. It is known that a general antibody, such as an IgG antibody, exhibits antigen-binding activity in a state where a variable region is formed by pairing of VH and VL, while the self-domain structure of a single-domain antibody can exhibit antigen-binding activity on its own without pairing with another domain. Generally, a single-domain antibody has a relatively low molecular weight and exists in a monomeric form.

[0462] Examples of single-domain antibodies include, but are not limited to, antigen-binding molecules that are congenitally lacking a light chain, such as VHHs of camelids and shark VNARs, and antibody fragments containing all or part of an antibody VH domain or all or part of an antibody VL domain. Examples of single-domain antibodies that are antibody fragments containing all or part of an antibody VH or VL domain include, but are not limited to, artificially prepared single-domain antibodies derived from a human antibody VH or a human antibody VL, as described in U.S. Patent No. 6,248,516B1, etc. In some embodiments of the present disclosure, a single-domain antibody has three CDRs (CDR1, CDR2, and CDR3).

[0463] In one embodiment, the single-domain antibody of the present disclosure includes scFv-Fc or VHH-Fc.

[0464] Variable region

[0465] In one embodiment, the polypeptide of the present disclosure may be a polypeptide having a variable region.

[0466] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in antibody binding to an antigen. The variable domains of the heavy and light chains of a naturally occurring antibody (VH and VL, respectively) generally have similar structures, each containing four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, for example, Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain can confer antigen-binding specificity. In addition, an antibody that binds a specific antigen can be isolated using the VH or VL domain from the antibody that binds the antigen, respectively, to screen a library of complementary VL or VH domains. See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0467] HVR or CDR

[0468] In one embodiment, the polypeptide of the present disclosure is an antibody and may have HVRs or CDRs.

[0469] As used herein, the term "hypervariable region" or "HVR" refers to each of the regions in the variable domain of an antibody that are hypervariable ("complementary determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). Hypervariable regions (HVRs) are also known as "complementary determining regions" (CDRs), and these terms are used interchangeably herein to refer to the variable region portions that form the antigen-binding region. Typically, an antibody contains six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Exemplary HVRs herein include:

[0470] (a) hypervariable loops at amino acid residues 26 - 32 (L1), 50 - 52 (L2), 91 - 96 (L3), 26 - 32 (H1), 53 - 55 (H2), and 96 - 101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901 - 917 (1987));

[0471] (b) CDRs at amino acid residues 24 - 34 (L1), 50 - 56 (L2), 89 - 97 (L3), 31 - 35b (H1), 50 - 65 (H2), and 95 - 102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991));

[0472] (c) antigen contact points at amino acid residues 27c - 36 (L1), 46 - 55 (L2), 89 - 96 (L3), 30 - 35b (H1), 47 - 58 (H2), and 93 - 101 (H3) (MacCallum et al. J. Mol. Biol. 262:732 - 745 (1996)); and

[0473] (d) A combination of (a), (b) and / or (c), comprising HVR amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3) and 94-102 (H3).

[0474] Unless otherwise specified, HVR residues and other residues (e.g., FR residues) in the variable domains are numbered herein according to Kabat et al., loc. cit.

[0475] HVR-H1, HVR-H2, HVR-H3, HVR-L1, HVR-L2 and HVR-L3 are also referred to as "H-CDR1", "H-CDR2", "H-CDR3", "L-CDR1", "L-CDR2" and "L-CDR3", respectively.

[0476] Framework

[0477] In one embodiment, the polypeptide of the present disclosure is an antibody and may have a variable region containing a framework.

[0478] In one embodiment, the polypeptide of the present disclosure may have a variable region containing a framework.

[0479] "Framework" or "FR" refers to the variable domain residues other than the hypervariable region (HVR) residues. The FR of the variable domain generally consists of the following four FR domains: FR1, FR2, FR3 and FR4. Thus, the HVR and FR sequences generally occur in VH (or VL) in the following sequence: FR1-H1 (L1)-FR2-H2 (L2)-FR3-H3 (L3)-FR4.

[0480] Antibody

[0481] In one embodiment, the polypeptide of the present disclosure is an antibody. The term "antibody" herein is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies and antibody fragments, as long as they exhibit the required antigen-binding activity.

[0482] Antibody class

[0483] In one embodiment, the polypeptide of the present disclosure is an antibody and is not limited to a specific class of antibody.

[0484] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five main classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of them can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are designated α, δ, ε, γ, and μ, respectively.

[0485] EU number

[0486] Unless otherwise specified, the numbering of amino acid residues in the light chain constant regions herein is according to Kabat et al., and the numbering of amino acid residues in the heavy chain constant regions is according to the EU numbering system, also known as the EU index, as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0487] Chimeric antibody

[0488] In one embodiment, the polypeptide of the present disclosure can be a chimeric antibody.

[0489] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. Similarly, the term "chimeric antibody variable domain" refers to an antibody variable domain in which a portion of the heavy and / or light chain variable region is derived from a particular source or species, while the remainder of the heavy and / or light chain variable region is derived from a different source or species.

[0490] Humanized antibody

[0491] In one embodiment, the polypeptide of the present disclosure can be a humanized antibody.

[0492] A "humanized" antibody is a chimeric antibody that contains amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will generally contain all or substantially all of at least one, and usually two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to the HVRs of a non-human antibody, and all or substantially all of the FRs correspond to the FRs of a human antibody. A humanized antibody optionally can contain at least a portion of the antibody constant region derived from a human antibody.

[0493] Human antibody

[0494] In one embodiment, the polypeptide of the present disclosure can be a human antibody.

[0495] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or an amino acid sequence of a non-human-derived antibody derived from a human antibody library or other human antibody-encoding sequences.

[0496] Nucleic acid (polynucleotide)

[0497] In one embodiment, the present disclosure relates to nucleic acids encoding the polypeptides of the present disclosure.

[0498] In one embodiment, the nucleic acid is RNA, DNA, or a vector or plasmid carrying the nucleic acid.

[0499] As used interchangeably herein, "nucleic acid" or "polynucleotide" refers to a polymer of nucleotides of any length and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides can contain modified nucleotides such as methylated nucleotides and their analogs. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can contain modifications made after synthesis, such as conjugation to a label.

[0500] In one embodiment, the RNA of the present disclosure includes, for example, mRNA, genomic RNA, and circular RNA.

[0501] The polynucleotides of the present disclosure can contain a 5' cap structure. "5' cap structure" refers to, for example, a 5'-modified nucleotide, particularly a guanine nucleotide, located at the 5' end of an RNA (such as mRNA). The 5′ cap structure can be linked via a 5′-5′ triphosphate bond. Examples of 5′ cap structures are cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose in the nucleotide adjacent to m7GpppN), cap2 (additional methylation of the ribose in the second nucleotide downstream of m7GpppN), cap3 (additional methylation of the ribose in the third nucleotide downstream of m7GpppN), cap4 (additional methylation of the ribose in the fourth nucleotide downstream of m7GpppN), ARCA (reverse cap analog), modified ARCA (e.g., phosphorothioate-modified ARCA), inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0502] The polynucleotides of the present disclosure may comprise at least one poly(A) sequence and / or at least one poly(C) sequence and / or at least one histone step-loop sequence / structure.

[0503] The polynucleotides of the present disclosure may comprise a 5' untranslated region (UTR) and / or a 3' untranslated region (UTR). In one embodiment, the untranslated region may include structures that facilitate translation, such as an IRES (internal ribosome entry site). In one embodiment, the nucleic acid encoding the polypeptide of the present disclosure is RNA (e.g., mRNA). In one embodiment, the RNA comprises one or more of the following elements: a coding region (coding sequence; CDS), a 5′ UTR, a 3' UTR, a Cap, and a poly-A tail. In one embodiment, the coding region comprises a start codon and a stop codon. In the RNA of the present disclosure, in one embodiment, one or more or all of the uridines may be replaced by pseudouridine (N1-methylpseudouridine).

[0504] In one embodiment, the nucleic acid of the present disclosure is DNA. In one embodiment, the DNA is configured such that the aforementioned RNA of the present disclosure is transcribed. In one embodiment, the DNA of the present disclosure is vector DNA or plasmid DNA. In one embodiment, the vector or plasmid comprises one or more of the following elements: an ORF (region encoding the polypeptide of the present disclosure), a promoter, a Kozak, a marker gene (in one embodiment, a drug selection gene, a fluorescent marker gene, a reporter gene, etc.).

[0505] The polynucleotides of the present disclosure may be formulated with a transporter. The transporter is, for example, a liposome, a liposome, a cationic lipid complex, a lipid nanoparticle, a polymer, a peptide, a protein, a cell, a nanoparticle mimic, a nanotube, or a conjugate. The transporter may contain lipids. The lipids may be selected from the group consisting of cholesterol, coprostanol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, α-tocopherol, and mixtures thereof.

[0506] Vector

[0507] In one embodiment, the present disclosure relates to a vector carrying the nucleic acid of the present disclosure.

[0508] As used herein, the term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of the host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids operably linked thereto. Such vectors are referred to herein as "expression vectors". A vector can be introduced into a host cell using viral or electroporation methods. However, the introduction of a vector is not limited to in vitro methods. For example, in vivo methods can also be used to directly introduce a vector into a subject.

[0509] Host cell

[0510] In one embodiment, the present disclosure relates to a host cell into which a nucleic acid of the present disclosure has been introduced. In one embodiment, the host cell is a cell that expresses a polypeptide of the present disclosure. The cell can be a eukaryotic cell or a prokaryotic cell.

[0511] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to, for example, a cell into which an exogenous nucleic acid has been introduced, including progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived from the primary transformed cells, regardless of the number of passages. The progeny may not be identical in nucleic acid content to the parental cell, but may contain mutations.

[0512] Furthermore, in one embodiment, the present disclosure relates to a polypeptide expressed by the aforementioned host cell.

[0513] Fc region

[0514] In the present disclosure, the terms "Fc region" or "Fc domain" are used interchangeably and refer to a region containing a fragment composed of a hinge or a part thereof and the CH2 and CH3 domains in an antibody molecule. The Fc region of the IgG class refers to, but is not limited to, the region from, for example, cysteine 226 (EU numbering) to the C-terminus or from proline 230 (EU numbering) to the C-terminus. The Fc region can preferably be obtained by partially digesting, for example, an IgG1, IgG2, IgG3, or IgG4 monoclonal antibody with a proteolytic enzyme such as pepsin, followed by eluting the fraction adsorbed on a protein A column or a protein G column. The proteolytic enzyme is not particularly limited as long as the enzyme can digest the whole antibody under appropriate set reaction conditions (e.g., pH) of the enzyme to restrictively form Fab or F(ab′)2. Examples thereof can include pepsin and papain.

[0515] In one embodiment, an Fc region derived from, for example, a naturally occurring IgG can be used as the "Fc region" prior to modification. In this case, a naturally occurring IgG means a polypeptide containing an amino acid sequence identical to that of an IgG found in nature and belonging to the antibody class substantially encoded by the immunoglobulin gamma gene. A naturally occurring human IgG means, for example, naturally occurring human IgG1, naturally occurring human IgG2, naturally occurring human IgG3, or naturally occurring human IgG4. Naturally occurring IgG also includes variants spontaneously derived therefrom, etc. Multiple allotypic sequences based on gene polymorphisms are described for the constant regions of human IgG1, human IgG2, human IgG3, and human IgG4 antibodies in Sequences of proteins of immunological interest, NIH Publication No. 91-3242, any of which can be used in the present disclosure. In particular, the sequence of human IgG1 can have DEL or EEM as the amino acid sequence at EU numbering positions 356 to 358.

[0516] In one embodiment described herein, the Fc region is an IgG Fc region. In one embodiment, the Fc region is the Fc region of IgG1, IgG2, IgG3, or IgG4. Further, in one embodiment, the Fc region is a human IgG1 Fc region.

[0517] In one embodiment, a polypeptide having the Fc region of the present disclosure shows a binding affinity for human FcRn or FcγR comparable to that of a naturally occurring (wild-type) human IgG1 Fc region.

[0518] In one embodiment, a polypeptide having the Fc region of the present disclosure shows a binding affinity for human FcRn or FcγR similar to that of the polypeptide prior to modification.

[0519] In one embodiment, the Fc region (Fc domain) exhibits a binding affinity for Fc receptors similar to that of a naturally occurring IgG1 Fc region. In one embodiment, the Fc region (or a polypeptide having the Fc region) exhibits a binding affinity for Fc receptors of 80% or higher, preferably 90% or higher, more preferably 95% or higher, and most preferably 98% or higher of that of a naturally occurring IgG1 Fc region (or a polypeptide containing a naturally occurring IgG1 Fc region).

[0520] In one embodiment, the Fc region (or a polypeptide having the Fc region) binds to an Fc receptor. In one embodiment, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activating Fc receptor. In a specific embodiment, the Fc receptor is an activating human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa.

[0521] In one embodiment, the Fc region (Fc domain) of the polypeptide of the present disclosure comprises modifications that do not reduce the binding affinity of the Fc region for Fc receptors. In one embodiment, the modifications of the polypeptide of the present disclosure do not reduce the binding affinity of the Fc region for human FcRn. In embodiments, such modifications include those shown in Table 8.

[0522] The modified Fc region (mutant Fc domain) can be prepared by genetic or chemical methods well known in the art, by amino acid deletion, substitution, insertion, or modification. Genetic methods can include site-directed mutagenesis of the coding DNA sequence, PCR, gene synthesis, etc. Correct nucleotide changes can be verified, for example, by sequencing.

[0523] Binding to Fc receptors can be readily determined, for example, by ELISA or by surface plasmon resonance (SPR) using standard instrumentation such as a BIAcore instrument (GE Healthcare), and Fc receptors, etc. can be obtained by recombinant expression.

[0524] Fc receptor

[0525] The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a naturally occurring human FcR. In some embodiments, the FcR is an FcR that binds IgG antibodies (a gamma receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced forms of those receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), both of which have similar amino acid sequences, differing mainly in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See, for example, Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). For example, the following references review FcR: Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). The term "Fc receptor" herein encompasses other FcRs, including those to be identified in the future.

[0526] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and the homeostatic regulation of immunoglobulins. Methods for measuring binding to FcRn are known (see, for example, Ghetie and Ward, Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.).

[0527] Fcγ receptor

[0528] Fcγ receptors refer to receptors that can bind to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies and include all members belonging to the protein family substantially encoded by the Fcγ receptor genes. In humans, this family includes FcγRI (CD64), which includes the isotypes FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes the isotypes FcγRIIa (including the allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes the isotypes FcγRIIIa (including the allotypes V158 and F158) and FcγRIIIb (including the allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2); as well as all uncharacterized human Fcγ receptors, Fcγ receptor isotypes, and their allotypes. However, Fcγ receptors are not limited to these examples. Fcγ receptors include those derived from humans, mice, rats, rabbits, and monkeys, but are not limited thereto. Fcγ receptors can be derived from any organism. Mouse Fcγ receptors include, but are not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as all uncharacterized mouse Fcγ receptors, Fcγ receptor isotypes, and their allotypes. Such preferred Fcγ receptors include, for example, human FcγRI (CD64), FcγRIIA (CD32), FcγRIIB (CD32), FcγRIIIA (CD16), and / or FcγRIIIB (CD16). The polynucleotide sequences and amino acid sequences of FcγRI are shown in RefSeq accession numbers NM_000566.3 and RefSeq accession number NP_000557.1, respectively; the polynucleotide sequences and amino acid sequences of FcγRIIA are shown in RefSeq accession numbers BC020823.1 and RefSeq accession number AAH20823.1, respectively; the polynucleotide sequences and amino acid sequences of FcγRIIB are shown in RefSeq accession numbers BC146678.1 and RefSeq accession number AAI46679.1, respectively; the polynucleotide sequences and amino acid sequences of FcγRIIIA are shown in RefSeq accession numbers BC033678.1 and RefSeq accession number AAH33678.1, respectively; and the polynucleotide sequences and amino acid sequences of FcγRIIIB are shown in RefSeq accession numbers BC128562.1 and RefSeq accession number AAI28563.1, respectively.In addition to the above FACS and ELISA formats, the binding activity of Fcγ receptors to the Fc domain of monoclonal IgG1, IgG2, IgG3, or IgG4 antibodies can also be evaluated by ALPHA screen (amplified luminescent proximity homogeneous assay), the BIACORE method based on surface plasmon resonance (SPR), etc. (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0529] Fcγ receptor binding activity

[0530] The binding activity of the Fc region (Fc domain) to any one of Fcγ receptors FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB can be evaluated by using the above FACS and ELISA formats, as well as ALPHA screen (amplified luminescent proximity homogeneous assay), the BIACORE method based on surface plasmon resonance (SPR) (Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005-4010).

[0531] ALPHA screen is performed by ALPHA technology using two types of beads: donor beads and acceptor beads based on the principle described below. A luminescence signal is detected only when the molecule linked to the donor bead biologically interacts with the molecule linked to the acceptor bead and when the positions of the two beads are in close proximity. Excited by a laser beam, the photosensitizer in the donor bead converts the oxygen around the bead into excited singlet oxygen. When the singlet oxygen diffuses around the donor bead and reaches the acceptor bead in close proximity, a chemiluminescence reaction is induced within the acceptor bead. This reaction ultimately causes luminescence. If the molecule linked to the donor bead does not interact with the molecule linked to the acceptor bead, the singlet oxygen generated by the donor bead will not reach the acceptor bead, and no chemiluminescence reaction will occur.

[0532] For example, a biotinylated antigen-binding molecule or antibody is immobilized to a donor bead, and a glutathione S-transferase (GST)-tagged Fcγ receptor is immobilized to a receptor bead. In the absence of a polypeptide comprising a competitive mutant Fc domain, the Fcγ receptor interacts with a polypeptide comprising a wild-type Fc region, thereby inducing a signal at 520 nm to 620 nm. A polypeptide having an unlabeled mutant Fc region competes with a polypeptide comprising a wild-type Fc region for interaction with the Fcγ receptor. The relative binding affinity can be determined by quantifying the fluorescence decrease resulting from the competition. Methods for biotinylating polypeptides such as antibodies using sulfo-NHS-biotin are known. Suitable methods for adding a GST tag to the Fcγ receptor include methods involving: fusing in-frame a polypeptide encoding the Fcγ receptor and GST, expressing the fusion gene using cells introduced with a vector carrying the gene, and then purifying using a glutathione column. The induced signal can preferably be analyzed, for example, by fitting to a single-site competition model using software such as GRAPHPAD PRISM (GraphPad; San Diego) based on non-linear regression analysis.

[0533] One of the substances used to observe their interaction is immobilized to the gold thin layer of the sensor chip as a ligand. When light is incident on the back surface of the sensor chip such that total internal reflection occurs at the interface between the gold thin layer and the glass, the intensity of the reflected light is partially decreased at a certain site (SPR signal). The other substance used to observe their interaction is injected onto the surface of the sensor chip as an analyte. When the analyte binds to the ligand, the mass of the immobilized ligand molecules increases. This changes the refractive index of the solvent on the surface of the sensor chip. The change in refractive index causes a shift in the position of the SPR signal (conversely, dissociation shifts the signal back to the original position). In the Biacore system, the above-mentioned shift amount (i.e., the mass change on the surface of the sensor chip) is plotted on the vertical axis, and thus the change in mass over time is shown as measurement data (sensing diagram). The kinetic parameters (association rate constant (ka) and dissociation rate constant (kd)) are determined from the curve of the sensing diagram, and the affinity (KD) is determined from the ratio between these two constants. Inhibition assays are preferably used for the BIACORE method. Examples of such inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103(11), 4005 - 4010.

[0534] Maintenance / reservation of Fc function and physicochemical properties

[0535] In one embodiment, a polypeptide having a modified Fc region of the present disclosure retains the function of Fc.

[0536] In one embodiment, a polypeptide having a modified Fc region of the present disclosure maintains the physicochemical properties of a polypeptide having a wild-type Fc region (in one embodiment, a wild-type IgG antibody, e.g., wild-type IgG1). In one embodiment, the physicochemical properties include the monomer ratio of the polypeptide, thermal stability (e.g., the thermal transition midpoint (Tm) value), antibody yield, and the like. In one embodiment, compared with the polypeptide (antibody) before modification, the monomer ratio, Tm value, and polypeptide (antibody) yield of the polypeptide (antibody) having a modified Fc region of the present disclosure do not change, do not decrease, or do not decrease significantly.

[0537] In one embodiment, "the monomer ratio of the polypeptide does not decrease significantly" means that compared with the polypeptide having a wild-type Fc region, the monomer ratio does not decrease by 50% or more, preferably 30% or more, more preferably 10% or more, or even more preferably 5% or more.

[0538] The monomer ratio of the polypeptide is measured by the following method: It is evaluated by size exclusion chromatography (SEC) using ACQUITY UPLC H-Class (Waters), where 50 mM phosphate buffer pH 7.0 (Isekyu) containing 300 mM sodium chloride is used as the running buffer, and a TSKgel SuperSW3000 custom column (4.6 mm x 15 cm, 4 μm, GelLot 89R) (TOSOH) is used as the analytical column. The chromatogram is recorded at a wavelength of UV 215 nm. The sample is diluted to 0.1 mg / mL and 10 μL is injected. The column temperature is set at 30 °C, and the measurement is performed at a flow rate of 0.35 mL / min for 10 minutes. The data is analyzed using Empower3 (Waters). The peak area ratios (%) of the monomer, related multimers, and degradation products of the antibody estimated from an antibody reference sample (e.g., tocilizumab) are calculated, and the monomer ratio is determined.

[0539] In one embodiment, "the Tm of the polypeptide does not decrease significantly" means that compared with the polypeptide having a wild-type Fc region, the Tm does not decrease by 30 °C or more, preferably 20 °C or more, more preferably 10 °C or more, even more preferably 5 °C or more, or even more preferably 2 °C or more.

[0540] The Tm of the polypeptide (antibody Fc region) was evaluated by differential scanning fluorimetry (DSF). It has been reported that the Tm determined by this method has a good correlation with the Tm determined by differential scanning calorimetry, a well-known method for evaluating the thermal stability of antibodies (Journal of Pharmaceutical Science 2010; 4: 1707-1720). A 5000x concentrate of SYPRO Orange protein gel stain (Invitrogen) was diluted 50-fold with 0.5 M PBS (Sigma), and 18 μL of the Fc-containing polypeptide solution diluted to 0.1 mg / mL was mixed with 2 μL of this detection dye. The 20-μL mixture was dispensed into tubes for measurement, and its temperature was increased from 30 °C to 99 °C at a rate of 240 °C / hr using a Rotor-Gene Q (QIAGEN). Fluorescence changes were observed at 470 nm (excitation wavelength) / 555 nm (fluorescence wavelength) as the temperature increased. The data obtained were used to determine the temperature at which a fluorescence transition was observed using Rotor-Gene Q series software (QIAGEN), and the lowest value (Tm1) was determined as the Tm value derived from the Fc region. However, if the Tm value derived from the polypeptide domain fused to the Fc region is equivalent to or lower than the Tm value of Fc, the Tm1 value cannot be called the Tm derived from Fc. In this case, it is necessary to separately assign the Tm derived from the Fc region (CH3, CH2) by preparing and comparing the individual Fc domains or enzymatically cleaving the polypeptide domain fused to the Fc region before measurement.

[0541] In one embodiment, "the yield of the polypeptide (antibody) is not significantly reduced" means that the yield of the polypeptide (antibody) is not reduced by 95% or more, preferably 80% or more, more preferably 50% or more, even more preferably 30% or more, or more preferably 10% or more, compared to the polypeptide having a wild-type Fc region.

[0542] The yield of the polypeptide (antibody) can be determined by the following method: Transfect 1 mL of Expi293-F cells (Thermo Fisher Scientific) at a density of 2E+6 cells / mL with a total of 1 μg of plasmid encoding the full-length heavy and light chains of the antibody at a mass ratio of 1:1 or 1:2, and allow transient expression. For transfection, use the ExpiFectamine 293 transfection kit (Thermo Fisher Scientific). Four days after transfection, collect the culture supernatant and purify it using MonoSpin ProA (GL science) or MonoSpin ProG (GL science). The amount (mg) of the antibody thus purified is determined as the yield of the polypeptide (antibody). Polypeptides having a wild-type Fc region and polypeptides having amino acid modifications in the Fc region can be expressed and purified by the same method, and their yields can be compared to calculate the expression reduction ratio.

[0543] In one embodiment, the expression level of the polypeptide (in one embodiment, the antibody) of the present disclosure having a modified Fc region is comparable to the expression level of the wild-type polypeptide (e.g., IgG1). The monomer formation ratio is also high, and the Tm of the Fc region is also comparable.

[0544] In one embodiment, the polypeptide of the present disclosure having a modified Fc region maintains the Fc function of the polypeptide having a wild-type Fc region (in one embodiment, a wild-type IgG antibody, such as wild-type IgG1). In one embodiment, the Fc function includes the ability to bind to FcRn or FcγR. In one embodiment, compared to the polypeptide (antibody) before modification, as a result of the modification in the CH3 interface, the FcRn binding ability and / or FcγR binding ability of the polypeptide (in one embodiment, the antibody) of the present disclosure having a modified Fc region do not change, do not decrease, or do not decrease significantly.

[0545] In one embodiment, "the FcRn binding ability of the polypeptide does not decrease significantly" means that the KD value of FcRn does not become 50 times or more, preferably 10 times or more, more preferably 2 times or more, or even more preferably 1.1 times or more of the KD value of the polypeptide having a wild-type Fc region.

[0546] The KD value of FcRn can be determined by the following method: Binding to human neonatal Fc receptor (FcRn) was evaluated using a Biacore T200 (Cytiva). Evaluation was performed at 25 °C using 50 mM phosphate buffer, 150 mM NaCl, 0.05 w / v%-P20 (pH 6.0) as the running buffer. rProtein L (BioVision) was immobilized as a ligand capture molecule onto a Series S CM4 (Cytiva). However, in the case of antibody molecules that do not bind to Protein L, other capture molecules such as Protein A and Protein G can be used. An antibody solution prepared with the running buffer was allowed to interact with the CM4 sensor chip to capture approximately 400 RU of antibody. The human FcRn protein used in this measurement was prepared by the method described in WO2010107110. Human FcRn was diluted to 0, 250, 500, 1000, 2000, and 4000 nM with the running buffer and allowed to bind to the captured antibody. The chip was regenerated using 10 mM glycine-HCl (pH 1.5) and repeatedly used for antibody capture for measurement. The KD (M) of each antibody for FcRn was calculated using Biacore T200 evaluation software 3.2.1 and the steady-state model. The binding ability to human FcRn can be compared by calculating the ratio of the KD value of a polypeptide having an amino acid modification in the Fc region to the KD value of a polypeptide having a wild-type Fc region.

[0547] In one embodiment, "the FcγR binding ability of the polypeptide is not significantly reduced" means that due to the modification in the CH3 interface, the binding level to FcγR / the amount of captured antibody does not become 1 / 10 or less, preferably 1 / 5 or less, more preferably 1 / 2 or less, or even more preferably 2 / 3 or less of that of a polypeptide having a wild-type Fc region.

[0548] The binding level to FcγR / amount of captured antibody can be measured by the following method: The binding activity of the generated Fc-modified antibody to each human Fcγ receptor was evaluated using a Biacore T200 (Cytiva). The evaluation was performed at 25 °C using 50 mM phosphate buffer, 150 mM NaCl, 0.05 w / v%-P20 (pH 7.4) as the running buffer. rProtein L (BioVision) was immobilized as a ligand capture molecule onto a Series S CM4 (Cytiva). An antibody solution prepared with the running buffer was allowed to interact with the CM4 sensor chip to capture approximately 500 RU of antibody in the case of measuring human FcγRIa and 2000 RU of antibody in the case of measuring other human FcγRs. The human FcγR protein for this measurement was prepared by the method described in WO2022220275. In the case of FcγRIa, the human FcγR was diluted to 8 nM with the running buffer, or in the case of other FcγRs, diluted to 1000 nM, and allowed to bind to the captured antibody. The chip was regenerated using 10 mM glycine-HCl (pH 1.5) and repeatedly used to capture antibody for measurement. The binding activity of each antibody to each FcγR was evaluated by calculating the FcγR binding (RU) level / amount of antibody per unit using the Biacore T200 evaluation software version 3.2.1. The binding ability to human FcγR can be compared by calculating the ratio of the FcγR binding (RU) level / amount of antibody per unit of the polypeptide having an amino acid modification in the Fc region to the FcγR binding (RU) level / amount of antibody per unit of the polypeptide having a wild-type Fc region.

[0549] In one embodiment, the polypeptide (in one embodiment, an antibody) of the present disclosure having a modified Fc region has both an FcRn binding ability and an FcγR binding ability comparable to that of a wild-type polypeptide (e.g., IgG1).

[0550] Affinity

[0551] In one embodiment, the polypeptide of the present disclosure has an affinity between polypeptides (e.g., between CH3s).

[0552] In one embodiment, the polypeptide of the present disclosure binds to FcRn or FcγR. This binding can be evaluated from the affinity of the polypeptide of the present disclosure for FcRn or FcγR.

[0553] "Affinity" refers to the strength of the non-covalent interaction between a single binding site of a molecule (e.g., a polypeptide or an antibody) and its binding partner (e.g., FcRn). The affinity of molecule X for its partner Y can generally be expressed by the dissociation constant (KD), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). Affinity can be measured by well-established methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0554] In one embodiment, KD is measured by a radiolabeled antigen binding assay (RIA). In one implementation, the Fab form of the target antibody and its antigen are used to perform the RIA. For example, the solution binding affinity of a polypeptide for an assay target is measured by equilibrating the polypeptide with a minimal concentration ( 125 I) of radiolabeled antigen in the presence of a series of unlabeled antigen titrations, and then capturing the bound assay target with a plate coated with an anti-polypeptide antibody (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)).

[0555] According to another embodiment, a surface plasmon resonance assay, such as an assay using or (BIAcore, Inc., Piscataway, NJ) is used to measure Kd.

[0556] Control of association

[0557] In one embodiment, the control of the association of the present disclosure is to control the association between polypeptides of the same type. In one embodiment, it is the control of the association between heavy chains of the same type or between CH3 domains of the same type.

[0558] In one embodiment, the association between a polypeptide having a modified Fc region and a polypeptide having a wild-type Fc region is controlled.

[0559] In one embodiment, the association between a modified CH3 and a wild-type CH3 is controlled.

[0560] In one embodiment, the control of the association of the present disclosure includes controlling the association between any polypeptides, such as the association between any polypeptides containing an Fc region.

[0561] In one embodiment, the associated "control" refers to promoting the association between polypeptides into which the modifications of the present disclosure have been introduced, or promoting or increasing the formation of homodimers of the polypeptides of the present disclosure. (The formation of the homodimers mentioned herein refers to the formation of dimer molecules through the association of two identical polypeptides. In the case of antibodies, it particularly refers to a molecule comprising components in which any two identical polypeptides comprising an Fc region are associated.) In one embodiment, the associated "control" is suppressing or inhibiting the formation of heterodimers of the polypeptides of the present disclosure. The formation of the heterodimers mentioned herein refers to dimer molecules formed by the association of two different polypeptides. In the case of antibodies, it particularly refers to a molecule comprising components in which any two different polypeptides comprising an Fc region are associated. Alternatively, it may be referred to as a heteromultimer, as components in which multiple different polypeptides are associated.

[0562] Polypeptide into which a modification has been introduced

[0563] In one embodiment, the present disclosure relates to polypeptides comprising an Fc region into which a modification has been introduced (also referred to as "the polypeptides of the present disclosure").

[0564] In one embodiment, the polypeptides of the present disclosure are antibodies and may have variable regions or variable domains.

[0565] In one embodiment, the present disclosure relates to polypeptides having a modification introduced into the heavy chain Fc region or the heavy chain CH3.

[0566] In one embodiment, the polypeptides of the present disclosure have a heavy chain Fc region. In one embodiment, the polypeptide comprises, for example, a CH3 region.

[0567] In one embodiment, the polypeptides of the present disclosure are antibody heavy chains. The polypeptides of the present disclosure may have an antigen-binding domain, a heavy chain variable region, and / or a light chain variable region.

[0568] In one embodiment, the polypeptide comprising an Fc region into which a modification has been introduced according to the present disclosure associates more readily with a polypeptide having the modification than with a polypeptide not having the modification.

[0569] In one embodiment, the polypeptide comprising an Fc region into which a modification has been introduced according to the present disclosure associates less readily with a polypeptide not having the modification than with a polypeptide having the modification.

[0570] In one embodiment, the polypeptide comprising an Fc region into which a modification has been introduced according to the present disclosure is less likely to form a heterodimer with another polypeptide not having the modification. In one embodiment, the polypeptides of the present disclosure are less likely to form heterodimers compared to homodimers. In one embodiment, the polypeptides of the present disclosure are more likely to form homodimers compared to heterodimers.

[0571] In one embodiment, compared to a polypeptide without a modification, a polypeptide according to the present disclosure comprising an Fc region into which a modification has been introduced has an increased ability to form associated multimers with a polypeptide having the modification.

[0572] In one embodiment, a polypeptide according to the present disclosure comprising an Fc region into which a modification has been introduced associates with a polypeptide having the modification.

[0573] In one embodiment, the polypeptides of the present disclosure form homomers (homodimers, homodimerized molecules).

[0574] In one embodiment, the polypeptides of the present disclosure exhibit a stronger ability to promote homodimerization than a control (e.g., a polypeptide before modification, wild-type IgG, etc.).

[0575] In one embodiment, the polypeptides of the present disclosure are polypeptides in which the association between polypeptides is controlled. In one embodiment, the association is an association between Fc regions or between CH3 domains.

[0576] In one embodiment, the polypeptides of the present disclosure do not associate or are less likely to associate with endogenous IgG or antibody fragments thereof containing an Fc region.

[0577] Modification to be introduced

[0578] In one embodiment, due to one of the following effects caused by the introduced modification, the association of the polypeptides of the present disclosure with a polypeptide having the modification is easier than the association with a polypeptide without the modification: (1) steric complementarity (also referred to as "steric hindrance"), (2) disulfide bonding (also referred to as "disulfide bond"), and (3) electrostatic charge (also referred to as "charge").

[0579] In one embodiment, the modification introduced into the polypeptides of the present disclosure causes (1) steric complementarity (also referred to as "steric hindrance"), (2) disulfide bonding (also referred to as "disulfide bond"), or (3) electrostatic charge (also referred to as "charge").

[0580] Spatial complementarity (steric hindrance control)

[0581] In one embodiment, the modifications according to the present disclosure that cause steric complementarity (steric hindrance) include modifying amino acid residues in the region where the association between polypeptides occurs (e.g., the CH3 interface) into bulky amino acids and / or small amino acids. In one embodiment, the modification into bulky amino acids and small amino acids is carried out simultaneously. In one embodiment, such modifications include replacing an original small amino acid or non-bulky amino acid with a bulky amino acid or replacing an original bulky amino acid or non-bulky amino acid with a small amino acid before the modification.

[0582] In one embodiment, "bulky amino acids" include those amino acids having a larger molecular weight than the amino acid prior to modification. Bulky amino acids include, for example, tyrosine (Y), tryptophan (W), arginine (R), histidine (H), phenylalanine (F), leucine (L), valine (V), isoleucine (I), methionine (M), serine (S), threonine (T), cysteine (C), asparagine (N), glutamine (Q), lysine (K), aspartic acid (D), and glutamic acid (E).

[0583] In one embodiment, "small amino acids" include those amino acids having a smaller molecular weight than the amino acid prior to modification. Smaller amino acids include, for example, alanine (A), threonine (T), leucine (L), valine (V), asparagine (N), serine (S), isoleucine (I), methionine (M), glycine (G), cysteine (C), glutamine (Q), lysine (K), arginine (R), histidine (H), aspartic acid (D), and glutamic acid (E).

[0584] In one embodiment, the modification introduced into the polypeptides of the present disclosure gives rise to steric complementarity (steric hindrance). In one embodiment, the steric complementarity is generated by introducing a knob and a hole into the polypeptides of the present disclosure.

[0585] In one embodiment, the modification is a so-called "knob and hole" modification, including a protrusion ("knob") modification and a cavity ("hole") modification in the Fc region. The knob and hole structure technology is described, for example, in US 5,731,168; US 7,695,936; Ridgway et al., Prot Eng 9, 617 - 621 (1996) and Carter, J Immunol Meth 248, 7 - 15 (2001). The protrusion (knob) is constructed, for example, by replacing a small amino acid side chain at the interface of the polypeptide of the present disclosure with a larger side chain (such as tyrosine or tryptophan). A compensatory cavity (hole) having the same or similar size as the protrusion is created in the interface of the polypeptide of the present disclosure by, for example, replacing a large amino acid side chain with a smaller amino acid side chain (such as alanine or threonine).

[0586] In one embodiment, the steric complementarity is generated by the presence of the knob and the hole in the Fc region. In one embodiment, the modification introduced into the polypeptides of the present disclosure is the introduction of the knob and the hole into the Fc region. The knob and the hole in the Fc region induce or promote the formation of homodimers of the polypeptides of the present disclosure.

[0587] In one embodiment, both the knob and the hole are introduced into the same molecule of the polypeptides of the present disclosure.

[0588] In one embodiment, in the CH3 domain of the Fc region of a polypeptide, an amino acid residue is replaced with an amino acid residue having a larger side chain volume (the pestle structure), and another amino acid position in the same polypeptide is replaced with an amino acid residue having a smaller side chain volume to form a cavity (the mortar structure).

[0589] The protrusion and the cavity can be prepared by altering the nucleic acid encoding the polypeptide (e.g., by site-directed mutagenesis or by peptide synthesis).

[0590] In one embodiment, the amino acid positions modified for spatial complementarity (steric hindrance control) include, for example, the following positions on the Fc region:

[0591] Positions 347, 349, 350, 351, 354, 357, 364, 366, 368, 370, 392, 394, 399, 405, 407, and 409 (EU numbering).

[0592] In one embodiment, the amino acid modifications for spatial complementarity (steric hindrance control) include, for example, the following modifications (amino acid substitutions):

[0593] Q347E, T350V, L351Y, S354Y, Y349T, E357N, S364H, T366W, T366Y, T366V, T366L, L368A, K370E, K392L, T394W, T394F, D399V, D399M, F405A, F405L, F405T, Y407V, Y407T, Y407A, K409R, K409W, and K409V.

[0594] In one embodiment, one or more of the above modifications are combined.

[0595] In one embodiment, the combinations of amino acid modifications for spatial complementarity (steric hindrance control) include, for example, the following:

[0596] T394W / F405A (where " / " means that both the foregoing and following modifications are included; the same applies hereinafter), T366W / L368A / Y407V, T366Y / Y407T, T366W / Y407A, T366Y / T394W / F405A / Y407T, E357N / K370E / D399V / F405T / K409W, Y349T / S364H / T394F / F405A, T394F / F405A, Q347E / S354Y / T366Y / Y407T, T350V / L351Y / T366L / T394W / F405A, K392L / T394W / F405A / Y407V, and T350V / L351Y / T366L / K392L / T394W / F405A / Y407V.

[0597] Disulfide bonding (disulfide control)

[0598] In one embodiment, the modifications introduced into the polypeptides of the present disclosure result in disulfide bonding (disulfide bridges) between the modified polypeptides.

[0599] Modifications that introduce disulfide bonding in the polypeptides of the present disclosure include, for example, replacing an amino acid other than cysteine with cysteine.

[0600] In one embodiment, the modification is the introduction of one or more cysteines into the Fc region. In one embodiment, the modification is the replacement of one or more amino acids in the Fc region with cysteine.

[0601] In one embodiment, disulfide bonding is formed between the modified polypeptides, thereby stabilizing the homodimer.

[0602] In one embodiment, at least one amino acid substitution having a cysteine residue is introduced into a polypeptide comprising an Fc region, and disulfide bonding is formed between the introduced cysteine and an existing cysteine.

[0603] In one embodiment, at least two amino acid substitutions having cysteine residues are introduced into a polypeptide comprising an Fc region, and disulfide bonding is formed between the introduced cysteine residues.

[0604] In one embodiment, disulfide bonding is formed between the polypeptides of the present disclosure.

[0605] In one embodiment, disulfide bonding is formed between a modified (introduced) cysteine in one of the polypeptides forming a homodimer and a cysteine at the corresponding identical position in the other polypeptide.

[0606] In one embodiment, a disulfide bond is formed between a modified (introduced) cysteine in one of the polypeptides forming a homodimer and a cysteine at a different position in the other polypeptide.

[0607] In one embodiment, amino acid positions for modification (substitution with cysteine) for disulfide bonding (disulfide bond control) include, for example, the following positions on the Fc region:

[0608] 349, 351, 354, 356, 357, 392, 394, 397, and 399 (EU numbering).

[0609] In one embodiment, modifications (substitution with cysteine) at one or more of the above positions are combined.

[0610] In one embodiment, amino acid combinations for modification (substitution with cysteine) for disulfide bonding (disulfide bond control) include, for example, the following:

[0611] K392C / D399C, Y349C / E356C, Y349C / E357C, L351C / S354C, and T394C / V397C.

[0612] In one embodiment, these modifications are combined.

[0613] In one embodiment, combined modifications (substitution with cysteine) include, for example, the following:

[0614] Y349C / E357C / K392C / D399C, Y349C / L351C / S354C / E357C, Y349C / E357C / T394C / V397C, Y349C / E357C / K392C / T394C / V397C / D399C, K392C / T394C / V397C / D399C, and L351C / S354C / K392C / D399C.

[0615] Electrostatic charge (charge control)

[0616] In one embodiment, the modification introduced into the polypeptides of the present disclosure is the introduction of a charged amino acid. In one embodiment, such modifications include substituting an amino acid residue in the polypeptides of the present disclosure with a charged amino acid residue.

[0617] In one embodiment, the effect of the electrostatic charge is generated by one or more charged amino acids modified (introduced) in the Fc region.

[0618] In one embodiment, the "charged amino acid residue" (charged amino acid) is, for example, selected from amino acid residues in any of the following groups:

[0619] (a) Glutamic acid (E) and aspartic acid (D); and

[0620] (b) Lysine (K), arginine (R) and histidine (H).

[0621] In one embodiment, the modification introduced into the polypeptides of the present disclosure is the introduction of two or more amino acids with opposite charges.

[0622] The phrase "with opposite charges" means that, for example, when at least one of two or more amino acid residues is selected from the amino acid residues included in any one of the above groups (a) and (b), the remaining amino acid residues are selected from the amino acid residues included in the other group.

[0623] In one embodiment, the modification of the polypeptides of the present disclosure introduces positively charged amino acids and / or negatively charged amino acids.

[0624] Generally, lysine (K), arginine (R) and histidine (H) are referred to as positively charged amino acids (positively charged amino acids). Glutamic acid (E) and aspartic acid (D) are referred to as negatively charged amino acids (negatively charged amino acids).

[0625] In one embodiment, the above-mentioned positively charged amino acids are selected from lysine (K), arginine (R) and histidine (H), and / or the above-mentioned negatively charged amino acids are selected from aspartic acid (D) and glutamic acid (E).

[0626] In one embodiment, the amino acid positions modified for electrostatic charge (charge control) include, for example, the following positions on the Fc region:

[0627] Positions 345, 347, 351, 356, 357, 360, 366, 370, 392, 399, 409 and 439 (EU numbering).

[0628] In one embodiment, the modifications at one or more of the above positions are combined.

[0629] In one embodiment, the positions to be modified into positively charged amino acids include, for example, the following:

[0630] Positions 345, 347, 351, 356, 357, 366 and 399.

[0631] In one embodiment, the positions to be modified into negatively charged amino acids include, for example, the following:

[0632] Positions 351, 360, 370, 392, 409 and 439.

[0633] In one embodiment, the combinations of amino acid positions to be modified to a positively charged amino acid or a negatively charged amino acid include, for example, the following:

[0634] Modifying position 345 to a positively charged amino acid and modifying position 360 to a negatively charged amino acid;

[0635] Modifying position 347 to a positively charged amino acid and modifying position 360 to a negatively charged amino acid;

[0636] Modifying position 357 to a positively charged amino acid and modifying position 370 to a negatively charged amino acid;

[0637] Modifying position 399 to a positively charged amino acid and modifying position 409 to a negatively charged amino acid;

[0638] Modifying position 399 to a positively charged amino acid and modifying position 392 to a negatively charged amino acid;

[0639] Modifying position 366 to a positively charged amino acid and modifying position 351 to a negatively charged amino acid;

[0640] Modifying position 351 to a positively charged amino acid and modifying position 366 to a negatively charged amino acid; and

[0641] Modifying position 356 to a positively charged amino acid and modifying position 439 to a negatively charged amino acid.

[0642] In one embodiment, one or more of the above modifications are combined. In one embodiment, the above modifications can be combined with further modifications to individual positively charged amino acids or negatively charged amino acids.

[0643] For example, the combined modifications include, for example, the following:

[0644] "Modifying position 357 to a positively charged amino acid and modifying position 370 to a negatively charged amino acid" and "modifying position 399 to a positively charged amino acid and modifying position 409 to a negatively charged amino acid";

[0645] "Modifying position 356 to a positively charged amino acid and modifying position 439 to a negatively charged amino acid" and "modifying position 399 to a positively charged amino acid and modifying position 409 to a negatively charged amino acid";

[0646] "Modifying position 356 to a positively charged amino acid and modifying position 439 to a negatively charged amino acid" and "modifying position 357 to a positively charged amino acid and modifying position 370 to a negatively charged amino acid";

[0647] "Modifying position 356 to a positively charged amino acid and modifying position 439 to a negatively charged amino acid" and "modifying position 399 to a positively charged amino acid and modifying position 392 to a negatively charged amino acid";

[0648] "Modifying position 357 to a positively charged amino acid and modifying position 370 to a negatively charged amino acid" and "modifying position 399 to a positively charged amino acid and modifying position 392 to a negatively charged amino acid";

[0649] "Modifying position 399 to a positively charged amino acid and modifying position 409 to a negatively charged amino acid" and "modifying position 392 to a negatively charged amino acid";

[0650] "Modifying position 399 to a positively charged amino acid and modifying position 409 to a negatively charged amino acid", "modifying position 392 to a negatively charged amino acid" and "modifying position 356 to a positively charged amino acid"; and

[0651] "Modifying position 356 to a positively charged amino acid and modifying position 439 to a negatively charged amino acid", "modifying position 357 to a positively charged amino acid and modifying position 370 to a negatively charged amino acid" and "modifying position 399 to a positively charged amino acid and modifying position 409 to a negatively charged amino acid".

[0652] In one embodiment, amino acid modifications for electrostatic charge (charge control) include, for example, the following modifications:

[0653] E345K / K360E, E345R / K360E, Q347R / K360D, Q347R / K360E, E357K / K370E, D399K / K409E, D399K / K409D, D399R / K409E, D399R / K409D, K392E / D399K, K392D / D399K, K392E / D399R, K392D / D399R, L351D / T366K, L351E / T366K, L351K / T366D, L351K / T366E and E356K / K439E.

[0654] In one embodiment, two or more of these modifications can be combined. Examples include the following:

[0655] E357K / K370E / D399K / K409E, E356K / D399K / K409E / K439E, E356K / E357K / K370E / K439E, E356K / K392D / D399K / K439E, E357K / K370E / K392D / D399K, K392D / D399K / K409D, E356K / K392D / D399K / K409D, and E356K / E357K / K370E / D399K / K409E / K439E.

[0656] Combination of introduced modifications

[0657] In one embodiment, the modifications introduced into the polypeptides of the present disclosure can be a combination of two or more different types of modifications.

[0658] In one embodiment, the combination of two or more different types of modifications is a combination selected from the foregoing (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0659] In one embodiment, the combination is a combination of (1) steric complementarity and (2) disulfide bonding, a combination of (1) steric complementarity and (3) electrostatic charge, or a combination of (2) disulfide bonding and (3) electrostatic charge.

[0660] In one embodiment, the modifications introduced into the polypeptides of the present disclosure include introducing all of the modifications of (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0661] In the case of the combination of (1) steric complementarity and (3) electrostatic charge, in one embodiment, examples of the modifications include, but are not limited to, the following:

[0662] Q347R / K360E / D399V / F405T / K409W, E345R / Q347R / K360D / T366V / D399M / Y407A / K409V, and E345R / K360E / D399M / Y407A / K409V.

[0663] In the case of the combination of (2) disulfide bonding and (3) electrostatic charge, in one embodiment, examples of the modifications include, but are not limited to, the following:

[0664] Y349C / E357C / K392D / D399K, E357K / K370E / T394C / V397C, Y349C / E357C / K370E, E357K / K370E / K392C / D399C, and K392D / T394C / V397C / D399K.

[0665] In one embodiment, different modifications can be introduced into each of the plurality of polypeptides of the present disclosure to control the association of each polypeptide (in one embodiment, to promote the formation of homodimers of each polypeptide).

[0666] In one embodiment, the modifications in the polypeptides of the present disclosure promote the formation of homodimers of the polypeptides. Amino acid modifications that exhibit strong homodimer formation promoting ability include, but are not limited to, the following (the category of modification effect is shown in parentheses):

[0667] T394W / F405A (steric hindrance)

[0668] T366W / L368A / Y407V (steric hindrance)

[0669] Q347R / K360E / D399V / F405T / K409W (steric hindrance + charge)

[0670] T394F / F405A (steric hindrance)

[0671] E356K / D399K / K409E / K439E (charge)

[0672] Q347E / S354Y / T366Y / Y407T (steric hindrance)

[0673] K392L / T394W / F405A / Y407V (steric hindrance)

[0674] E356K / K392D / D399K / K439E (charge)

[0675] K392D / D399K / K409D (charge)

[0676] Y349C / E357C / K392D / D399K (charge + disulfide bonding)

[0677] E345R / Q347R / K360D / T366V / D399M / Y407A / K409V (steric hindrance + charge)

[0678] E356K / K392D / D399K / K409D (charge)

[0679] E345R / K360E / D399M / Y407A / K409V (steric hindrance + charge)

[0680] T350V / L351Y / T366L / K392L / T394W / F405A / Y407V (steric hindrance).

[0681] In addition, in one embodiment, such modifications include those shown in Tables 16 - 18. In one embodiment, the amino acid modifications disclosed herein can also be combined. Examples include combinations of amino acid modifications shown in Tables 19 - 21.

[0682] In one embodiment, the modifications introduced into the plurality of polypeptides of the present disclosure are selected from the above (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge. In one embodiment, when a modification directed to (1) steric complementarity, (2) disulfide bonding, or (3) electrostatic charge is introduced into a first polypeptide, a modification different from the modification in the first polypeptide is introduced into a second polypeptide.

[0683] In one embodiment, the combination of modifications introduced into the first polypeptide and the second polypeptide includes combinations shown in Table 5 in which the amount of heterodimer is less than 52%.

[0684] In one embodiment, when two antibodies are co - expressed, those combinations of modifications can be used. As shown in Example 6, the modifications associated with inhibiting heterodimers in the experimental results are not expected to cause hetero - oligomeric associations, regardless of how many of them are combined. Thus, such pairs can be used not only for co - expressing two antibodies, but also for co - expressing three, four, five, or more antibodies.

[0685] Modification of amino acid residues

[0686] "Modification" of an amino acid residue in the present disclosure specifically refers to replacing the original amino acid residue with another amino acid residue, deleting the original amino acid residue, adding a new amino acid residue, etc. In one embodiment, the modification refers to replacing the original amino acid residue with another amino acid residue.

[0687] The modifications introduced into the polypeptides of the present disclosure are not limited to only the modifications directed to the above (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge, but in one embodiment, can further include modifications other than these modifications.

[0688] In one embodiment, when the polypeptide of the present disclosure is an antibody, the constant region of the antibody, particularly the heavy - chain constant region, can be modified as needed to improve antibody function and / or stability. Examples of modifications that improve antibody function include modifications that enhance or weaken the binding of the antibody to the Fcγ receptor (FcγR), modifications that enhance or weaken the binding of the antibody to FcRn, and modifications that enhance or weaken the cytotoxic activity of the antibody (e.g., ADCC activity, CDC activity, etc.). In addition, in one embodiment, modifications that improve the heterogeneity of the antibody and / or improve immunogenicity and / or pharmacokinetics can be included. In addition, in one embodiment, modifications that promote antibody hexamerization and modifications to the glycosylation sequence can be included.

[0689] Regarding the heterogeneity of the C-terminal sequence of the IgG antibody heavy chain, deletion of the C-terminal lysine residue and amidation of the C-terminal carboxyl group due to deletion of both of the two C-terminal amino acids, glycine and lysine, have been reported (Anal Biochem. January 1, 2007; 360(1):75-83). Thus, when the polypeptide of the present disclosure is an antibody, in one embodiment, IgG having a C-terminal lysine or deletion of C-terminal lysine and glycine can be used to reduce the C-terminal heterogeneity of the heavy chain Fc region.

[0690] Interface

[0691] In one embodiment, the above-described modification is introduced into the polypeptide region that forms the interface. In one embodiment, the above-described modification is introduced into the polypeptide region of the Fc region that forms the interface and is included in the Fc region. In one embodiment, the modification is introduced into the region that forms the CH3 interface.

[0692] The "interface" in the present disclosure generally refers to the surface where association (interaction) occurs. The amino acid residues that form the interface generally refer to one or more amino acid residues included in the polypeptide region that undergoes the association, and in one embodiment, refer to the amino acid residues that are close to and participate in the interaction when the association occurs.

[0693] In the present disclosure, the amino acid residues in the polypeptide that undergo modification are not limited to the amino acid residues in the Fc region or the CH3 region. In one embodiment, the amino acid residues in the region that forms the interface between the polypeptides are modified. Those skilled in the art can identify the amino acid residues that form the interface by using commercially available software such as homology modeling. Then, the amino acid residues at these positions can be modified to control the association.

[0694] In one embodiment, the amino acid residues to be modified are the amino acid residues that are close to each other when the association occurs between the polypeptide regions that form the interface.

[0695] Antibody format undergoing modification

[0696] In one embodiment, the antibody format to be subjected to the modifications of the present disclosure is the IgG format. For example, wild-type IgG without modifications in the Fc or IgG containing modifications in the Fc can be used. In one embodiment, various antibody formats can be used, such as VHH or scFv linked to the Fc, and antibody formats in which amino acid modifications for altering Fc function have been introduced. In one embodiment, an IgG format having a deglycosylated Fc region and / or containing modifications for enhancing binding to FcRn, inhibiting binding to FcγR, and / or promoting hexamer formation can be used. In one embodiment, an Ig format containing an Fc linked to a polypeptide containing a Fab, VHH, or scFv or to a polypeptide other than a Fab, VHH, or scFv can also be used. In one embodiment, an IgG format containing amino acid modifications that do not affect the Fc interface can be used.

[0697] Co-expression of homodimers and heteromultimers

[0698] In one embodiment, the amino acid modifications for promoting the formation of homodimers of the present disclosure can be used in combination with the amino acid modifications for promoting the formation of heterodimers (e.g., knob-into-hole modifications).

[0699] In one embodiment, the present disclosure relates to a method for co-expressing a homodimer and a heteromultimer.

[0700] In one embodiment, the method includes:

[0701] the step of obtaining a nucleic acid encoding a first polypeptide;

[0702] the step of obtaining a nucleic acid encoding a second polypeptide;

[0703] the step of obtaining a nucleic acid encoding a third polypeptide; and / or

[0704] the step of expressing the nucleic acid.

[0705] Here, in one embodiment, the first polypeptide has an Fc region into which modifications have been introduced. Due to the modifications introduced into the Fc region, the first polypeptide associates with a first polypeptide having modifications via the Fc region more readily than with a first polypeptide containing an Fc region into which modifications have not been introduced.

[0706] Here, in one embodiment, the second polypeptide associates with the third polypeptide more readily than with the second polypeptide.

[0707] Here, in one embodiment, the second polypeptide has an Fc region into which modifications have been introduced. Due to the modifications introduced into the Fc region, the second polypeptide associates with the third polypeptide via the Fc region more readily than with the second polypeptide.

[0708] In one embodiment, the amino acid modifications for promoting heterodimer formation include the modifications shown in Example 13 described hereinafter.

[0709] Combination of modifications showing strong heteromeric association inhibition

[0710] In one embodiment, the above-mentioned Fc region into which the modification has been introduced includes an Fc region into which a modification (e.g., a combination of modifications) showing strong heteromeric association inhibition has been introduced. Combinations of modifications showing strong heteromeric association inhibition include, for example, the amino acid modifications shown in Tables 19-21.

[0711] In one embodiment, the above-mentioned Fc region into which the modification has been introduced contains an amino acid modification at one position combination or two or more position combinations selected from the position combinations shown in the following (a) to (d):

[0712] (a) Positions 394 and 405;

[0713] (b) Positions 366, 368 and 407;

[0714] (c) Positions 347, 360, 399, 405 and 409; and

[0715] (d) Positions 356, 392, 399 and 439, according to EU numbering.

[0716] In one embodiment, the above-mentioned Fc region into which the modification has been introduced contains at least one amino acid selected from the group consisting of:

[0717] (a) W, F or Y at position 394, and

[0718] A, S, T, C, G or V at position 405;

[0719] (b) W, Y or F at position 366,

[0720] A, S, T, C, V or G at position 368, and

[0721] V, L, I, M, A, S, T, C, N or Q at position 407;

[0722] (c) R, K, Y or H at position 347,

[0723] E or D at position 360,

[0724] V, L, I, M, S, T, C, H, A, N, Q or G at position 399,

[0725] T, A, V, S, C, N, D or G at position 405, and

[0726] W, F, Y or H at position 409; and

[0727] (d) K or R at position 356,

[0728] D or E at position 392,

[0729] K or R at position 399, and

[0730] E or D at position 439,

[0731] According to EU numbering.

[0732] In one embodiment, the modified Fc region into which the above has been introduced contains at least one amino acid selected from the group consisting of:

[0733] (a) W or F at position 394, and

[0734] A, S, T or G at position 405;

[0735] (b) W, Y or F at position 366,

[0736] A, T, C, V or G at position 368, and

[0737] V, L, I, M, A or C at position 407;

[0738] (c) R, K, Y or H at position 347,

[0739] E or D at position 360,

[0740] V, L, I, M, S, T, C, H, A, N or G at position 399,

[0741] T, A or V at position 405, and

[0742] W or F at position 409; and

[0743] (d) K at position 356,

[0744] D at position 392,

[0745] K at position 399, and

[0746] E at position 439,

[0747] According to EU numbering.

[0748] In one embodiment, the modified Fc region into which the modification has been introduced contains an amino acid modification at one combination of positions or two or more combinations of positions selected from the combinations of positions shown in (a) to (d) below:

[0749] (a) Positions 345, 347, 360, 366, 399, 407, and 409;

[0750] (b) Positions 356, 399, 409, and 439;

[0751] (c) Positions 356, 392, 399, and 409; and

[0752] (d) Positions 392, 399, and 409, according to EU numbering.

[0753] In one embodiment, the modified Fc region into which the modification has been introduced contains at least one amino acid selected from the group consisting of:

[0754] (a) R or E at position 345,

[0755] R or K at position 347,

[0756] D or E at position 360,

[0757] V at position 366,

[0758] M, Q, N, H, I, F, Y, T, S, V, or L at position 399,

[0759] A at position 407, and

[0760] V, Q, N, H, L, I, F, Y, T, or S at position 409;

[0761] (b) K, R, or H at position 356,

[0762] K, R, or H at position 399,

[0763] E or D at position 409, and

[0764] E or D at position 439,

[0765] (c) K, R, or H at position 356,

[0766] D or E at position 392,

[0767] K, R, or H at position 399,

[0768] D or E at position 409; and

[0769] (d) D or E at position 392,

[0770] K or R at position 399, and

[0771] D or E at position 409,

[0772] According to EU numbering.

[0773] Purification of polypeptide

[0774] The polypeptides (in one embodiment, antibodies) prepared as described herein can be purified by techniques known in the art, such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The actual conditions for purifying a particular protein will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. For affinity chromatography purification, an antibody, ligand, receptor, or antigen that binds to the polypeptide can be used. For example, for affinity chromatography purification of the polypeptides (e.g., antibodies) of the present disclosure, a matrix with protein A or G can be used. Sequential protein A or G affinity chromatography and size exclusion chromatography can be used to separate the polypeptides. The purity of the polypeptides of the present disclosure can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, etc.

[0775] Composition

[0776] In one embodiment, the present disclosure relates to a composition comprising a nucleic acid encoding a polypeptide of the present disclosure.

[0777] In one embodiment, the composition comprises a nucleic acid encoding a first polypeptide, wherein the first polypeptide has a modified Fc region into which modifications have been introduced, and wherein due to the modifications introduced into the Fc region, the first polypeptide associates more readily with a polypeptide having the modification than with a polypeptide not having the modification.

[0778] In one embodiment, the composition of the present disclosure comprises a nucleic acid encoding a second polypeptide, wherein the second polypeptide has a modified Fc region into which modifications have been introduced, and wherein due to the modifications introduced into the Fc region, the second polypeptide associates more readily with a polypeptide having the modification than with a polypeptide not having the modification.

[0779] In one embodiment, the composition of the present disclosure may further comprise nucleic acids encoding a plurality of different polypeptides (in one embodiment, a third polypeptide and / or a fourth polypeptide) that are different from the above-mentioned first polypeptide or second polypeptide.

[0780] In some embodiments, the composition of the present disclosure comprises:

[0781] A nucleic acid encoding a first polypeptide;

[0782] a nucleic acid encoding a second polypeptide; and

[0783] a nucleic acid encoding a third polypeptide.

[0784] Herein,

[0785] In one embodiment, the first polypeptide has a modified Fc region introduced therein, and due to the modification introduced into the Fc region, the first polypeptide associates via the Fc region more readily with the first polypeptide having the modification than with the first polypeptide comprising an Fc region into which the modification has not been introduced.

[0786] In one embodiment, the second polypeptide associates more readily with the third polypeptide than with the second polypeptide.

[0787] In one embodiment, the second polypeptide has a modified Fc region introduced therein, and due to the modification introduced into the Fc region, the second polypeptide associates via the Fc region more readily with the third polypeptide than with the second polypeptide.

[0788] Pharmaceutical composition

[0789] In one embodiment, the present disclosure provides a pharmaceutical composition comprising a nucleic acid or polypeptide (in one embodiment, an antibody) of the present disclosure. In one embodiment, the present disclosure relates to a composition (pharmaceutical composition) comprising a nucleic acid or polypeptide of the present disclosure and a pharmaceutically acceptable carrier.

[0790] In one embodiment, a pharmaceutical preparation of the present disclosure in the form of a lyophilized preparation or an aqueous solution is prepared by mixing a polypeptide (in one embodiment, an antibody) having a desired degree of purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. Ed. (1980)). Acceptable carriers are non-toxic to the recipient at the dosages and concentrations employed; and include, but are not limited to: buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoic acid, such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol, and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG).

[0791] The polypeptides (in one embodiment, antibodies), pharmaceutical compositions, etc. of the present disclosure are administered, for example, orally or parenterally to a subject (in one embodiment, a patient). For example, parenteral administration is preferred. Specifically, such administration methods include injection, nasal administration, pulmonary administration, and transdermal administration. Injection includes, for example, intravenous injection, intramuscular injection, intraperitoneal injection, and subcutaneous injection. For example, the pharmaceutical compositions of the present disclosure can be administered locally or systemically by injection. In addition, an appropriate administration method can be selected according to the age and symptoms of the subject (patient). For each administration, the administered dose can be selected, for example, from the range of 0.0001 mg / kg body weight to 1,000 mg / kg body weight. Alternatively, the dose can be selected, for example, from the range of 0.001 mg / body to 100,000 mg / body for each patient. However, the dose of the pharmaceutical compositions of the present disclosure is not limited to these doses.

[0792] In one embodiment, the pharmaceutical compositions of the present disclosure contain the nucleic acids of the present disclosure. In one embodiment, the pharmaceutical composition contains the nucleic acids of the present disclosure encapsulated in vesicles. Such vesicles include, for example, lipid nanoparticles (LNP), viruses, extracellular vesicles (EV), and liposomes.

[0793] Direct expression in a subject

[0794] If desired, a vector of a nucleic acid molecule encoding a polypeptide (in one embodiment, an antibody) of the present disclosure can be introduced into a subject to directly express the polypeptide of the present disclosure within the subject. Subjects include, for example, humans, non-human animals, isolated cells, and in vitro cells. Examples of vectors that can be used are adenoviruses, but are not limited thereto. A nucleic acid molecule encoding a polypeptide (in one embodiment, an antibody) of the present disclosure can also be directly administered to a subject, including (encapsulated) nucleic acid molecules in vesicles, transferring a nucleic acid molecule encoding a polypeptide of the present disclosure to a subject via electroporation, or administering cells containing a nucleic acid molecule encoding a polypeptide of the present disclosure to be expressed to a subject to express the polypeptide (in one embodiment, an antibody) of the present disclosure in the subject.

[0795] In one embodiment, the present disclosure relates to the expression of a polypeptide in a living organism in which the association between Fc regions is controlled. In one embodiment, the polypeptide of the present disclosure is an antibody that does not associate with wild-type IgG, and thus when it is desired to express an antibody in a living organism, a therapeutic agent based on homogeneous, safe, and multiple antibodies can be provided. In one embodiment, when expressing a therapeutic antibody in a living organism, the fact that they do not associate with endogenous IgG will result in reduced side effects.

[0796] In one embodiment, messenger RNA (mRNA) encoding a polypeptide of the present disclosure can be introduced into a subject (mammal, human, non-human animal, etc.) to promote the formation of a homodimer of the polypeptide of the present disclosure.

[0797] In one embodiment, lipid nanoparticles encapsulating mRNA (mRNA-LNP) can be used to introduce into a subject.

[0798] Method for obtaining a polypeptide with controlled association

[0799] In one embodiment, the present disclosure relates to a method for obtaining a polypeptide in which the association between polypeptides is controlled.

[0800] In one embodiment, the method includes:

[0801] The step of obtaining a nucleic acid encoding the polypeptide; and

[0802] The step of expressing the nucleic acid.

[0803] In one embodiment, the above polypeptide has an Fc region, and due to a modification introduced into the Fc region, the association of the polypeptide with a polypeptide having the modification is easier than the association with a polypeptide not having the modification.

[0804] In one embodiment, the above expression is in vivo, ex vivo, or in vitro expression.

[0805] Here, the term "step" can be used interchangeably with "process" and "phase".

[0806] Methods for production and for controlling association

[0807] In one embodiment, the present disclosure relates to a method for generating a polypeptide with controlled association. In one embodiment, the generating method is a method for generating a polypeptide with controlled association of the Fc region.

[0808] In one embodiment, the generating method comprises the following steps:

[0809] (a) obtaining a nucleic acid encoding a polypeptide in which a modification has been introduced into the Fc region;

[0810] (b) introducing the nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and

[0811] (c) recovering the polypeptide from the culture of the host cell.

[0812] In one embodiment, due to at least one of the following effects, the association of the above polypeptide with a polypeptide having a modification is easier than the association with a polypeptide not having a modification: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0813] In one embodiment, the above generating method further comprises the step of introducing a modification into the Fc region such that the effects of (1) steric complementarity, (2) disulfide bonding, and / or (3) electrostatic charge occur in the above polypeptide.

[0814] In one embodiment, the generating method comprises the following steps:

[0815] (a) modifying a nucleic acid encoding a polypeptide comprising an Fc region such that, due to at least one of the following effects: the association of the polypeptide with a polypeptide having a modification is easier than the association with a polypeptide not having a modification: (1) steric complementarity, (2) disulfide bonding and (3) electrostatic charge;

[0816] (b) introducing the modified nucleic acid into a host cell and culturing the host cell to express the nucleic acid; and

[0817] (c) recovering the polypeptide from the culture of the host cell.

[0818] In one embodiment, the present disclosure relates to a method for controlling the association between polypeptides.

[0819] In one embodiment, the method is a method for controlling the association between polypeptides comprising an Fc region. In one embodiment, the method is a method for controlling the association between CH3 domains.

[0820] In one embodiment, the method includes modifying a polypeptide such that the polypeptide associates more readily with the modified polypeptide than with the unmodified polypeptide due to at least one of the following effects: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

[0821] In one embodiment, a method for controlling the association of homomers of a polypeptide includes:

[0822] obtaining a nucleic acid encoding the polypeptide; and

[0823] expressing the nucleic acid,

[0824] wherein the polypeptide has an Fc region, and

[0825] wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with the modified polypeptide than with a polypeptide comprising an Fc region into which the modification has not been introduced.

[0826] In one embodiment, the present disclosure relates to a method for promoting the expression of homomers of a polypeptide.

[0827] In one embodiment, the method includes obtaining a nucleic acid encoding a polypeptide; and expressing the nucleic acid,

[0828] wherein the polypeptide comprises an Fc region, and

[0829] wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with the modified polypeptide than with a polypeptide comprising an Fc region into which the modification has not been introduced.

[0830] All prior art documents cited herein are incorporated herein by reference.

[0831] The examples provided below are one embodiment of the present invention, and the present invention is not limited to these embodiments. In the sequence listing, SEQ ID NOs: 1-60, 78-101, and 104-114 are full-length heavy chains, SEQ ID NO: 61 is a light chain, and SEQ ID NOs: 62-77 and 102-103 are Fc fragments. (For SEQ ID NOs: 115-620, see Table 14 and subsequent tables.)

[0832] The value "0" in the tables provided in the examples is interpreted as having the same number of significant digits as the other values in the same column. For example, when the other values in the same column have one decimal place of significant digits, "0" is interpreted as "0.0".

[0833] [Examples]

[0834] [Example 1] Search for amino acid modifications that promote the formation of heavy chain homodimers

[0835] To express multiple antibodies in cells simultaneously, first, search for amino acid modifications located at the CH3 interface that are unfavorable for heterodimer formation between the heavy chains of two antibodies in terms of electrostatic forces, disulfide bond formation, or steric hindrance, but are favorable for homodimer formation. The concept is shown in Figure 1 . To date, many amino acid modifications for promoting the heterodimerization of the heavy chains of antibodies A and B have been reported (listed below). Therefore, it is considered that similar residues may also be able to be used as residues that promote homodimerization.

[0836] -WO2006106905 - Method for producing polypeptides by adjusting assemblies - An efficient route to human bispecific IgG (Nat Biotechnol. July 1998; 16(7): 677 - 81. doi:10.1038 / nbt0798 - 677.)

[0837] -Efficient generation of stable bispecific IgG1 by controlled Fab - arm exchange (Proc Natl Acad Sci U S A. March 26, 2013; 110(13): 5145 - 50. doi:10.1073 / pnas.1220145110.)

[0838] -Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins (Front Immunol. October 6, 2016; 7: 394. doi:10.3389 / fimmu.2016.00394.eCollection 2016.)

[0839] However, since the modifications for promoting the formation of homodimers of the heavy chain need to be achieved between one type of heavy chain, the degree of freedom of available amino acid modifications is lower than that when using two different heavy chains to promote heterodimer formation. In particular, promoting the formation of disulfide bonds between the same heavy chains and control methods based on physical hindrance via protrusions and cavities are expected to be difficult because they require a more stringent orientation than electrostatic interactions (which can act at a relatively long distance of about 5-10), and thus the modifications for heterodimers may not be compatible. In fact, only a limited combination of charged amino acid modifications for promoting homodimer formation has been reported so far (WO2013157953A1, WO2014015804A1, WO2017205014, J Biol Chem. October 27, 2017; 292(43):17885-17896). In these cases, the present inventors conducted a large-scale study on the combination of amino acid modifications at the CH3 interface to find amino acid modifications that promote homodimer formation. The list of amino acid modifications studied and the expected modification effects are shown in Table 1. The amino acid numbers in Table 1 conform to the EU numbering (Sequences of proteins of immunological interest, NIH Publication No. 91-3242). Subsequent amino acid numbers also conform to the EU numbering. In the screening, the heavy chain used was the human IgG1 sequence.

[0840] Plasmids expressing full-length heavy chains with these amino acid modifications and Fc fragments below the hinge region without modification were prepared by methods known to those skilled in the art. When these two plasmids and the plasmid encoding the light chain are co-expressed in mammalian cells, homodimers of the full-length IgG heavy chain, homodimers of the Fc fragment, and heterodimers in which the two associate will be produced. The expression ratios of these three can be analyzed by size exclusion chromatography (SEC) because they have different molecular weights. To confirm whether the formation of heavy chain homodimers is promoted, the peak area ratio of each is calculated and compared with unmodified (WT) human IgG1. If the formation of heavy chain homodimers is promoted, the area ratio of the heterodimer is expected to decrease compared to WT-IgG1. The gene combinations of each antibody used in the screening and their SEQ ID NO are shown in Table 1.

[0841] The antibodies listed in Table 1 were transiently expressed in mammalian cells using genes generated by methods known to those skilled in the art, and then purified by methods known to those skilled in the art. Specifically, the antibodies in Table 1 were transiently expressed by transfecting 1 mL of 2E+6 cells / mL of Expi293F (ThermoFisher Scientific) with plasmids encoding the full-length heavy chain, light chain, and Fc fragment at a mass ratio of 1:2:1. Four days after transfection, the supernatant was recovered and purified using MonoSpin ProA (GL science).

[0842] The purified antibodies were evaluated by size exclusion chromatography (SEC) analysis using ACQUITY UPLC H-Class (Waters). 50 mM phosphate buffer pH 7.0 (Isekyu) containing 300 mM sodium chloride was used as the running buffer, and a TSKgel SuperSW3000 custom column (4.6 mm x 15 cm, 4 μm, Gel Lot 89R) (TOSOH) was used as the analytical column. Chromatograms were recorded at a wavelength of UV 215 nm. Data were analyzed using Empower3 (Waters). For the analysis of the antibodies in Table 1, the area ratios of the three components, namely the homodimer component of the full-length heavy chain, the heterodimer component with the Fc fragment, and the homodimer component of the Fc fragment, were calculated separately and presented as percentages, as shown in the representative example of Figure 2 . In Figure 2 , reference samples prepared in Sample Nos. 61 and 62 (Table 2) were also shown, thereby confirming that these three components could be correctly separated in the SEC analysis. The analysis results are shown in Table 1. In this analysis, the ratio of the heteropolymer of WT-IgG1 was 51.7%, indicating that amino acid modifications exhibiting this ratio or lower ratios would promote the homodimerization of the heavy chain.

[0843]

[0844]

[0845]

[0846] [Example 2] Expression and Purification of Antibodies

[0847] Next, to examine the effects of these amino modifications in the CH3 interface on the physicochemical properties of the antibodies, the antibodies in Table 2 were prepared. Genes generated by methods known to those skilled in the art were used to transiently express the antibodies in mammalian cells by the method described in Example 1, and then purified. Plasmids encoding the full-length heavy chain and light chain were used for transfection at a mass ratio of 1:1 or 1:2.

[0848] (Table 2) Modifications of antibodies used in the analysis and SEQ ID NO

[0849]

[0850]

[0851]

[0852] [Example 3] Evaluation of the physicochemical properties of antibodies with amino acid modifications introduced at the CH3 interface

[0853] The effects of CH3 interface modifications on antibody expression level, monomer content (%), and midpoint temperature of thermal denaturation (Tm) were compared. The yields of the antibodies prepared in Example 2 are shown in Table 3.

[0854] In addition, the monomer content in the purified antibodies was evaluated by the SEC analysis method described in Example 1. The results of the SEC analysis were analyzed to calculate the monomer content (%), where the components eluting on the side with a molecular weight higher than that of the monomer were collectively referred to as associated multimers, and the components eluting on the side with a molecular weight lower than that of the monomer were collectively referred to as degradation products. The analysis results are shown in Table 3.

[0855] The Tm of the antibodies undergoing amino acid modifications was evaluated by differential scanning fluorimetry (DSF). It has been reported that the Tm determined by this method has a good correlation with the Tm determined by differential scanning calorimetry, a well-known method for evaluating the thermal stability of antibodies (Journal of Pharmaceutical Science 2010; 4: 1707-1720).

[0856] A 5000x concentrate of SYPRO Orange Protein Gel Stain (Invitrogen) was diluted with PBS (Sigma), and the antibody solution was mixed with this detection dye. A 20-μL mixture was dispensed into tubes for measurement, and its temperature was increased from 30 °C to 99 °C at a rate of 240 °C / hr using Rotor-Gene Q (QIAGEN). Fluorescence changes with increasing temperature were observed at 470 nm (excitation wavelength) / 555 nm (fluorescence wavelength).

[0857] The data obtained were used to determine the temperature at which fluorescence transitions were observed using Rotor-Gene Q Series Software (QIAGEN), and this value was determined as the Tm value. For the antibodies used in this test, the Tm of Fab was approximately 95 °C, significantly higher than the Tm of CH2 and CH3. Therefore, in this test, the Tm value observed at the lowest temperature was considered to be the Tm change caused by Fc modification and was used for comparison. The results are shown in Table 3.

[0858] (Table 3) Analysis Results of Antibodies with Amino Acid Modifications

[0859]

[0860]

[0861]

[0862] [Example 4] Selection and Discussion of Modifications for Promoting Heavy Chain Homodimerization

[0863] As shown in Table 1, in the results of SEC analysis performed after expressing the full-length heavy chain, light chain, and Fc fragment, all modification pairs that exhibited a heterodimer formation ratio lower than that of WT-IgG1 (51.7%) can be said to provide a certain degree of promotion for the formation of heavy chain homodimers. As initially expected, modifications previously reported for promoting heteromeric association of heavy chains do not necessarily cause promotion of heavy chain homodimer formation. In particular, sample numbers 24, 33, and 57 led to a significant promotion of heavy chain heterodimerization, producing undesirable effects. They also had difficulty maintaining good physicochemical properties of the antibody. After SEC analysis, it was found that some modifications significantly reduced the monomer ratio, decreased the Tm value, or decreased the antibody yield. This experimental fact indicates that the modification pairs for promoting heavy chain heterodimerization are not necessarily the same as those for promoting heavy chain homodimerization, and it is important to find amino acid modification pairs that promote homodimerization between one type of heavy chain and maintain good physicochemical properties. The present inventors evaluated various antibodies shown in Table 1 and thereby found amino acid modifications that particularly strongly inhibit heavy chain heterodimerization and promote heavy chain homodimerization. These selected antibodies are shown in Table 4. Compared with WT-IgG1, all of these antibodies showed comparable antibody expression levels, high monomer formation ratios, and comparable Tm values in the Fc region (however, only a decrease in antibody yield was observed for the antibody of sample number 112 compared with WT-IgG1).

[0864] (Table 4) Amino Acid Modifications Exhibiting Strong Heavy Chain Homodimerization Promotion Ability

[0865]

[0866] [Example 5] Identification of Amino Acid Modification Pairs Allowing Co-Expression of Multiple Antibodies

[0867] Next, to identify amino acid modification pairs that allow co-expression of multiple antibodies, genes were prepared such that the amino acid modifications shown in Table 4 were also introduced into the Fc fragment of IgG1. These elaborate combinations were examined to find modification pairs that promote the formation of heavy chain homodimers between the modified Fc regions. For expression, the light chain of SEQ ID NO:61 was used. The antibodies were expressed by the same method as described in Example 1. The ability of each modification pair to promote homodimerization was determined based on whether the heterodimer level decreased compared to the same modification pair after SEC analysis. (For example, when expressing the WT-IgG pair, the heterodimer level was 52%. Thus, combinations showing a heterodimer level lower than 52% can be determined as pairs with the ability to promote homodimerization.) The SEQ ID NOs and their combinations used in the experiment, as well as their heterodimer formation ratios, are shown in Table 5. Modification pairs related to the inhibition of heterodimerization in the experimental results are not expected to cause heteroassociation, regardless of how many of them are combined. Therefore, they can be used not only for the co-expression of two antibodies, but also for the co-expression of three, four, five, six, seven, eight, nine, or more antibodies.

[0868] More specifically, amino acid modification pairs showing a heteromultimer level lower than 10% in Table 5 can be used in combination. Table 6 shows the effective amino acid modification pairs composed of these combinations (<10%, Combinations 1 - 3512).

[0869] In addition, amino acid modification pairs composed only of pairs with a stronger ability to promote heavy chain homomeric association (amino acid modification pairs showing a heteromultimer level of 0% in Table 5) (0%, Combinations 1 - 744) are shown in Table 7. In Tables 5, 6, and 7, no introduced modification is denoted as WT.

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[1258] [Example 6] Confirmation of co-expression of multiple antibodies by ion exchange chromatography (IEC)

[1259] Example 5 identified pairs that allow co-expression of two or more antibodies. To confirm whether they can actually promote the formation of homodimers between multiple IgG antibodies, Expi293F was allowed to express multiple modified IgG antibodies. For expression, plasmids encoding the heavy chain and plasmids encoding the light chain were transfected at a mass ratio of 1:1. When multiple heavy chains were used for expression, they were used such that the mass ratio of each heavy chain was equal. Each antibody after protein A purification was analyzed by ion exchange chromatography (IEC). Specifically, the resulting antibodies were analyzed by cation exchange chromatography (CIEX) using an Alliance system (Waters). A YMC-BioPro SP-F 5μm 4.6x100 (YMC) was used as the analytical column, CX-1 pH gradient buffer A pH 5.6 (Thermo) was used as mobile phase A, and CX-1 pH gradient buffer B pH 10.2 (Thermo) was used as mobile phase B for the dual-solvent gradient method. Samples were detected by absorption at a wavelength of 280 nm (represented as AU on the vertical axis in the figure). Empower3 (Waters) was used to analyze the data to calculate the ratio of each detected peak.

[1260] The modifications used in antibody expression and the corresponding SEQ ID NOs are shown in Table 8 (no introduced modification is indicated as "-"). In the expression, heavy chains of four different antibodies with different isoelectric points (pI) were used, and for simplicity of analysis, a common light chain (SEQ ID NO: 61) was used. Antibodies before modification and singly modified antibodies were also analyzed to assign the peaks detected when expressing multiple heavy chains. The analysis results, as well as the assignment and area ratio of each peak, are shown in Figures 3-1 to 3-47 In these figures, peak assignments are represented using abbreviations characteristic of heavy chain association. The abbreviations and the correspondence between the template heavy chain sequence and the modifications are shown in Table 9 (no introduced modification is indicated as "-"). For example, an antibody with a homodimerizing identical heavy chain with SEQ ID NO: 1 and a common light chain is denoted as "aa". All implemented combinations were found to have a strong ability to promote heavy chain homopolymeric association. Similarly, the combinations shown in Tables 6 and 7 are also considered to exhibit heavy chain homopolymeric association ability.

[1261]

[1262]

[1263]

[1264]

[1265] [Example 7] Evaluation of human FcRn binding of antibodies with modifications introduced into the CH3 region

[1266] Next, the effect of modifications for promoting heavy chain homooligomerization on Fc function was examined.

[1267] Binding to human neonatal Fc receptor (FcRn) was evaluated using a Biacore T200 (Cytiva). Evaluation was performed at 25 °C using 50 mM phosphate buffer, 150 mM NaCl, 0.05 w / v%-P20 (pH 6.0) as the running buffer. rProtein L (BioVision) was immobilized as a ligand capture molecule onto a Series S CM4 (Cytiva). An antibody solution prepared with the running buffer was allowed to interact with this CM4 sensor chip to capture approximately 400 RU of antibody. The human FcRn protein used in this measurement was prepared by the method described in WO2010107110. Human FcRn was diluted to 0, 250, 500, 1000, 2000, and 4000 nM with the running buffer and allowed to bind to the captured antibody. The chip was regenerated using 10 mM glycine-HCl (pH 1.5) and repeatedly used for antibody capture for measurement. The FcRn binding activity of each antibody was evaluated by calculating KD (M) using the Biacore T200 evaluation software 3.2.1 and the steady-state model. The KD values were similar in the presence and absence of modifications at the CH3 interface. Table 10 shows the KD (M) between human FcRn and each antibody (absence of introduced modification is denoted as "-").

[1268] (Table 10) Evaluation of human FcRn binding activity of antibodies with modifications introduced into the CH3 region

[1269]

[1270] [Example 8] Evaluation of human Fcγ receptor binding of antibodies with modifications introduced into the CH3 region

[1271] The binding activity of the generated modified antibodies to each human Fcγ receptor (hereinafter referred to as FcγR) was evaluated using a Biacore T200 (Cytiva). The evaluation was carried out at 25 °C using 50 mM phosphate buffer, 150 mM NaCl, 0.05 w / v%-P20 (pH 7.4) as the running buffer. rProtein L (BioVision) was immobilized as a ligand capture molecule onto a Series S CM4 (Cytiva). The antibody solution prepared with the running buffer was allowed to interact with the CM4 sensor chip to capture approximately 500 RU of antibody in the case of measuring human FcγRIa and 2000 RU of antibody in the case of measuring other human FcγRs. The human FcγR proteins for this measurement were prepared by the method described in WO2022220275. In the case of FcγRIa, the human FcγR was diluted to 8 nM with the running buffer, or in the case of other FcγRs, diluted to 1000 nM, and allowed to bind to the captured antibody. The chip was regenerated using 10 mM glycine-HCl (pH 1.5) and repeatedly used for capturing antibodies for measurement. The binding activity of each antibody to each FcγR was evaluated by calculating the FcγR binding (RU) level / unit amount of antibody using the Biacore T200 evaluation software version 3.2.1.

[1272] Table 11 shows the binding levels per unit amount of antibody, and Table 12 shows the relative levels (%) of the binding levels to the Fc control antibody (sample number 61) (representation without introduced modification is "-"). Antibodies with CH3 modifications that promote heavy-chain homodimer formation generally showed comparable or slightly higher binding activity to each human FcγR compared to the control. However, sample number 117 showed a binding level to some FcγRs that was half or lower than that of the control. Therefore, if Fc function comparable to the control antibody is required for the intended use, CH3 modifications other than those of sample number 117 should be used.

[1273]

[1274]

[1275] [Example 9] Evaluation of ECM binding of antibodies with modifications introduced into the CH3 region

[1276] Next, the effect of the heavy-chain homomeric association-promoting modification on the non-specific binding of the antibody was examined.

[1277] The system for evaluating the binding to the extracellular matrix (ECM) is called an in vitro evaluation system for non-specific binding. This assay system was used for the evaluation (US Patent 2014 / 0080153). The measurement results are in Figure 4Shown in the CH3 interface, the presence and absence of the modification produce similar levels of ECM binding.

[1278] [Example 10] Evaluation of the ability to form heavy chain homodimers between different IgG subclasses

[1279] In Examples 1-9, antibodies of the IgG1 subclass were used for evaluation. However, other subclasses (IgG2 and IgG4) are also important for therapeutic antibodies. In addition, to prevent association with endogenous antibodies in living organisms, it is important not to form heavy chain heteromers between different subclasses. Here, some of the modifications showing strong heavy chain homodimer formation ability in IgG1 were applied to different IgG subclasses, and their heavy chain homodimer formation ability was examined by SEC analysis. The correspondence between the template subclass for antibody expression, amino acid modification, SEQ ID NO, and area ratio is shown in Table 13 (absence of introduced modification is indicated as "-"). Antibody expression and analysis were performed according to the method described in Example 1. However, pairs containing IgG3 were purified using MonoSpin ProG (GL science) because it is difficult to perform affinity purification of them using protein A.

[1280] The results in Table 13 indicate that all pairs of subclasses of IgG1, IgG2, and IgG4 have the ability to form heavy chain homodimers.

[1281] In addition, analysis of WT-IgG1, IgG2, and IgG4 paired with WT-IgG3 revealed that WT-IgG1, IgG2, and IgG4 are inherently less likely to form pairs with IgG3. This indicates that these amino acid-modified antibodies do not form heavy chain heterodimers between IgG1, IgG2, and IgG4, and are even less likely to form heavy chain heteropolymers with WT-IgG3.

[1282]

[1283]

[1284]

[1285]

[1286]

[1287]

[1288] [Example 11] Evaluation of the ability to form heavy chain homodimers in antibodies containing different IgG formats and Fc modifications

[1289] The evaluations of Examples 1 - 10 were performed using the basic IgG format and the Fc of wild - type IgG (without other modifications except for the CH3 interface control). However, to prepare and express more effective therapeutic antibodies, it is desirable to apply to various antibody formats, such as formats in which VHH or scFv is linked to Fc, and antibodies in which amino acid modifications for altering Fc function are introduced. Here, some of the modifications in IgG that exhibit strong heavy - chain homodimer - forming ability were applied to various antibody formats and Fc - modified antibodies, and their heavy - chain homodimer - forming ability was examined by SEC analysis. The correspondence between the heavy - chain framework or amino acid modifications (and their functions) contained in the template antibodies for antibody expression, the amino acid modifications for promoting heavy - chain homodimer formation, SEQ ID NO, and the area ratio is shown in Table 14 (no modification introduced is denoted as “ - ”). Antibody expression and analysis were performed according to the method described in Example 1. However, for antibodies containing the E345R / E430G / S440Y modification that promotes hexamer formation of IgG, a multimer component was detected in the SEC analysis. In addition, for pairs containing the scFv.LH - Fc or scFv.HL - Fc format, it was difficult to completely separate each homopolymer and heteropolymer. Therefore, for these samples, a qualitative evaluation of the effect of suppressing heteropolymer formation was performed, focusing on the peak of the heteropolymer component ( Figures 5-1 to 5-14 ).

[1290] Based on the results in Table 14 and Figures 5-1 to 5-14 , it was revealed that even when the binding region of the antibody is VHH or scFv, and even when the Fc region is deglycosylated or contains modifications for enhancing FcRn binding, inhibiting FcγR binding, or promoting hexamer formation, the heavy - chain homodimer - forming ability (or the effect of suppressing heteropolymer formation) is still maintained.

[1291] In addition, since the heavy - chain homodimer - forming ability (or the effect of suppressing heterodimer formation) is maintained in all formats, it can also be expected that this ability is maintained even when c is linked to polypeptides other than Fab, VHH, and scFv, and is widely maintained even in the presence of amino acid modifications that do not affect the Fc interface.

[1292]

[1293]

[1294]

[1295]

[1296]

[1297] [Example 12] Further search for modifications that promote the formation of heavy chain homodimers

[1298] The above examples revealed that the amino acid modifications in Table 4 have a particularly strong ability to promote heavy chain homodimerization and have broad applicability regardless of the antibody subclass, format, and Fc modification in the template antibody. However, the amino acid modifications surrounding these effective modification sets have not been fully retrieved, and there may be other effective modifications in addition to the modification sets in Table 4. Therefore, for the modifications of T394W / F405A (T394F / F405A), T366W / L368A / Y407V, Q347R / K360E / D399V / F405T / K409W, E345R / Q347R / K360D / T366V / D399M / Y407A / K409V, E356K / D399K / K409E / K439E, E356K / K392D / D399K / K409E, E356K / K392D / D399K / K409D, K392D / D399K / K409D, and E356K / K392D / D399K / K439E in Table 4, large-scale amino acid modifications were performed at each modification position. In addition, their heavy chain homodimer formation ability was evaluated by SEC analysis. The correspondence between the functional category of the modifications used for antibody expression, amino acid modifications, SEQ ID NO, and area ratio is shown in Table 15 (no modification introduced is denoted as "-"). Antibody expression and analysis were performed according to the method described in Example 1. Homodimers containing each Fc modification were prepared according to the method described in Example 3, and the monomer ratio, Tm, and yield (mg) in SEC analysis were evaluated by a similar method and shown together in Table 15. Therefore, the modifications with a heteromultimer (%) lower than the control (51.7%) in Table 15 were evaluated as effective modifications. These effective modifications at each position can also be used in combination.

[1299] In this test, for samples with a low monomer ratio, the heteromultimer peak in the homodimer analysis contains half-antibodies (monomers composed of one heavy chain and one light chain) derived from the homodimer. Therefore, the apparent ratio of heteromultimers in these samples may be higher than the true value. However, since this is considered not to affect the determination of amino acid modifications with strong homodimerization ability and good physical properties, a single criterion of 51.7% or lower was used to evaluate the ability to inhibit the formation of heteromultimers.

[1300] Modifications that show a heteromultimer ratio (%) of 10% or less in Table 15 and are thus considered more effective are shown in Table 16. In addition, modifications that show a heteromultimer ratio (%) of 0% in Table 15 and are thus considered more effective are shown in Table 17. In addition, modifications that show a heteromultimer ratio (%) of 0% in Table 15 and for which the monomer ratio, Tm, and yield (mg) are 95% or higher, 65 °C or higher, and 0.15 mg or higher, respectively, in SEC analysis when preparing a homodimer and are thus particularly advantageous in terms of heavy-chain homodimer formation ability and the physicochemical properties of the homodimer are shown in Table 18. At the same time, the samples in Table 4 with a heteromultimer ratio (%) of 0% and a monomer ratio, Tm, and yield (mg) of the homodimer of 95% or higher, 65 °C or higher, and 0.15 mg or higher, respectively, are sample numbers 80, 87, 92, 95, 97, 100, 102, 109, 111, 119, and 121. The modifications contained in these also have excellent heavy-chain homodimer formation ability and physicochemical properties.

[1301] The amino acid modifications and their combinations at the respective positions shown as effective in Tables 15 - 18 are expected to have similar properties and can thus be used as alternatives to the modification sets of T394W / F405A (T394F / F405A), T366W / L368A / Y407V, Q347R / K360E / D399V / F405T / K409W, E345R / Q347R / K360D / T366V / D399M / Y407A / K409V, E356K / D399K / K409E / K439E, E356K / K392D / D399K / K409D, K392D / D399K / K409D, and E356K / K392D / D399K / K439E shown in Tables 6 and 7 and can be applied to Fc modifications expressing multiple homodimers.

[1302] In addition, in the combinations of each modification, amino acids shown to be effective at the same modification position can also be used in combination. The modification combinations showing strong heteroassociation inhibition in Table 1 and the amino acid modifications in Table 15 that inhibit the heteromultimer ratio to less than WT (51.7%) are shown in Table 19. For example, for modification set number 1, the effects are present for W, F, Y, and H at EU number position 394 and for A, S, T, C, V, and G at position 405. Therefore, 4 x 6 = 24 combinations of the modifications at each position are also considered to play a role. Similarly, for other modification sets, the combinations of the modifications shown in the table can also exhibit a certain heteromultimer inhibition ability.

[1303] Similarly, the amino acid modifications that suppress the heteromultimer ratio to 10% or less in Table 15 are shown in Table 20. Combinations of these corresponding amino acids can be more preferred heteromultimer-suppressing modifications.

[1304] In addition, the amino acid modifications that suppress the heteromultimer ratio to 0% in Table 15 are shown in Table 21. Combinations of these corresponding amino acids can be even more preferred heteromultimer-suppressing modifications.

[1305]

[1306]

[1307]

[1308]

[1309]

[1310]

[1311]

[1312]

[1313]

[1314]

[1315]

[1316]

[1317]

[1318]

[1319]

[1320]

[1321]

[1322]

[1323]

[1324]

[1325]

[1326] (Table 16) Amino acid modifications showing strong heavy-chain homodimerization promoting ability with a heteromultimer ratio (%) of 10% or lower in Table 15

[1327]

[1328]

[1329]

[1330]

[1331]

[1332]

[1333]

[1334]

[1335]

[1336]

[1337] (Table 17) Amino acid modifications showing strong heavy-chain homodimerization promoting ability with a heteromultimer ratio (%) of 0% in Table 15

[1338]

[1339]

[1340]

[1341]

[1342]

[1343] (Table 18) Amino acid modifications showing a heteromultimer ratio (%) of 0%, a homodimer monomer ratio of 95% or higher, a Tm of 65°C or higher, and a yield (mg) of 0.15 mg or higher in Table 15

[1344]

[1345]

[1346]

[1347]

[1348]

[1349] (Table 19) Modification combinations showing strong hetero - oligomeric association inhibition in Table 1 and amino acid modifications at the corresponding residue positions in Table 15 that inhibit the heteromultimer ratio to less than 51.7%

[1350]

[1351]

[1352] (Table 20) Modification combinations showing strong hetero - oligomeric association inhibition in Table 1 and amino acid modifications at the corresponding residue positions in Table 15 that inhibit the heteromultimer ratio to 10% or less

[1353]

[1354]

[1355] (Table 21) Modification combinations showing strong hetero - oligomeric association inhibition in Table 1 and amino acid modifications at the corresponding residue positions in Table 15 that inhibit the heteromultimer ratio to 0%

[1356]

[1357]

[1358]

[1359] [Example 13] Evaluation of Homodimers and Heteromultimers

[1360] The amino acid modifications promoting homodimer formation found in the present invention may be able to be used simultaneously with amino acid modifications (e.g., mortise - and - tenon modifications) promoting heterodimer formation. To verify this possibility, antibodies containing some of the modifications showing strong homodimer - forming ability in IgG and antibodies containing mortise - and - tenon modifications were co - expressed, and the formation efficiencies (%) of the target homodimers and heterodimers were compared by IEC analysis when co - expressed with WT - IgG1. Antibody preparation and IEC analysis were carried out according to the method described in Example 6. Samples were detected at an excitation wavelength of 280 nm and a fluorescence wavelength of 330 nm (represented as EU on the vertical axis in the figure).

[1361] The correspondence between amino acid modifications that promote homodimer formation and heterodimer formation, SEQ ID NOs, and area ratios is shown in Table 22 (absence of modification is denoted as "-"). This test was conducted using two pairs of template antibodies for homodimers and two pairs for heteropolymers (in Table 22, the template antibody group "hetero1+homo1" refers to a heterodimer containing SEQ ID NOs: 561 and 562 and a homodimer pair containing SEQ ID NO: 563, and the template antibody group "hetero2+homo2" refers to a heterodimer containing SEQ ID NOs: 567 and 568 and a homodimer containing SEQ ID NO: 78. The corresponding homodimer formation-promoting modifications were introduced into the homodimers.). The chromatograms for each antibody analysis are shown in Figures 6-1 to 6-10 . The elution positions of the expected heavy-chain heterodimers and heavy-chain homodimers and the area ratios of the unexpected multimers were calculated by assigning peaks based on the elution positions of the corresponding reference samples.

[1362] This result indicates that the amino acid modifications that promote homodimer formation found in the present invention can be used simultaneously with amino acid modifications that promote heteropolymers (e.g., heterodimer formation). It also shows that this effect allows for more efficient expression of the target homodimers and heteropolymers compared to the homodimer expression of WT-IgG.

[1363]

[1364]

[1365]

[1366] The amino acid modifications contained in the heavy chain (denoted as heavy chain abbreviations in the table) are as follows:

[1367] KT366W

[1368] HT366S / L368A / Y407V

[1369] K'Y349C / T366W

[1370] H'E356C / T366S / L368A / Y407V

[1371] e-

[1372] ET394W / F405A

[1373] E'E356K / K392D / D399K / K439E

[1374] E”Q347R / K360E / D399V / F405T / K409W

[1375] f-

[1376] FT394W / F405A

[1377] F'E356K / K392D / D399K / K439E

[1378] F”Q347R / K360E / D399V / F405T / K409W

[1379] In this test, two types of knobs-in-hole modifications were used as heteromultimer-promoting modifications; however, other reported modifications can also be used. For example, modifications that can be used include strand exchange engineered domain (SEED) bodies, which utilize Ig-class, heterodimeric T cell receptor (TCR) alpha and beta chains (BEAT Fc), T366W / S354C-T366S / L368A / Y407V / Y349C, K409D / K392D-D399K / E356K, S364H / F405A-Y349T / T394F, D221E / P228E / L368E-D221R / P 228R / K409R, F405L-K409R, T350V / T366L / K392L / T394W-T350V / L351Y / F405A / Y407V, K360E / K409W-Q347R / D399V / F405T, K360E / K409W / Differences in association preferences between Y349C-Q347R / D399V / F405T / S354C, K370E / K409W-E357N / D399V / F405T, K360D / D399M / Y407A-E345R / Q347R / T366V / K409V, Y349S / K370Y / T366M / K409V-E356G / E357D / S364Q / Y407A, L351D / L368E-L351K / T366K and L368D / K370S-E357Q / S364K (separation between two different heavy chains is indicated as "-"). In this case, as modifications for promoting homodimer formation, it is more preferred to use, for example, a modification set that has no modification at the same residue number as the amino acid modification for promoting heteromeric association, or a modification set that is greatly different in amino acid properties (in terms of size or presence / absence of charge) from the amino acid modification for promoting heteromeric association, because it is expected that this will be less likely to lead to promotion of unintended association due to the two types of modifications.

[1380] In this test, a heteromultimer and a homomultimer were expressed; however, the number of samples to be co-expressed is not limited to this. For example, when expressing multiple heteromultimers, modifications at residue numbers that are different from each other are preferably combined. Multiple homomultimers with amino acid modifications for promoting homomeric association can be further expressed, and the amino acid modification is different from those for promoting the association of multiple heteromultimers in terms of amino acid residue number, or the amino acid modification contains modifications at the same residue number but composed of amino acids with different properties. This will enable the simultaneous expression of multiple heteromultimers and multiple homomultimers with high purity.

[1381] [Example 14] Evaluation of the ability to promote heavy chain homodimer formation in a mammalian expression system using mRNA

[1382] Next, it was examined whether transfection of mammalian cells with messenger RNA (mRNA) instead of plasmid would also have an effect of promoting homodimer formation. The antibodies listed in Table 25 were transiently expressed in mammalian cells using mRNA containing sequences encoding the heavy and light chains of the antibodies listed in Tables 23 and 24 by methods known to those skilled in the art, and purified by methods known to those skilled in the art. Specifically, plasmid DNA containing sequences encoding the heavy and light chains of the antibodies listed in Tables 23 and 24 was used as a template and PCR was performed to add a T7 promoter sequence and a poly A sequence. The constructed PCR fragment was used as a template to prepare mRNA by an in vitro transcription (IVT) method using the MEGAscript T7 Transcription Kit (Thermo Fisher Scientific, AM1333). In the transcription, ARCA (TriLink BioTechnologies, N-7003-1) was added as a cap structure, and 1-methylpseudouridine-5'-triphosphate (TriLink BioTechnologies, N-1081-1) was added as a modified nucleic acid. The prepared mRNA was transfected into 1 mL of Expi293 cells at 2.5E+6 cells / mL at the mass ratios listed in Table 25 for transient expression of the antibody. Four days after transfection, the culture supernatant was collected and the antibody was purified by methods known to those skilled in the art.

[1383] The prepared antibodies were analyzed by the IEC method described in Example 6. The corresponding area ratios generated by triple transfection are shown in Table 25. Constructs containing amino acid modifications for promoting homodimer formation showed a significant decrease in the heteromultimer ratio.

[1384] The above results confirmed that even when mRNA is used for expressing antibodies in mammalian cells, the amino acid modifications for promoting homodimer formation are effective.

[1385]

[1386]

[1387] [Example 15] Preparation of mRNA-encapsulated lipid nanoparticles (mRNA-LNP)

[1388] Lipid nanoparticles (mRNA-LNP) encapsulating the mRNAs shown in Tables 23 and 24 were prepared using the general lipid compositions and preparation methods described in the references, etc. (Hou X et al., Nat Rev Mater. 2021;6(12):1078-1094, Weng Y et al., Biotechnol Adv. May-Jun 2020;40:107534, Webb C et al., Mol Pharm. Apr 4, 2022;19(4):1047-1058). Specifically, cationic lipid, DOPE, cholesterol, and PEG lipid were dissolved in EtOH at a molar ratio of 35:16:46.5:1.5 and a total lipid concentration of about 1.3 mg / mL. The mRNA untranslated region (UTR) was designed, and full-length mRNA was synthesized using 5-methoxyuridine as the modified nucleic acid and CleanCap AG as the cap structure by Trilink. The prepared mRNA was diluted to about 0.02 mg / mL in 25 mM sodium acetate buffer (pH 4.0). mRNA-LNP was prepared using NanoAssemblr Ignite (Precision NanoSystems) by mixing the lipid ethanol solution and the aqueous mRNA solution at a ratio of 1:3 (vol / vol). The prepared mRNA-LNP was diluted with approximately three times its volume of PBS. Then, the external buffer components were replaced with PBS using an Amicon 100 kDa centrifugal filter (Millipore), and the mRNA-LNP was concentrated to the desired concentration. The resulting mRNA-LNP was filtered through a 0.22 μm sterile filter. The final mRNA-LNP was stored at -80 °C until further use.

[1389] The particle size, polydispersity index (PdI), and ζ potential of the mRNA-LNP were measured using a Zetasizer NanoZS (Malvern Panalytical). Before measuring the particle size and PdI, the mRNA-LNP sample was diluted 20-fold in PBS, or diluted 170-fold in 10 mM phosphate buffer (pH 7.2) before measuring the ζ potential.

[1390] By fluorescence-based assay ( Reagents, Thermo Fisher Scientific) to determine the total RNA concentration and free RNA in the mRNA-LNP sample. The encapsulation efficiency was calculated as (total RNA - free RNA) / total RNA. The mRNA-LNP sample was appropriately diluted with 1x TBE buffer containing 1% Triton-X100 and reagent to determine the total RNA, or diluted with 1x TBE buffer containing only reagent to determine the free RNA. The starting RNA solution used for preparing the mRNA-LNP was diluted with 1x TBE buffer containing reagent + / - 1.0% Triton-X100 to generate a calibration curve. Samples of the mixture containing Triton-X100, reagent, buffer and the mRNA-LNP sample were incubated in the dark at room temperature for about 5 minutes and measured using a SpectraMax M3 microplate reader (Molecular Devices), where the excitation wavelength, auto cut-off wavelength and emission wavelength were set at 488 nm, 515 nm and 525 nm, respectively. The total RNA and free RNA were determined from the appropriate calibration curve.

[1391] When preparing LNP in a typical manner, the encapsulation, particle size and PdI were >90%, <100 nm and <0.1, respectively.

[1392] The LNPs encapsulating the corresponding mRNAs were mixed to obtain the combinations of mRNAs 1-3 shown in Table 26, and the in vivo test of Example 16 was carried out.

[1393]

[1394] [Example 16] Evaluation of the ability to promote heavy chain homodimer formation in mouse in vivo expression using mRNA-LNP

[1395] To test the in vivo effect of the amino acid modification for promoting heavy chain homodimer formation, the mRNA-LNP generated in Example 15 was intravenously administered to C57BL6 / J mice (male, 6 weeks old) at 1.0 mg RNA / kg. The correspondence between the names of the mRNA-LNPs used in the in vivo test, the mRNAs contained therein, their mass ratios, and the abbreviations of the expression samples is shown in Table 26.

[1396] Whole blood of the mice was collected from the jugular vein 3 and 7 days after administration. After centrifugation at 12,000 rpm for 5 minutes at 4°C, plasma was collected.

[1397] Measure the homodimers and heterodimers in the collected plasma. This measurement is performed using a Gyrolab xP workstation or Gyrolab xPand (Gyros Protein Technologies).

[1398] First, label the human GPC3 core protein, TIA0124-rabbit Fc (anti-idiotypic (ID) antibody against anti-CD3 antibody (TR01H113-G1T6 / L0011-kT0 and TR01H113-G1T6v1 / L0011-kT0)), rAQ8-mIgG2b (anti-ID antibody against anti-FIXa antibody (Q499-G4d / L404-kT0 and Q499-G4dv3 / L404-kT0)) described in WO2016047652, and rAJ540-rbtIgG (anti-ID antibody against anti-FX antibody (J327-G4d / L404-kT0 and J327-G4dv4 / L404-kT0)) with biotin and Alexa Fluor 647 according to the manufacturer's protocol using EZ-Link Sulfo-NHS-biotin (21217, Thermo Fisher Scientific) and Alexa Fluor 647 Antibody Labeling Kit (A20186, Life Technologies).

[1399] When measuring homodimers, mix the collected plasma or plasma diluted 2 to 200-fold with pooled plasma from C57BL / 6J (male, 8 weeks old, Jackson Laboratory Japan) with an equal volume of Rexxip A-max (P0004821, Gyros Protein Technologies). Mix the plasma mixed with an equal volume of Rexxip A-max with biotin-labeled antibody and Alexa Fluor 647-labeled antibody in Rexxip A (P0004820, Gyros Protein Technologies) at 1:1:1 (the final concentrations of the biotin-labeled antibody and Alexa Fluor 647-labeled antibody are 1 μg / mL), and incubate at room temperature for 2 hours.

[1400] When measuring anti-CD3 homodimers, mix the plasma sample with biotin-labeled TIA0124-rabbit Fc and Alexa Fluor 647-labeled TIA0124-rabbit Fc at 1:1:1.

[1401] When measuring anti-GPC3 homodimer, plasma samples were mixed with biotin-labeled human GPC3 core protein and Alexa Fluor 647-labeled human GPC3 core protein at a ratio of 1:1:1.

[1402] When measuring anti-FIXa homodimer, plasma samples were mixed with biotin-labeled rAQ8-mIgG2b and Alexa Fluor 647-labeled rAQ8-mIgG2b at a ratio of 1:1:1.

[1403] When measuring anti-FX homodimer, plasma samples were mixed with biotin-labeled rAJ540-rbtIgG and Alexa Fluor 647-labeled rAJ540-rbtIgG at a ratio of 1:1:1.

[1404] The incubated samples were measured using a Gyrolab xP workstation or Gyrolab xPand. In this measurement, a Gyrolab Bioaffy 1000 (P0004253, Gyros Protein Technologies) was used as the measurement disk. The incubation mixture of the above biotin-labeled antibody, Alexa Fluor 647-labeled antibody, and plasma sample was added to the measurement disk, and the biotin-labeled antibody-homodimer-Alexa Fluor 647-labeled antibody complex was captured on the reaction layer in the disk. The amount of the captured complex was detected by the fluorescence signal of Alexa647 (detection PMT 1%). The fluorescence signal of Alexa647 was analyzed using a Gyro Evaluator (version 3.7.2.5976, Gyros Protein Technologies).

[1405] When measuring heterodimer, the collected plasma was diluted 3 to 5 times with mixed plasma from C57BL / 6J (male, 8 weeks old, Jackson Laboratory Japan) and mixed with an equal volume of Rexxip A-max (P0004821, Gyros Protein Technologies).

[1406] Measure the mixed plasma using a Gyrolab xP workstation or Gyrolab xPand. In this measurement, a Gyrolab Bioaffy 1000 (P0004253, Gyros Protein Technologies) is used as the measurement disk. Add the biotinylated antibody diluted to 25 μg / mL with PBS 0.05% Tween 20 (P3563, SIGMA-ALDRICH) to the measurement disk and fix it to the reaction layer in the disk. Add the above-prepared plasma sample into it and add the Alexa Flour 647-labeled antibody diluted to 5 μg / mL with Rexxip F (P0004825, Gyros Protein Technologies). Detect the captured heterodimer with the fluorescence signal of Alexa647 (detection PMT 1%). Analyze the fluorescence signal of Alexa647 using a Gyro Evaluator (version 3.7.2.5976, Gyros Protein Technologies).

[1407] When measuring the anti-CD3 / GPC3 heterodimer, use the biotinylated human GPC3 core protein as the immobilized antigen and the Alexa Fluor 647-labeled TIA0124-rabbit Fc as the detection antibody.

[1408] When measuring the anti-FIXa / FX heterodimer, use the biotinylated rAQ8-mIgG2b as the immobilized antibody and the Alexa Fluor 647-labeled rAJ540-rbtIgG as the detection antibody.

[1409] As a result of this analysis, the plasma concentration of each antibody is shown in Figure 7-1 and 7-2 and the amino acid sequences of Figures 7-3 and 7-4.

[1410] On the 3rd day after administration, for the WT antibodies without homopolymeric association-promoting modifications (CD3-WT x GPC3-WT and FIXa-WT x FX-WT), the expression of two types of homodimers and heterodimers was detected. For the engineered antibodies containing homopolymeric association-promoting modifications in either or both antibodies (CD3-WT x GPC3-Design 2, CD3-Design 1 x GPC3-WT, CD3-Design 1 x GPC3-Design 2, FIXa-WT x FX-Design 4, FIXa-Design 3 x FX-WT, and FIXa-Design 3 x FX-Design 4), the expression level of homodimers was detected to be similar to or slightly increased compared to the WT antibodies. On the other hand, the heterodimer expression of the engineered antibodies was significantly suppressed in all samples. Compared to the heterodimer expression in the WT antibodies, the anti-CD3 / GPC3 heterodimer expression was suppressed by 72 to 878-fold or more, and the anti-FIXa / FX heterodimer expression was suppressed by 241 to 446-fold or more.

[1411] The suppression of heterodimer expression continued even on the 7th day after administration, indicating an 84 to 717-fold or more suppression of anti-CD3 / GPC3 heterodimer expression and a 184 to 375-fold or more suppression of anti-FIXa / FX heterodimer expression.

[1412] This demonstrates that the engineered antibodies can inhibit the expression of heterodimers in vivo.

[1413] This test demonstrated the in vivo expression of two antibodies containing the Q347R / K360E / D399V / F405T / K409W or E356K / K392D / D399K / K439E modifications in one or both heavy chains, and the in vivo expression of two antibodies containing the T394W / F405A or T366W / L368A / Y407V modifications in one or both heavy chains. Similar suppression of heterodimer expression was also expected for other amino acid modifications that promoted homodimer formation, which were confirmed in in vitro tests using plasmids, in other molecular formats and functional Fcs, and in combinations of three or more antibodies.

[1414] Examples 1-16 above found many modifications with a strong ability to promote heavy chain homodimerization and also excellent physicochemical properties and Fc functions for therapeutic antibodies. In addition, an exhaustive study of combinations of modified Fc regions found that many combinations allowed heavy chain homodimerization to be maintained between the modified Fc regions. Since these are antibodies that do not associate with WT-IgG, they can provide homogeneous and safe therapeutic drugs based on multiple antibodies when in vivo antibody expression is expected.

[1415] [Industrial Applicability]

[1416] The present disclosure provides polypeptides in which the association between polypeptides is controlled, methods for producing polypeptides with controlled association, methods for controlling the association of polypeptides, nucleic acids encoding polypeptides with controlled association, compositions containing such nucleic acids, and the like. Due to the controlled association between polypeptides, the polypeptides of the present disclosure can be particularly used to produce antibodies and express therapeutic drugs in living organisms.

Claims

1. A nucleic acid encoding a first polypeptide, wherein the first polypeptide comprises an Fc region into which a modification has been introduced, wherein due to the modification introduced into the Fc region, the first polypeptide associates via the Fc region more readily with a first polypeptide having the modification than with a first polypeptide comprising an Fc region into which the modification has not been introduced.

2. The nucleic acid according to claim 1, wherein due to at least one of the following effects resulting from the introduced modification, the first polypeptide associates via the Fc region more readily with a first polypeptide having the modification than with a first polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

3. A composition comprising: the nucleic acid according to claim 1; and a nucleic acid encoding a second polypeptide, wherein the second polypeptide comprises an Fc region into which a modification has been introduced, wherein due to the modification introduced into the Fc region, the second polypeptide associates via the Fc region more readily with a second polypeptide having the modification than with a second polypeptide comprising an Fc region into which the modification has not been introduced.

4. A composition comprising: the nucleic acid according to claim 1; a nucleic acid encoding a second polypeptide; and a nucleic acid encoding a third polypeptide, wherein the second polypeptide associates more readily with the third polypeptide than with the second polypeptide.

5. A composition comprising: the nucleic acid according to claim 2; and a nucleic acid encoding a second polypeptide, wherein the second polypeptide does not have the modification that the first polypeptide has.

6. The composition according to claim 3, wherein the modification introduced into the first polypeptide is different from the modification introduced into the second polypeptide.

7. The composition according to claim 3, wherein due to at least one of the following effects resulting from the introduced modification, the second polypeptide associates via the Fc region more readily with a second polypeptide having the modification than with a second polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

8. The nucleic acid according to claim 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 2.

9. The nucleic acid according to claim 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 4.

10. The nucleic acid according to claim 6, wherein the modification introduced into the first polypeptide and the modification introduced into the second polypeptide are at least one of the combinations of the modifications listed in Table 6.

11. The nucleic acid according to claim 1, wherein the polypeptide is an antibody.

12. The nucleic acid according to claim 1, wherein the Fc region is the Fc region of IgG.

13. The nucleic acid according to claim 1, wherein the polypeptide into which the modification has been introduced retains the function of the polypeptide before the modification.

14. The nucleic acid according to claim 12, wherein the Fc region into which the modification has been introduced retains the function of the Fc of IgG.

15. The nucleic acid according to claim 1, wherein the Fc region is derived from any one of IgG1, 2, 3, and 4.

16. A host cell into which the nucleic acid according to any one of claims 1, 2, and 8 to 14 or the composition according to any one of claims 3 to 7 has been introduced.

17. A polypeptide expressed by the nucleic acid according to claim 1.

18. A composition comprising a nucleic acid encoding a polypeptide, wherein the polypeptide comprises an Fc region into which a modification has been introduced, wherein the modification is at least one of the modifications listed in Table 2.

19. A polypeptide comprising an Fc region into which a modification has been introduced, wherein due to at least one of the following effects resulting from the introduced modification, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced: (1) steric complementarity, (2) disulfide bonding, and (3) electrostatic charge.

20. A method for obtaining a polypeptide with controlled association, the method comprising: obtaining a nucleic acid encoding the polypeptide; and expressing the nucleic acid, wherein the polypeptide comprises an Fc region, and wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced.

21. A method for controlling the association of a homopolymer of a polypeptide, the method comprising: obtaining a nucleic acid encoding the polypeptide; and expressing the nucleic acid, wherein the polypeptide comprises an Fc region, and wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced.

22. A method for promoting the expression of a homopolymer of a polypeptide, the method comprising: obtaining a nucleic acid encoding the polypeptide; and expressing the nucleic acid, wherein the polypeptide comprises an Fc region, and wherein due to the modification introduced into the Fc region, the polypeptide associates more readily via the Fc region with a polypeptide having the modification than with a polypeptide comprising an Fc region into which the modification has not been introduced.

23. The composition according to claim 4, wherein the second polypeptide comprises an Fc region into which a modification has been introduced, wherein due to the modification introduced into the Fc region, the second polypeptide associates more readily via the Fc region with the third polypeptide than with itself.

24. The composition according to claim 1, wherein the Fc region into which the modification has been introduced comprises amino acid modifications at one or two or more combinations of positions selected from the following combinations of positions shown in (a) to (d): (a) positions 394 and 405; (b) positions 366, 368, and 407; (c) positions 347, 360, 399, 405, and 409; and (d) positions 356, 392, 399, and 439, according to EU numbering.

25. The composition according to claim 24, wherein the Fc region into which the modification has been introduced comprises at least one amino acid selected from the group consisting of: (a) W, F or Y at position 394, and A, S, T, C, G or V at position 405; (b) W, Y or F at position 366, A, S, T, C, V or G at position 368, and V, L, I, M, A, S, T, C, N or Q at position 407; (c) R, K, Y or H at position 347, E or D at position 360, V, L, I, M, S, T, C, H, A, N, Q or G at position 399, T, A, V, S, C, N, D or G at position 405, and W, F, Y or H at position 409; and (d) K or R at position 356, D or E at position 392, K or R at position 399, and E or D at position 439, According to EU numbering.

26. The composition according to claim 24, wherein the Fc region into which the modification has been introduced comprises at least one amino acid selected from the group consisting of: (a) W or F at position 394, and A, S, T or G at position 405; (b) W, Y or F at position 366, A, T, C, V or G at position 368, and V, L, I, M, A or C at position 407; (c) R, K, Y or H at position 347, E or D at position 360, V, L, I, M, S, T, C, H, A, N or G at position 399, T, A or V at position 405, and W, F or Y at position 409; and (d) K at position 356, D at position 392, K at position 399, and E at position 439, According to EU numbering.

27. The nucleic acid according to claim 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 15.

28. The nucleic acid according to claim 1, wherein the modification introduced into the Fc region is at least one of the modifications listed in Table 17.

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