Fc variants with enhanced thermal stability and reduced effector function

Abstract

A polypeptide comprising one or more variant Fc regions, each of the variant Fc regions comprising two CH2 domains, at least one of the CH2 domains in at least one of the variant Fc regions is a variant CH2 domain, and the amino acid residues at positions 234, 235, and 265 (all positions represented by EU numbering) of the variant CH2 domain are Ala, Ala, and Gly, or Ala, Ala, and Asn, respectively.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to Japanese Patent Application No. 2021-144352, filed on September 3, 2021, the disclosure of which is incorporated by reference in its entirety into this specification for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in pdf format and is incorporated herein by reference in its entirety. A copy of the pdf was created on August 30, 2022, is named ZYME086WO_Sequence Listing.pdf, and is 716,800 bytes in size.

[0003] Field The present disclosure relates to the field of Fc variants, in particular to polypeptides comprising variant IgG Fc regions, molecules comprising said polypeptides, and polypeptide-drug conjugates in which said polypeptides are conjugated to drugs, which are useful as pharmaceuticals. [Background technology]

[0004] background The Fc region of an antibody confers a long serum half-life and effector functions such as antibody-dependent cellular cytotoxicity (ADCC) to the antibody. When the Fc region is derived from the IgG subclass used in most antibody drugs, the effector functions depend on the binding of the Fc region to a family of receptors called Fcγ receptors (FcγR). However, binding activity to FcγR has been suggested to be involved in safety risks such as acute infusion reactions (see J Immunotoxicol; 5(1):11-5 (2008) (Non-Patent Document 1)), and therefore, in some circumstances, may be an undesirable property for antibodies used as medicines.

[0005] Various amino acid substitutions in the Fc region of IgG antibodies that reduce the binding activity to human FcγR have been reported. For example, see International Publication Nos. WO1988 / 07089 (Patent Document 1); WO1999 / 51642 (Patent Document 2); WO2000 / 42072 (Patent Document 3); WO2013 / 092001 (Patent Document 4); WO2015 / 109131 (Patent Document 5) and WO2020 / 086776 (Patent Document 6), as well as Protein Eng Des Sel; 29(10): 457-66 (2016) (Non-Patent Document 2), and J Biol Chem; 292(5): 1865-75 (2017) (Non-Patent Document 3).

[0006] Amino acid substitutions that reduce the binding of the Fc region to FcγR can result in reduced thermal stability of the Fc region (see, for example, International Publication No. WO2020 / 086776 (Patent Document 6) and J Biol Chem; 292(5):1865-75 (2017) (Non-Patent Document 3)).

[0007] International Publication No. WO2013 / 118858 (Patent Document 7) describes amino acid substitutions in the Fc region that improve thermal stability and also increase or decrease effector function. WO2013 / 118858 (Patent Document 7) shows that combinations of amino acid substitutions each independently improve thermal stability, but do not necessarily improve thermal stability additively. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO1988 / 07089 [Patent Document 2] WO1999 / 51642 [Patent Document 3] WO2000 / 42072 [Patent Document 4] WO2013 / 092001 [Patent Document 5] WO2015 / 109131 [Patent Document 6] WO2020 / 086776 [Patent Document 7] WO2013 / 118858 [Non-patent literature]

[0009] [Non-Patent Document 1] J Immunotoxicol;5(1):11-5(2008) [Non-Patent Document 2] Protein Eng Des Sel;29(10):457-66(2016) [Non-Patent Document 3] J Biol Chem;292(5):1865-75(2017) Summary of the Invention

[0010] overview Described herein are Fc variants with increased thermal stability and attenuated effector function. One aspect of the disclosure relates to a polypeptide comprising one or more variant Fc regions, each of the variant Fc regions comprising two CH2 domains, at least one CH2 domain in at least one of the variant Fc regions is a variant CH2 domain comprising amino acid substitutions at positions 234, 235 and 265 (all positions represented by EU numbering), wherein the amino acid residues at positions 234, 235 and 265 of the variant CH2 domain are Ala, Ala and Gly, respectively, or Ala, Ala and Asn, respectively, and each of the one or more variant Fc regions is a variant IgG1 Fc region, a variant IgG4 Fc region or a variant IgG1 / IgG4 Fc region.

[0011] Another aspect of the disclosure pertains to molecules, including the polypeptides described herein.

[0012] Another aspect of the present disclosure relates to polypeptide-drug conjugates comprising a polypeptide described herein conjugated to one or more drugs or modifying agents.

[0013] Another aspect of the present disclosure pertains to nucleic acids encoding nucleotide sequences that encode the polypeptides described herein or portions thereof.

[0014] Another aspect of the present disclosure pertains to a host cell comprising a nucleic acid comprising a nucleotide sequence encoding a polypeptide described herein or a portion thereof.

[0015] Another aspect relates to a method for producing a polypeptide described herein, comprising culturing a host cell comprising a nucleic acid comprising a nucleotide sequence encoding the polypeptide or a portion thereof in a medium under conditions suitable for expression of the polypeptide, and recovering the polypeptide from the medium or the host cell.

[0016] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a polypeptide described herein, a molecule comprising such a polypeptide, or a polypeptide-drug conjugate comprising such a polypeptide conjugated to a drug or modifier, and a pharma- ceutically acceptable carrier.

[0017] Another aspect relates to the therapeutic use of a polypeptide described herein, a molecule comprising said polypeptide, or a polypeptide-drug conjugate comprising the polypeptide conjugated to a drug or modifier. [Brief description of the drawings]

[0018] [Figure 1] 1 is a schematic diagram showing the structure of a general IgG antibody. An IgG antibody is composed of a heavy chain each composed of VH, CH1, hinge, CH2, and CH3, and a light chain each composed of VL and CL. [Figure 2A]FIG. 1 shows the binding activity to human FcγRI of IgG1 WT obtained by replacing the IgG2 Fc region of the anti-EGFR antibody panitumumab with the human wild-type IgG1 Fc region; a variant obtained by introducing L234A / L235A (LALA) mutations into the Fc region of IgG1 WT; a variant obtained by introducing L234A / L235A / D265A (LALA-DA) mutations into the Fc region of IgG1 WT; and a variant obtained by introducing L234A / L235A / P329G (LALA-PG) mutations into the Fc region of IgG1 WT, evaluated by the SPR method. [Figure 2B] 2B shows the binding activity of the antibodies shown in FIG. 2A to human FcγRIIa, evaluated by the SPR method. [Figure 2C] 2B shows the binding activity of the antibodies shown in FIG. 2A to human FcγRIIb / c, evaluated by the SPR method. [Figure 2D] 2B shows the binding activity of the antibodies shown in FIG. 2A to human FcγRIIIa, evaluated by the SPR method. [Figure 2E] 2B shows the binding activity of the antibodies shown in FIG. 2A to human FcγRIIIb, evaluated by the SPR method. [Diagram 3] 2B shows the binding activity of the antibodies shown in FIG. 2A to cynomolgus monkey FcγRI, evaluated by the SPR method. [Figure 4A] 1 shows the temperature-dependent heat capacity change of the anti-EGFR antibody panitumumab IgG1 WT as measured by DSC. The numbers near each peak represent the temperature of the peak top (Tm). [Figure 4B] 1 shows the temperature-dependent heat capacity change of the LALA variant of the anti-EGFR antibody panitumumab as measured by DSC. The numbers near each peak represent the Tm. [Figure 4C] 1 shows the temperature-dependent heat capacity change of the LALA-DA variant of the anti-EGFR antibody panitumumab, as measured by DSC. The numbers near each peak represent the Tm. [Figure 4D]1 shows the temperature-dependent heat capacity change of the LALA-PG variant of the anti-EGFR antibody panitumumab, as measured by DSC. The numbers near each peak represent the Tm. [Diagram 5] The figure shows the binding activity to human FcγRI of a variant obtained by replacing the IgG2 Fc region of the anti-EGFR antibody panitumumab with a human wild-type IgG1 Fc region, the variant obtained by introducing L234A / L235A / D265A (LALA-DA) mutations into the Fc region of IgG1 WT; the variant obtained by introducing L234A / L235A / D265G (LALA-DG) mutations into the Fc region of IgG1 WT; the variant obtained by introducing L234A / L235A / D265N (LALA-DN) mutations into the Fc region of IgG1 WT; the variant obtained by introducing L234A / L235A / D265Q (LALA-DQ) mutations into the Fc region of IgG1 WT; and the variant obtained by introducing L234A / L235A / D265T (LALA-DT) mutations into the Fc region of IgG1 WT, evaluated by the SPR method. [Figure 6] 5B shows the binding activity of the antibodies shown in FIG. 5A to cynomolgus monkey FcγRI, assessed by the SPR method. [Figure 7A] 1 shows the temperature-dependent heat capacity change of the LALA-DG variant of the anti-EGFR antibody panitumumab, as measured by DSC. The numbers near each peak represent the Tm. [Figure 7B] 1 shows the temperature-dependent heat capacity change of the LALA-DN variant of the anti-EGFR antibody panitumumab, as measured by DSC. The numbers near each peak represent the Tm. [Figure 7C] 1 shows the temperature-dependent heat capacity change of the LALA-DQ variant of the anti-EGFR antibody panitumumab as measured by DSC. The numbers near each peak represent the Tm. [Figure 7D] 1 shows the temperature-dependent heat capacity change of the LALA-DT variant of the anti-EGFR antibody panitumumab, as measured by DSC. The numbers near each peak represent the Tm. [Figure 8]This shows the time course of blood concentrations of the anti-EGFR antibody panitumumab after intravenous (iv) administration of IgG1 WT, LALA variant, and LALA-DG variant to mice. Error bars in the figure show standard deviation (n=3). [Figure 9A] 1 shows the binding activity to human FcγRI of the LALA-DG variant of the anti-EGFR antibody panitumumab, and a variant obtained by introducing L234A / L235A / D265G / P329A (LALA-DGPA) mutations into the Fc region of IgG1 WT, evaluated by the SPR method. [Figure 9B] 9B shows the binding activity of the antibodies shown in FIG. 9A to cynomolgus monkey FcγRI, assessed by the SPR method. [Figure 10A] 1 shows the binding activity of the LALA and LALA-DG variants of the anti-CD70 IgG1 antibody borsetuzumab to human FcγRI, evaluated by the SPR method. [Figure 10B] 1 shows the binding activity of the LALA and LALA-DG variants of the anti-LPS antibody O11-1111, an IgG1 antibody, to human FcγRI, evaluated by the SPR method. [Figure 11A] The amino acid sequence of the wild-type human IgG1 (G1m17) constant region is shown (SEQ ID NO: 1). The underlined, double underlined and boxed portions indicate the positions of the hinge region, CH2 and CH3 domains, and amino acid substitutions (mutations), respectively. [Figure 11B] The amino acid sequence of the wild-type human IgG1 (G1m3) constant region is shown (SEQ ID NO: 2). Notation is the same as in FIG. 11A. [Figure 11C] The amino acid sequence of the wild-type human IgG4 constant region is shown (SEQ ID NO: 3). Notation is the same as in Figure 11A. [Figure 12A]

[0036] Figure 11 shows the amino acid sequence of the LALA-DG variant of the human IgG1 (G1m17) constant region (SEQ ID NO: 4). Notation is the same as in Figure 11A. [Figure 12B] 1 shows the amino acid sequence of the LALA-DG variant of the human IgG1 (G1m3) constant region (SEQ ID NO:5). Notation is the same as in FIG. 11A. [Figure 13A] 1 shows the amino acid sequence of the LALA-DN variant of the human IgG1 (G1m17) constant region (SEQ ID NO: 6). Notation is the same as in FIG. 11A. [Figure 13B] 1 shows the amino acid sequence of the LALA-DN variant of the human IgG1 (G1m3) constant region (SEQ ID NO: 7). Notation is the same as in FIG. 11A. [Figure 14A] 1 shows the amino acid sequence of the LALA-DGPA variant of the human IgG1 (G1m17) constant region (SEQ ID NO: 8). Notation is the same as in FIG. 11A. [Figure 14B] 1 shows the amino acid sequence of the LALA-DGPA variant of the human IgG1 (G1m3) constant region (SEQ ID NO: 9). Notation is the same as in FIG. 11A. [Figure 15A]

[0036] Figure 10 shows the amino acid sequence of the FALA-DG variant of the human IgG4 constant region (SEQ ID NO: 10). Notation is the same as in Figure 11A. [Figure 15B] The amino acid sequence of the FALA-DN variant of the human IgG4 constant region is shown (SEQ ID NO: 11). Notation is the same as in Figure 11A. [Figure 15C] 1 shows the amino acid sequence of the FALA-DGPA variant of the human IgG4 constant region (SEQ ID NO: 12). Notation is the same as in FIG. 11A. [Figure 16A] 1 shows the amino acid sequence of the IgG1 WT light chain of the anti-EGFR antibody panitumumab (SEQ ID NO: 13). The underlined portions indicate the variable regions. [Figure 16B] 14 shows the amino acid sequence of the IgG1 WT heavy chain of the anti-EGFR antibody panitumumab (SEQ ID NO: 14). The underlined, double underlined, and boxed portions indicate the positions of the hinge region, CH2 and CH3 domains, and amino acid substitutions (mutations), respectively. [Figure 17A] 1 shows the amino acid sequence of the light chain of the LALA variant of the anti-CD70 antibody borsetuzumab (SEQ ID NO: 15). The underlined regions indicate the variable regions. [Figure 17B] 16 depicts the amino acid sequence of the heavy chain of the LALA variant of the anti-CD70 antibody borsetuzumab (SEQ ID NO: 16). Notation is the same as in FIG. 16B. [Figure 18A]1 shows the amino acid sequence of the IgG1 WT light chain of anti-LPS antibody O11-1111 (SEQ ID NO: 17). The underlined parts indicate the variable regions. [Figure 18B] 1 depicts the amino acid sequence of the IgG1 WT heavy chain of anti-LPS antibody O11-1111 (SEQ ID NO: 18). Notation is the same as in FIG. 16B. [Figure 19A] The amino acid sequence of human Fc gamma RI (Accession Number: P12314) is shown (SEQ ID NO: 19). The underlined part indicates the sequence of recombinant human Fc gamma RI / CD64 protein CF (R&D Systems). [Figure 19B] The amino acid sequence of human Fc gamma RIIa (R167) (Accession number: AAA35827) is shown (SEQ ID NO: 20). The underlined part indicates the sequence of recombinant human Fc gamma RIIa / CD32a (R167) protein (R&D Systems). [Figure 19C] The amino acid sequence of human Fc gamma RIIa (H167) (Accession No.: P12318-1 (P12318.4)) is shown (SEQ ID NO: 21). The underlined portion indicates the sequence of recombinant human Fc gamma RIIa / CD32a (H167) protein (R&D Systems). [Figure 19D] The amino acid sequence of human Fc gamma RIIb / c (Accession Number: P31994) is shown (SEQ ID NO: 22). The underlined part indicates the sequence of recombinant human Fc gamma RIIB / C (CD32b / c) protein CF (R&D Systems). [Figure 19E] The amino acid sequence of human Fc gamma RIIIa (accession number: AAH17865) is shown (SEQ ID NO: 23). The underlined part indicates the sequence of recombinant human Fc gamma RIIIa / CD16a protein CF (R&D Systems). [Figure 19F] The amino acid sequence of human Fc gamma RIIIb (Accession Number: O75015) is shown (SEQ ID NO: 24). The underlined portion indicates the sequence of recombinant human Fc gamma RIIIB / CD16b protein (R&D Systems). [Figure 20A]This shows the amino acid sequence of cynomolgus monkey Fc gamma RI (accession number: NP_001270969) (SEQ ID NO: 25). The underlined part shows the sequence of recombinant cynomolgus monkey Fc gamma RI / CD64 protein CF (R&D Systems). [Figure 20B] This shows the amino acid sequence of cynomolgus monkey Fc gamma RIIa (accession number: NP_001270598) (SEQ ID NO: 26). The underlined part shows the sequence of recombinant cynomolgus monkey Fc gamma RIIa / CD32a protein CF (R&D Systems). [Figure 20C] This shows the amino acid sequence of cynomolgus monkey Fc gamma RIIb (accession number: NP_001271060) (SEQ ID NO: 27). The underlined part shows the sequence of recombinant cynomolgus monkey Fc gamma RIIB protein CF (R&D Systems). [Figure 20D] This shows the amino acid sequence of cynomolgus monkey Fc gamma RIII (accession number: NP_001270121) (SEQ ID NO: 28). The underlined part shows the sequence of recombinant cynomolgus monkey Fc gamma RIII / CD16 protein CF (R&D Systems). [Figure 21] This shows the amino acid sequence of human fetal Fc receptor large subunit p51 (accession number: P55899) (sequence number 29). [Figure 22] This shows the amino acid sequence of beta2-microglobulin (accession number: NP_004039.1) (SEQ ID NO: 30). [Diagram 23] The amino acid sequence of human Fc gamma RIIIa (V176F) (Accession Number: P08637) is shown (SEQ ID NO: 31). The underlined portion indicates the sequence of recombinant human Fc gamma RIIA / CD16a (V176F) protein (R&D Systems). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] Detailed Description definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0020] As used herein, the term "about" refers to approximately a ±10% variation from a given value. It is to be understood that such a variation is always included in any given value presented herein, whether or not that variation is specifically referred to.

[0021] The use of the words "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0022] As used herein, the terms "comprising," "having," "including," and "containing," as well as grammatical variations thereof, are inclusive, i.e., open ended, and do not exclude additional unrecited elements and / or method steps. The term "consisting essentially of," when used in connection with a composition, use, or method, indicates that additional elements and / or method steps may be present, but that these additions do not substantially affect the manner in which the recited composition, method, or use functions. The term "consisting of," when used in connection with a composition, use, or method, excludes the presence of additional elements and / or method steps. When a composition, use, or method described herein is said to include certain elements and / or steps, in certain embodiments it may consist essentially of those elements and / or steps, and in other embodiments it may consist of those elements and / or steps, whether or not those embodiments are specifically mentioned.

[0023] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0024] The present disclosure relates to polypeptides comprising one or more variant IgG1 Fc regions or variant IgG4 Fc regions, each of the variant Fc regions comprising two CH2 domains, one or both of the CH2 domains in at least one of the variant Fc regions being a variant CH2 domain, and the amino acid residues at positions 234, 235, and 265 (all positions represented by EU numbering) of the variant CH2 domain are Ala, Ala, and Gly, respectively (and optionally the amino acid residue at position 329 is Ala), or are Ala, Ala, and Asn, respectively (and optionally the amino acid residue at position 329 is Ala). The disclosure further relates to molecules comprising the polypeptides; polypeptide-drug conjugates in which the polypeptides are conjugated to a drug; pharmaceutical compositions comprising the polypeptides, molecules, or conjugates; and methods for treating human subjects with the polypeptides, molecules, conjugates, or pharmaceutical compositions. In certain embodiments, the present disclosure relates to a polypeptide comprising a variant IgG Fc region, a molecule comprising the polypeptide, and a polypeptide-drug conjugate comprising the polypeptide conjugated to a drug, which have reduced binding activity to Fcγ receptors and good thermal stability, and are useful as pharmaceuticals. For example, in one embodiment, a polypeptide comprising a variant CH2 domain, a molecule comprising the polypeptide, and a polypeptide-drug conjugate comprising the polypeptide conjugated to a drug have reduced binding activity to Fcγ receptors compared to a parent polypeptide, molecule, and conjugate comprising the parent CH2 domain (i.e., the CH2 domain before introducing mutations at positions 234, 235, and 265 (and optionally position 329) contained in the variant CH2 domain). In another embodiment, the polypeptide, molecule, and conjugate have improved or comparable thermal stability compared to a parent polypeptide, molecule, and conjugate comprising the parent CH2 domain, respectively.In another embodiment, the polypeptides, molecules, and conjugates maintain antigen binding activity and have no reduction in serum half-life compared to the parent polypeptides, molecules, and conjugates, respectively, that contain the parent CH2 domain. In another embodiment, the CH2 domain of the polypeptides, molecules, or conjugates has a small number of amino acid substitutions (e.g., 3 or 4 amino acid substitutions) relative to the parent polypeptides, molecules, and conjugates, respectively, that contain the parent CH2 domain, reducing the risk of immunogenicity that may occur with an increased number of amino acid substitutions.

[0025] As used herein, the numbering of amino acid residues in the Fc region is according to the EU index (Proceedings of the National Academy of Sciences of the United States of America, Vol. 63, No. 1 (May 15, 1969), pp. 78-85), unless otherwise specified.

[0026] As used herein, "polypeptide" typically refers to a molecule that contains about 10 or more amino acid residues, which are connected to each other by peptide bonds. A polypeptide according to the present disclosure can be derived from a natural polypeptide or a synthetic or recombinant polypeptide. A polypeptide according to the present disclosure can be, for example, an antibody or functional fragment thereof, a fusion protein, etc., as described herein.

[0027] In one embodiment, the present disclosure relates to a molecule comprising the above-mentioned polypeptide. Such molecules include, but are not limited to, bispecific antibodies, multispecific antibodies, soluble receptors, immunocytokines, and moieties that bind to antigens or targets other than antibodies or their functional fragments (e.g., non-immunoglobulin proteins, receptors, ligands, nucleic acid aptamers, antisense nucleic acid molecules, low molecular weight compounds, etc.). When a molecule comprising a polypeptide according to the present disclosure is itself a polypeptide, the molecule can be considered as a "polypeptide" according to the present disclosure, and therefore can be referred to herein as either a "polypeptide according to the present disclosure" or a "molecule comprising a polypeptide according to the present disclosure". The "fusion protein" described below can also be itself a polypeptide in some embodiments, and therefore can be referred to herein as either a "polypeptide according to the present disclosure" or a "molecule comprising a polypeptide according to the present disclosure".

[0028] In one embodiment, the present disclosure relates to a polypeptide-drug conjugate in which the above-mentioned polypeptide is conjugated to a drug. The drug can be, for example, a therapeutic drug or a diagnostic drug. When the polypeptide contained in the polypeptide-drug conjugate is an antibody targeting tumor cells, it is preferable, but not essential, that the antibody itself has an antitumor effect. The polypeptide-drug conjugate can usually be prepared by conjugating the polypeptide and the drug via a linker. Linkers known in the art, such as sugar linkers and / or peptide linkers, can be used. In addition, various pharmaceuticals can be used as the drug depending on the intended therapeutic purpose of the use of the polypeptide-drug conjugate. The polypeptide-drug conjugate in which the above-mentioned polypeptide is conjugated to a drug can be considered as one of the other embodiments of the "molecule comprising a polypeptide according to the present disclosure".

[0029] Variant Fc Regions Polypeptides according to the present disclosure include variant IgG1 Fc regions, variant IgG4 Fc regions, or variant IgG1 / IgG4 regions (collectively "variant Fc regions"). As used herein, "Fc region" refers to the C-terminal region of an antibody heavy chain and includes two CH2 domains and two CH3 domains. The Fc region is usually in the form of a homodimer or heterodimer in which the C-terminal regions of the two heavy chains are connected by a hinge region, but in some embodiments may be a single chain Fc (scFc) region. When the Fc region is derived from an IgG1 antibody, the Fc region generally refers to, but is not limited to, the region from the amino acid residue at position 231 to the C-terminus.

[0030] As used herein, the term "hinge" or "hinge region" refers to the portion of an IgG antibody that includes the C-terminus of the CH1 domain to the N-terminus of the CH2 domain. When the Fc region is derived from an IgG1 or IgG4 antibody, the hinge generally extends, without limitation, from an amino acid residue at about position 216 to an amino acid residue at about position 230. As noted above, when the Fc region is derived from an IgG1 antibody, the Fc region generally refers to the amino acid residue at position 231 to the C-terminus of the heavy chain, although in some embodiments the "hinge" may be included in the Fc region.

[0031] As used herein, "CH2 domain" includes, but is not limited to, the region extending from the point of connection with the hinge to the point of connection with the CH3 domain. When the Fc region is derived from an IgG1 or IgG4 antibody, the CH2 domain generally extends from an amino acid residue at about position 231 to an amino acid residue at about position 340. The amino acid residues at positions 234, 235, 265, and 329 of the Fc region of human wild-type IgG1 or IgG4 are present in the CH2 domain. The CH2 domain usually has a loop structure in the Fc region, but is not limited to such a structure.

[0032] As used herein, "CH3 domain" includes, but is not limited to, the region that extends from the point of connection with the CH2 domain of the Fc region to the C-terminus of the heavy chain. When the Fc region is derived from an IgG1 or IgG4 antibody, the CH3 domain generally extends from the amino acid residue at about position 341 to the amino acid residue at about position 447 of IgG1, but is not limited to this. The CH3 domain usually has a loop structure in the Fc region, but is not limited to such a structure.

[0033] As used herein, a "variant IgG1 Fc region" or a "variant IgG4 Fc region" refers to an IgG1 Fc region or an IgG4 Fc region in which one or both of the two CH2 domains are variant CH2 domains. Variant IgG1 Fc regions and variant IgG4 Fc regions are sometimes collectively referred to herein as "variant Fc regions". The term "variant Fc region" also encompasses a mixed IgG1 / IgG4 Fc region in which one or both of the two CH2 domains are variant CH2 domains. A "variant CH2 domain" refers to a CH2 domain in which the amino acid residues at positions 234, 235, and 265 (all positions represented by EU numbering) are Ala, Ala, and Gly; or Ala, Ala, and Asn, respectively (i.e., 234A, 235A, and 265G; or 234A, 235A, and 265N). In one embodiment, the variant CH2 domain comprises amino acid residues at positions 234, 235, 265, and 329 substituted with Ala, Ala, Gly, and Ala, respectively (i.e., 234A, 235A, 265G, and 329A).

[0034] A polypeptide according to the present disclosure may include another (one or more) component(s) other than the variant Fc region, such as a portion, region, or domain of any one of the IgG subclasses: IgG1, IgG2, IgG3, or IgG4. The Fc region is preferably derived from a mammal. In some embodiments, the Fc region is derived from a human or a cynomolgus monkey (including, for example, a cynomolgus macaque, a rhesus monkey, a common marmoset, a squirrel monkey, etc.). In some embodiments, the Fc region is of human origin.

[0035] An example of the amino acid sequence of a wild-type human IgG1 Fc region is provided herein as the amino acid sequence consisting of amino acids 114 to 330 of SEQ ID NO: 1 or 2 (see Figures 11A and 11B), and an example of the amino acid sequence of a wild-type human IgG4 Fc region is provided herein as the amino acid sequence consisting of amino acids 111 to 327 of SEQ ID NO: 3 (see Figure 11C). The amino acid residues at positions 234, 235, 265, and 329 (all positions represented by EU numbering) of the human IgG1 Fc region prior to mutagenesis by the above-mentioned amino acid substitutions are Leu, Leu, Asp, and Pro, respectively (i.e., L234, L235, D265, and P329). Thus, the above-mentioned amino acid substitutions can be represented herein as L234A / L235A / D265G (LALA-DG), L234A / L235A / D265G / P329A (LALA-DGPA), and L234A / L235A / D265N (LALA-DN), and polypeptides containing these amino acid substitutions can be referred to herein as "LALA-DG variants", "LALA-DGPA variants" and "LALA-DN variants", respectively. The amino acid residues at positions 234, 235, 265, and 329 (all positions represented by EU numbering) of the human IgG4 Fc region before the mutations are introduced are the same as those of the IgG1 Fc region, except that the amino acid residue at position 234 is Phe (i.e., F234, L235, D265, and P329). Thus, where a "CH2 domain according to the present disclosure" is derived from an IgG4 antibody, the above-mentioned amino acid substitutions may be represented herein as F234A / L235A / D265G (FALA-DG), F234A / L235A / D265G / P329A (FALA-DGPA), and F234A / L235A / D265N (FALA-DN), and thus polypeptides comprising these amino acid substitutions may be referred to herein as the "FALA-DG variant," the "FALA-DGPA variant," and the "FALA-DN variant," respectively.

[0036] A "variant CH2 domain" according to the present disclosure comprises the specific amino acid substitutions at positions 234, 235, and 265 (and optionally position 329) described above, and may further comprise amino acid modification(s) (e.g., deletion, addition, substitution, or insertion) other than the specific amino acid substitutions described above. Similarly, a variant Fc region may comprise amino acid modification(s) (e.g., deletion, addition, substitution, or insertion) other than the specific amino acid substitutions described above. If the Fc region comprises additional amino acid modifications, the additional amino acid modifications may be present in the CH2 domain, the CH3 domain, the hinge region (if included), or a combination thereof. As described above, the Fc region comprises two CH2 domains and two CH3 domains. If the Fc region comprises additional amino acid modifications, the modifications may be symmetrical (the same modification is present in both CH2 domains and / or both CH3 domains) or asymmetrical (different modifications in each CH2 domain and / or CH3 domain, or present in only one of the CH2 domains and / or one of the CH3 domains).

[0037] An example of the amino acid sequence of a LALA-DG variant of a human IgG1 Fc region is provided herein as the amino acid sequence consisting of amino acids 114 to 330 of SEQ ID NO: 4 or 5 (see Figures 12A and 12B), an example of the amino acid sequence of a LALA-DN variant of a human IgG1 Fc region is provided herein as the amino acid sequence consisting of amino acids 114 to 330 of SEQ ID NO: 6 or 7 (see Figures 13A and 13B), and an example of the amino acid sequence of a LALA-DGPA variant of a human IgG1 Fc region is provided herein as the amino acid sequence consisting of amino acids 114 to 330 of SEQ ID NO: 8 or 9 (see Figures 14A and 14B). An example of the amino acid sequence of the FALA-DG variant of a human IgG4 Fc region is provided herein as the amino acid sequence consisting of amino acids 111 to 327 of SEQ ID NO: 10 (see FIG. 15A), an example of the amino acid sequence of the FALA-DN variant of a human IgG4 Fc region is provided herein as the amino acid sequence consisting of amino acids 111 to 327 of SEQ ID NO: 11 (see FIG. 15B), and an example of the amino acid sequence of the FALA-DGPA variant of a human IgG4 Fc region is provided herein as the amino acid sequence consisting of amino acids 111 to 327 of SEQ ID NO: 12 (see FIG. 15C). It should be understood that these sequences are provided by way of example only, and that the variant CH2 domain of the present disclosure is not limited thereto. In addition, it should be understood that the present disclosure also encompasses a "LALA-DNPA variant" and a "FALA-DNPA variant" in which the 265th Gly of the "LALA-DGPA variant" or "FALA-DGPA variant" is replaced with Asn, respectively.

[0038] The CH2 domain prior to the introduction of the above-mentioned specific amino acid substitutions (mutations) at positions 234, 235, and 265 (and optionally at position 329) is referred to herein as the "parent CH2 domain". The parent CH2 domain may be, but is not limited to, the wild-type CH2 domain of an IgG1 or IgG4 antibody. For example, if the CH2 domain already contains amino acid modification(s) prior to the introduction of the above-mentioned specific amino acid substitutions, the "parent CH2 domain" refers to the CH2 domain that contains such prior modification(s).

[0039] Similarly, an Fc region comprising the above-mentioned "parent CH2 domain" is referred to herein as a "parent Fc region," and a polypeptide comprising the above-mentioned "parent CH2 domain" or "parent Fc region" is referred to herein as a "parent polypeptide." A parent polypeptide typically refers to, but is not limited to, a polypeptide that is identical or equivalent to a polypeptide according to the present disclosure, except that it comprises a parent CH2 domain instead of a variant CH2 domain of the present disclosure.

[0040] The polypeptide according to the present disclosure comprises a variant Fc region in which the above-mentioned specific amino acid substitutions are introduced into the parent CH2 domain(s). As described above, since there are two CH2 domains in one Fc region, the amino acid substitutions of the variant Fc region may be introduced into only one of the two parent CH2 domains, or into both parent CH2 domains. In addition, when amino acid substitutions are introduced into both parent CH2 domains, the same amino acid substitutions may be introduced, or different amino acid substitutions may be introduced. For example, one of the two variant CH2 domains of the variant Fc region may include amino acid substitutions L234A / L235A / D265G, and the other may include amino acid substitutions L234A / L235A / D265N. Alternatively, one of the two variant CH2 domains of the variant Fc region may include amino acid substitutions L234A / L235A / D265G, and the other may include amino acid substitutions L234A / L235A / D265G / P329A. Other combinations are possible and are encompassed by the present disclosure. When the same amino acid substitutions are introduced into the two CH2 domains of the Fc region, the domain and the Fc region can be referred to as a "homodimer" formed by "homodimerization", and when different amino acid substitutions are introduced into the two CH2 domains of the Fc region, the domain and the Fc region can be referred to as a "heterodimer" formed by "heterodimerization". "Heterodimerization" can be achieved using a variety of known techniques, such as the "knobs-into-holes" technique (International Publication No. WO96 / 027011), "electrostatic steering" (J Biol Chem, 285:19637-46 (2010)), strand-exchange engineered domain (SEED) technique (Prot Eng Des Sel, 23(4):195-202 (2010)), Fab arm exchange (Proc Natl Acad Sci USA, 110(13):5145-50 (2013)), and approaches combining positive and negative design strategies to obtain stable asymmetrically modified Fc regions as described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. Furthermore, when the polypeptide of the present disclosure comprises multiple antibodies, at least one of the antibodies will comprise at least one variant CH2 domain.Furthermore, in some embodiments, one of the two CH2 domains of the variant Fc region may be derived from an IgG1 antibody and the other may be derived from an IgG4 antibody, or, in some embodiments, both CH2 domains of the variant Fc region may be derived from an IgG1 antibody or both may be derived from an IgG4 antibody.

[0041] In one embodiment, the variant CH2 domain in which the amino acid substitutions LALA-DG, LALA-DN or LALA-DGPA are introduced into the parent CH2 domain has improved thermal stability compared to the parent CH2 domain, i.e., the CH2 domain before the introduction of the above-mentioned specific amino acid substitutions at positions 234, 235 and 265 (and optionally at position 329). In a further embodiment, the variant CH2 domain has equivalent or improved thermal stability compared to the parent CH2 domain. In another embodiment, the variant CH2 domain in which the amino acid residues at positions 234, 235 and 265 (all positions are represented by EU numbering) are Ala, Ala and Gly, respectively, and the variant CH2 domain in which the amino acid residues at positions 234, 235, 265 and 329 (all positions are represented by EU numbering) are Ala, Ala, Gly and Ala, respectively, have equivalent thermal stability compared to the respective parent CH2 domain. In another embodiment, a variant CH2 domain in which the amino acid residues at positions 234, 235 and 265 (all positions represented by EU numbering) are Ala, Ala and Asn, respectively, has comparable thermal stability compared to a parent CH2 domain in which the amino acid residues at positions 234, 235 and 265 (all positions represented by EU numbering) are Leu, Leu and Asp, respectively.

[0042] As used herein, the term "thermostability" refers to the thermal denaturation midpoint temperature (T m The thermal denaturation midpoint temperature (Tm) can be measured, for example, by DSC (differential scanning calorimetry) or DSF (differential scanning fluorimetry).

[0043] In one embodiment, the thermal denaturation midpoint temperature (Tm) can be determined by DSC measurement, which observes the change in heat capacity with increasing temperature. For example, in measuring the thermal denaturation midpoint temperature (Tm) of a polypeptide according to the present disclosure, the thermal stability of the CH2 domain can be evaluated by preparing an IgG antibody, i.e., an antibody containing an Fc region (i.e., a dimer containing a hinge portion, a CH2 domain, and a CH3 domain) and a Fab region, and then evaluating the thermal stability of the antibody by DSC (see, for example, the Examples section of the present specification). When DSC analysis of an IgG antibody is performed, a peak of thermal denaturation with increasing temperature is generally observed in the order of the CH2 domain, the Fab region, and the CH3 domain. In the present disclosure, the thermal denaturation midpoint temperature (Tm) was determined by the peak temperature of thermal denaturation, and this was used to evaluate the thermal stability.

[0044] An example of a specific procedure for measuring Tm by DSC is as follows. First, a solution of the polypeptide according to the present disclosure (generally 0.1 μg / mL to 100 μg / mL) is prepared in a histidine buffer, a citrate buffer, TBS, or the like, and then the polypeptide solution is placed in the measurement pan of a DSC instrument. Thereafter, the temperature is increased at a constant rate, and the endothermic peak of the CH2 domain is observed, and Tm is calculated based on the observation. m DSC measurements of the parent polypeptide are performed in a similar manner to determine the Tm of the parent polypeptide, and then the Tm of the polypeptide of the present disclosure and the parent polypeptide are compared. m The difference in values ​​(ΔT m ) can be determined. A DSC measuring instrument commonly used in the art, such as MicroCal VP-Capillary DSC System (Malvern Panalytical Ltd., Malvern, UK), can be used. In one embodiment, the thermal stability of a polypeptide can be determined using the method described in Example 5, 11 or 14 of the present specification.

[0045] As used herein, "improved thermostability" refers to an increase in the thermal denaturation midpoint temperature (T m) is the thermal denaturation midpoint temperature (T m As used herein, "comparable thermostability" refers to a thermal stability that is at least 1°C higher than the thermal denaturation midpoint temperature (T m ) is not 1° C. higher or lower (i.e., within (±) 1° C.) than the reference CH2 domain (e.g., the parent CH2 domain). In one embodiment, when the variant CH2 domain is a LALA-DG variant, T m is at least 3° C. or at least 4° C. higher than the CH2 domain of the wild-type Fc region and at least 3° C. higher than the CH2 domain of the LALA variant (a variant in which the amino acid residues at positions 234 and 235 (all positions represented by EU numbering) of the human IgG1 Fc region are Ala and Ala, respectively). Thus, in one embodiment, the thermal stability of the LALA-DG variant is improved relative to both the wild-type CH2 domain and the CH2 domain of the LALA mutant, which differ from the LALA mutant by a single additional substitution at position 265. In one embodiment, the variant CH2 domain has a T of about 0.1° C., 0.5° C., 1° C. or higher than the parent CH2 domain. m In one embodiment, the variant CH2 domain has a T of about 1.5° C., 2° C., 2.5° C., 3° ​​C. or higher than the parent CH2 domain. m In one embodiment, the variant CH2 domain has a T of about 3.5° C., 4° C., 4.5° C., 5° C. or higher than the parent CH2 domain. m In addition to the specific amino acid substitutions at positions 234, 235, and 265 (optionally position 329) as described above, other additional amino acid modification(s) (such as deletion(s), addition(s), substitution(s)) may be introduced into the variant CH2 domain and / or CH3 domain. As used herein, the term "about" refers to ±10% of the specified value.

[0046] In one embodiment, the polypeptide according to the present disclosure has a reduced or weakened binding affinity to Fcγ receptors compared to the parent polypeptide. In one embodiment, the polypeptide according to the present disclosure has a reduced or weakened binding affinity to Fcγ receptors compared to the parent polypeptide, thereby reducing the effector function of the polypeptide compared to the parent polypeptide. Unless otherwise specified, "binding affinity" and "binding activity" as used herein have the same meaning. The term "Fcγ receptor" (also referred to as Fc gamma receptor or FcγR) refers to a receptor capable of binding to the Fc region of an IgG antibody. The FcγR may be from a mammal, such as a human, monkey, or rat. In one embodiment, the FcγR is a human FcγR or a cynomolgus monkey FcγR. In one embodiment, the FcγR is a human FcγR. The human receptor family includes FcγRI (CD64) (which includes isoforms FcγRIa, FcγRIb, and FcγRIc), FcγRII (CD32) (which includes isoforms FcγRIIa (allotypes H131 (H type) and R131 (R type)), FcγRIIb (which includes FcγRIIb-1 and FcγRIIb-2), and FcγRIIc (which includes FcγRIIc), and FcγRIII (CD16) (which includes isoform FcγRIIIa (allotypes V158 and F158) , FcγRIIIb (including allotypes FcγRIIIb-NA1 and FcγRIIIb-NA2), and FcγRIIIc). The cynomolgus monkey Fcγ receptor family includes, but is not limited to, the above FcγRI, FcγRII, and FcγRIII (including their isoforms), excluding FcγRIIc and FcγRIIIb. FcγR receptors may also include allelic variants and alternatively spliced ​​forms of the receptor.As used herein, "reduced binding affinity to FcγR" means that the activity to at least one of the nine isoforms of FcγRI, FcγRII, and FcγRIII may be reduced, for example, the binding affinity to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, or all of the FcγR isoforms may be reduced. In one embodiment, the polypeptide according to the present disclosure has reduced binding to one or more of FcγRI, FcγRII, or FcγRIII. In one embodiment, the polypeptide according to the present disclosure has reduced binding to FcγRI, FcγRII, and FcγRIII. The FcγRI, FcγRII, and / or FcγRIII may be human FcγRI, FcγRII, and / or FcγRIII, or cynomolgus monkey FcγRI, FcγRII, and / or FcγRIII. In one embodiment, the polypeptide according to the present disclosure has reduced binding to one or more of human FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa or FcγRIIIb. In one embodiment, the polypeptide according to the present disclosure has reduced binding to human FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa and FcγRIIIb. Examples of Fcγ receptors derived from human and cynomolgus monkeys include, but are not limited to, the receptors disclosed in the Comparative Examples and Examples of the present specification.

[0047] The term "effector function" as used herein refers to a biological activity attributable to or mediated by the Fc region of an antibody. Effector functions include, but are not limited to, complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), Fc receptor binding, C1q binding, downregulation of cell surface receptors (e.g., B cell receptors), and cytokine production. In one embodiment, a polypeptide according to the present disclosure may have reduced effector function by the above-mentioned specific amino acid substitutions at positions 234, 235, and 265 (and optionally at position 329), where the effector function is one or more of CDC activity, ADCC activity, ADCP activity, Fc receptor binding, C1q binding, downregulation of cell surface receptors, or excessive cytokine production, and may be useful for pharmaceutical applications where effector function is not required. Amino acid modifications that reduce effector function may be referred to herein as "effectorless mutations." Effector function can be assessed using assays known in the art, such as measuring binding affinity to Fcγ receptors, such as those disclosed herein.

[0048] "Binding affinity to Fcγ receptor" can be measured by known methods, for example, by surface plasmon resonance (SPR) method that measures interaction between ligand and analyte. This measurement can be performed, for example, using Biacore™ SPR system (Cytiva, Marlborough, MA). More specifically, in this method, each His-tagged FcγR is immobilized directly on a sensor chip as a ligand or immobilized by capture with anti-His tag antibody on the sensor chip, and then a polypeptide of the present disclosure is added to the sensor chip as an analyte. When the added analyte binds to the immobilized ligand, the apparent mass of the immobilized ligand molecule increases. The amount of position shift of the SPR signal, which depends on the change in refractive index of the solvent on the sensor chip surface, is measured. Based on the amount of this shift, the amount of analyte (antibody) bound to the ligand (FcγR) is determined as a binding response value (resonance unit, RU) (see, for example, Proc. Natl. Acad. Sci USA, 103(11):4005-4010 (2006)). Alternatively or additionally, the binding affinity to the Fcγ receptor can be measured by ELISA, FACS, or the like. In one embodiment, the "binding affinity to the Fcγ receptor" can be determined using the method described in Examples 1-4, 9-10, 12, or 13 of the present specification.

[0049] As used herein, "reduced or attenuated binding affinity to an Fcγ receptor" means that the binding affinity of a polypeptide according to the present disclosure to an Fcγ receptor is lower than the binding affinity of a reference polypeptide (e.g., parent polypeptide) to the same Fcγ receptor when determined by the same assay. The reduced or attenuated binding affinity can be determined, for example, by measuring whether the RU value of a polypeptide according to the present disclosure to at least one Fcγ receptor determined by the above-mentioned surface plasmon resonance assay is reduced compared to the RU value of a reference polypeptide (e.g., parent polypeptide) to the same Fcγ receptor determined by the same assay. In one embodiment, to effectively reduce effector function, when the binding affinity of a parent polypeptide comprising a parent CH2 domain to an Fcγ receptor is considered as 100%, the binding affinity of a polypeptide according to the present disclosure to the same Fcγ receptor is reduced to less than 80%, less than 70%, or less than 60%. In one embodiment, when the binding affinity of the parent polypeptide comprising the parent CH2 domain to an Fcγ receptor is considered as 100%, the binding affinity of the polypeptide according to the present disclosure to the same Fcγ receptor is reduced to less than 50%, less than 40%, less than 30%, less than 20%, or less than 10%. In one embodiment, when the binding affinity of the parent polypeptide comprising the parent CH2 domain to an Fcγ receptor is considered as 100%, the binding affinity of the polypeptide according to the present disclosure to the same Fcγ receptor is reduced to less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.1%.

[0050] As known in the art, amino acid substitutions in various regions of an antibody (including Fc region) can reduce the antigen-binding activity of the antibody.In one embodiment, the polypeptide according to the present disclosure does not show a significant reduction in "antigen-binding activity" compared with the parent polypeptide.The antigen-binding activity of a polypeptide can be measured by known methods, for example, SPR method (for example, see the method described in Example 6 of the present specification).

[0051] Also, as known in the art, amino acid substitutions in various regions of an antibody (including the Fc region) can reduce the serum half-life of the antibody. In one embodiment, the polypeptide according to the present disclosure does not show a significant reduction in "serum half-life" compared to the parent polypeptide. The serum half-life of a polypeptide can be determined by known methods, for example, using the method described in Example 8 herein. The serum half-life of an antibody or functional fragment thereof can be extended by chemical modification, for example, by covalent attachment to a polymer.

[0052] Also, as is known in the art, the binding activity of an antibody to the fetal Fc receptor FcRn can be reduced by amino acid substitution in various regions (including the Fc region) of the antibody. In one embodiment, with respect to the above-mentioned blood half-life, the polypeptide of the present disclosure does not show a significant reduction in "binding activity to fetal Fc receptor (FcRn)" compared to the parent polypeptide. As is known in the art, when an antibody is administered, it is non-specifically taken up into vascular endothelial cells and the like at a constant rate by pinocytosis. The antibody binds to FcRn in endosomes at low pH (about pH 6.0) and is transported out of the cell via FcRn, thereby avoiding lysosomal migration and antibody degradation. Thus, the binding of the antibody to FcRn contributes to prolonging the blood half-life of the antibody. The binding activity of a polypeptide to FcRn can be measured by known methods, for example, biolayer interferometry (see, for example, the method described in Example 7 of the present specification).

[0053] Antibodies and their functional fragments In one embodiment, the polypeptide according to the present disclosure may be an antibody or a fragment thereof (e.g., a functional fragment). An "antibody" is a molecule (immunoglobulin) that contains an antigen-binding site that immunospecifically binds to an antigen and generally contains a heavy chain variable region, a heavy chain constant region, a light chain variable region, and a light chain constant region. An "antibody" may be any one of a polyclonal antibody, a bispecific antibody, a multispecific antibody, a chimeric antibody, a humanized antibody, a human antibody, a functional fragment thereof, or a modification thereof. The antibody may be derived from a variety of species, including human, cynomolgus monkey, rat, mouse, camel, llama, shark, rabbit, and the like. In one embodiment, the antibody is derived from a human or cynomolgus monkey. In one embodiment, the antibody is of human origin. In one embodiment, when the antibody is derived from a non-human species, the antibody is chimerized or humanized using well-known techniques. In one embodiment, the antibody may be a polyclonal antibody or a monoclonal antibody. In one embodiment, the antibody is a monoclonal antibody.

[0054] In one embodiment, the polypeptide according to the present disclosure may be a "functional fragment of an antibody", which comprises a variant Fc region containing the above-mentioned specific amino acid substitutions. A "functional fragment of an antibody", also referred to as an "antigen-binding fragment of an antibody", refers to a partial fragment of an antibody that has antigen-binding activity, including, but not limited to, linear antibody fragments (see, e.g., U.S. Pat. No. 5,641,870) and multispecific antibody fragments. Antigen-binding fragments include fragments generated by treating a full-length antibody molecule with an appropriate enzyme, and proteins produced in a suitable host cell using engineered antibody genes (i.e., recombinant proteins). Functional fragments also include antigen-binding fragments that include, for example, asparagine (Asn297) and adjacent amino acids that are modified by N-linked glycosylation, which is well conserved in the Fc region of IgG heavy chains.

[0055] In one embodiment, the polypeptide may be a "bispecific antibody" or a "multispecific antibody." A multispecific antibody may be an antibody that includes multiple antigen-binding domains, each of which binds to a different antigen. A bispecific antibody is an antibody that includes two antigen-binding domains, each of which binds to a different antigen. In any of these cases, the amino acid substitution may be included in one or both of the two CH2 domains of the antibody Fc region, and if included in both, the same amino acid substitution may be included or different amino acid substitutions may be included. Alternatively, a multispecific antibody may be an artificial protein in which multiple antibodies each having a different antigen-binding domain are bound to each other, and a bispecific antibody may be an artificial protein in which two antibodies each having a different antigen-binding domain are bound to each other. In these cases, the amino acid substitution may be included in one or both of the two CH2 domains of the Fc region of at least one of the multiple antibodies, and if included in both, the same amino acid substitution may be included or different amino acid substitutions may be included. Introducing different amino acid substitutions into the two CH2 domains can be performed by using techniques that allow the production of antibody molecules from two different heavy chains (heterodimers), such as the "knobs-into-holes" technique (International Publication No. WO1998 / 050431), "electrostatic steering" (J Biol Chem, 285:19637-46 (2010)), strand-exchange engineered domain (SEED) technique (Prot Eng Des Sel, 23(4):195-202 (2010)), or the techniques described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. Bispecific or multispecific antibodies can also be produced as full-length antibodies or fragments thereof (see, for example, International Publication No. WO96 / 16673, and U.S. Pat. No. 5,837,234).Methods for making bispecific or multispecific antibodies are well known in the art and include, for example, coexpression of two immunoglobulin heavy chain light chain pairs, where the two chains have different specificities (see, e.g., Millstein et al., Nature, 305:537-539 (1983)); techniques for making bispecific antibodies from antibody fragments (see, Brennan et al., Science, 229:81 (1985)), and other methods known in the art.

[0056] In one embodiment, the antibody may be a "chimeric antibody." By "chimeric antibody" is meant an antibody that contains one or more regions derived from one antibody and one or more regions derived from one or more other antibodies. For example, the Fc sequence may be derived from a human antibody and the variable region sequence may be derived from a non-human species antibody, such as a cynomolgus monkey.

[0057] In one embodiment, the antibody may be a "human antibody." A "human antibody" includes all antibodies having one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all variable and constant domains of the antibody are derived from human immunoglobulin sequences (referred to as a "fully human antibody"). These antibodies can be prepared in a variety of ways, such as by immunization of non-human animals that have been genetically modified to express antibodies arising from genes encoding human heavy and / or light chains.

[0058] In one embodiment, the antibody may be a "humanized antibody." A "humanized antibody" is an antibody created by inserting an antigen-binding site (such as one or more CDRs) of an antibody produced in a non-human species into the sequence of a human antibody. Humanized antibodies include human immunoglobulins in which hypervariable region residues are replaced by residues from a hypervariable region of a non-human animal having the desired specificity, affinity, etc., or in which Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies are less likely to provoke an immune response and / or less likely to induce a severe immune response when administered to a human subject compared to antibodies from non-human species.

[0059] In one embodiment, the antibody can be a "single-chain antibody". "Single-chain antibody" refers to an antibody in which the heavy chain, which is originally a double chain, is connected by a linker to form a single chain (see, for example, Biomaterials, 117:24-31 (2017)). Thus, single-chain antibody also comprises two CH domains, and the above-mentioned specific amino acid substitutions can be introduced into one or both of the CH2 domains.

[0060] In general, antibodies can be obtained by immunizing an animal with a polypeptide that functions as an antigen and collecting and purifying the antibodies produced in vivo using methods commonly practiced in the art. The source of the antigen is not limited to humans, and antigens from non-human animals such as cynomolgus monkeys, mice, and rats can also be used to immunize animals and produce antibodies. The antigen can be one of a variety of antigens, such as a cytokine, a soluble or insoluble factor, a molecule expressed on a pathogen, a molecule expressed on a cell, or a molecule expressed on a cancer cell. In some embodiments, the antigen is a cancer antigen. Monoclonal antibodies can be obtained, for example, by fusing myeloma cells with antibody-producing cells to establish hybridomas using known methods (see, for example, Kohler and Milstein, Nature, 256:495-497 (1975); Kennett, R. ed., Monoclonal Antibodies, p. 365-367, Plenum Press, NY (1980)). Other methods are also known in the art. Antibodies applicable to human diseases can also be selected by testing the cross-reactivity of the resulting antibodies binding to heterologous antigens with human antigens.

[0061] It is known that antibodies produced in cultured mammalian cells have a deletion of the lysine residue at the carboxyl terminus of the heavy chain (Journal of Chromatography A, 705:129-134 (1995)), two amino acid residues, glycine and lysine, at the carboxyl terminus of the heavy chain, and a new amidation of the proline residue located at the carboxyl terminus (Analytical Biochemistry, 360:75-83 (2007)). However, these deletions or modifications of the heavy chain sequence do not affect the ability of the antibody to bind to an antigen or the effector function of the antibody (such as complement activation and antibody-dependent cellular cytotoxicity). Thus, the antibodies according to the present disclosure also include antibodies that have been subjected to such deletions or modifications and functional fragments of such antibodies, including antibodies having a deletion in which one or two amino acids are deleted at the carboxyl terminus of the heavy chain, and amidated antibodies having a deletion (for example, a heavy chain in which the proline residue at the carboxyl terminus is amidated and a heavy chain lacking a lysine residue at the carboxyl terminus). However, as long as the reduction in antigen-binding ability and effector function is maintained, the deletion of the carboxyl terminus of the heavy chain of the antibody is not limited to the above examples. The two heavy chains constituting the antibody may be derived from either one of the heavy chains of a full-length antibody, an antibody having the above deletion, or a combination of both. The ratio of each deletion may be influenced by the type of cultured mammalian cells producing the antibody and the culture conditions, but in one embodiment, the antibody has one amino acid residue deleted at the carboxyl terminus of both of the two heavy chains.

[0062] In one embodiment, when the polypeptide according to the present disclosure is an antibody, the antibody is an antibody that targets tumor cells or immune cells. In one embodiment, the antibody is an antibody that targets tumor cells. In one embodiment, the polypeptide is an antibody or a functional fragment thereof that binds to a tumor antigen. In one embodiment, when the polypeptide is an antibody that targets tumor cells, the antibody has one or more of the following characteristics: capable of recognizing tumor cells, capable of binding to tumor cells, capable of being taken up and internalized by tumor cells, and / or damaging tumor cells.

[0063] In one embodiment, where the polypeptide according to the disclosure is an antibody targeting a tumor cell, the antibody can be, for example, an anti-HER2 antibody, an anti-HER3 antibody, an anti-DLL3 antibody, an anti-FAP antibody, an anti-CDH11 antibody, an anti-CDH6 antibody, an anti-A33 antibody, an anti-CanAg antibody, an anti-CD19 antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD98 antibody, an anti-LPS antibody, an anti-TROP2 antibody, an anti-CEA antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, an anti-SLC44A4 antibody, an anti-mesothelin antibody, an anti-ENPP3 antibody, an anti-CD47 antibody, an anti-EGFR antibody, or an anti-DR5 antibody. In one embodiment, when the polypeptide according to the present disclosure is an antibody that targets tumor cells, the antibody can be an anti-CD70 antibody, an anti-LPS antibody or an anti-EGFR antibody. Such antibodies are prepared by known methods (see, for example, Proc.Natl.Acad.Sci.USA,81:6851-6855(1984);Nature 3211:522-525(1986) and International Publication No. WO90 / 07861). Also see anti-CD70 antibody (International Publication No. WO2004 / 073656 and WO2007 / 038637), anti-LPS antibody (International Publication No. WO2015 / 046505 and WO2019 / 065964), and anti-EGFR antibody (International Publication No. WO1998 / 050433 and WO2002 / 092771).

[0064] In one embodiment, when the polypeptide according to the present disclosure is an antibody that targets a tumor cell, the antibody is selected from the group consisting of panitumumab, alemtuzumab, atezolizumab, avelumab, belimumab, blinatumomab, glenbatumumab, ibritumomab, ipilimumab, nivolumab, brentuximab, canakinumab, cetuximab, daratumumab, denosumab, pidilisumab, mab, mogamulizumab, ramucirumab, rituximab, siltuximab, dinutuximab, durvalumab, elotuzumab, obinutuzumab, ofatumumab, olaratumab, pembrolizumab, pertuzumab, taborixizumab, tositumomab, trastuzumab, tremelimumab, varlilumab, borsetuzumab, or O11-1111. In one embodiment, when the polypeptide according to the present disclosure is an antibody that targets tumor cells, the antibody can be panitumumab, borsetuzumab, or O11-1111.

[0065] In one embodiment, a polypeptide according to the present disclosure may be an antibody that targets tumor cells and may be used, for example, to treat inflammatory diseases, immune diseases, infectious diseases, or in regenerative medicine.

[0066] Additionally, in some embodiments, the polypeptides according to the present disclosure can be any of a variety of antibodies, including, for example, monovalent antibodies, chain exchange engineered domains (SEEDs), triomabs, dual variable domain immunoglobulins (DVD-Ig), miniantibodies, dual affinity retargeting molecules (Fc-DART or Ig-DART), LUZ-Y antibodies, biclonic antibodies, dual targeting (DT) Ig antibodies, two-in-one antibodies, cross-linked Mabs, mAbs, 2 , CovX-body, Ts2Ab, BsAb, HERCULES antibody, TvAb, or SCORPION.

[0067] Fusion proteins Certain embodiments of the present disclosure relate to "fusion proteins" that include a variant Fc region or a polypeptide or antibody portion that includes a variant Fc region fused to another protein or polypeptide (a "heterologous" protein or polypeptide). Various heterologous proteins or polypeptides can be fused to a variant Fc region, polypeptide, or antibody portion using recombinant techniques to create a fusion protein. Examples include, but are not limited to, receptors or their target binding regions, adhesion molecules, ligands, enzymes, cytokines, immunocytokines, non-immunoglobulin proteins, chemokines, various protein domains, and the like. Other proteins of interest also include therapeutic agents that direct the fusion protein to a therapeutic target, where such a target may be a molecule associated with a disease, such as a receptor protein that binds to the target. Examples of fusion proteins that include a receptor protein that binds to a target include, but are not limited to, VEGFR-Fc fusions, TNFR-Fc fusions, CTLA4-Fc fusions, and the like. Fusion proteins may also include scFvs and can be constructed by fusing an Fc region to either the amino or carboxy terminus of an scFv (see, for example, Antibody Engineering, ed. Borrebaeck, 1995, Oxford University Press). It is understood that a "fusion protein" is one embodiment of the above-mentioned "molecule comprising a polypeptide according to the present disclosure."

[0068] Polypeptide-drug conjugates Certain embodiments of the present disclosure relate to polypeptide-drug conjugates in which the polypeptide according to the present disclosure is conjugated to one or more drugs or modifying agents. In one embodiment, the polypeptide-drug conjugate according to the present disclosure includes modified antibodies. For example, the antibody can be conjugated to chemotherapeutic agents, cytotoxic agents, or various non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene, or copolymers of polyethylene glycol and polypropylene glycol. Polypeptide-drug conjugates in which the polypeptide is conjugated to both drugs and polymers (modifying agents) are also included in certain embodiments.

[0069] Polypeptide-drug conjugates can be prepared by conjugating a polypeptide to a drug or modifier via a linker. The linker typically comprises a functional group capable of reacting with one or more targeting groups on an antibody and one or more functional groups capable of reacting with targeting groups on a drug or modifier. Suitable functional groups and linkers are known in the art and include, for example, those described in Bioconjugate Techniques (GTHermanson, 2013, Academic Press) and Antibody-Drug Conjugates: Methods in Molecular Biology (Ducry (Ed.), 2013, Springer).

[0070] Pharmaceutical Compositions In certain embodiments, the polypeptide, the molecule comprising the polypeptide, or the polypeptide-drug conjugate may be provided in a pharmaceutical composition comprising a pharma- ceutically acceptable carrier. Pharmaceutically acceptable carriers include excipients, inert diluents, granulating agents, disintegrating agents, binders, wetting agents, coloring agents, preservatives, aqueous vehicles and solvents, oily vehicles and solvents, surfactants, dispersants, sweeteners, flavorings, emulsifiers, lubricants, buffers, thickening agents, fillers, antioxidants, stabilizers, etc. Suitable pharma- ceutically acceptable carriers are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences" by EW Martin), Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0071] In one embodiment, the pharmaceutical composition may also include one or more other "therapeutic agents" or may be used or administered in combination with one or more other "therapeutic agents". For example, when used for the purpose of cancer treatment, examples of other therapeutic agents for cancer treatment include, but are not limited to, Abraxane, carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastine or drugs described in International Publication No. WO2003 / 038043, and LH-RH analogs (e.g., leuprorelin, goserelin), estramustine phosphate, estrogen antagonists (e.g., tamoxifen, raloxifene), aromatase inhibitors (e.g., anastrozole, letrozole, exemestane), immune checkpoint inhibitors (e.g., nivolumab, ipilimumab), etc. Routes of administration of the pharmaceutical composition include, but are not limited to, administration via intradermal, intramuscular, intraperitoneal, intravenous, or subcutaneous routes. When combined with other therapeutic agents, the pharmaceutical composition and one or more other therapeutic agents can be used or administered either as a single composition or separately, at the same time or at different times.

[0072] How to use In certain embodiments, the polypeptide, the molecule comprising it, or the polypeptide-drug conjugate can be used in a method for treating humans.For example, in one embodiment, the polypeptide, the molecule comprising it, or the polypeptide-drug conjugate can be used in the treatment of cancer or tumor, immune disease, inflammatory disease, or infectious disease.In one embodiment, the polypeptide, the molecule comprising it, or the polypeptide-drug conjugate can be used in a diagnostic method in humans.In one embodiment, the polypeptide, the molecule comprising it, or the polypeptide-drug conjugate can be used for the treatment of tumor or cancer in humans. Tumor diseases and cancers include, but are not limited to, lung cancer, kidney cancer, urothelial carcinoma, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, gastric cancer, esophageal cancer, uterine cancer, testicular cancer, cervical cancer, placental choriocarcinoma, glioblastoma multiforme, brain cancer, head and neck cancer, thyroid cancer, mesothelioma, gastrointestinal stromal tumor (GIST), gallbladder cancer, bile duct cancer, adrenal cancer, squamous cell carcinoma, leukemia, malignant lymphoma, myeloma, or sarcoma.

[0073] Additional Amino Acid Modifications In addition to the specific amino acid substitution(s) described above (e.g., the LALA-DG, LALA-DGPA, and LALA-DN mutations), a polypeptide may include additional amino acid modification(s) in the Fc region. Thus, a "variant Fc region" of a polypeptide according to the present disclosure may similarly include, in addition to the specific amino acid substitutions described above (e.g., the LALA-DG, LALA-DGPA, and LALA-DN), additional amino acid modification(s) in the Fc region. The additional amino acid modifications may be made to the Fc region of the polypeptide as well as to one or more other regions of the polypeptide.

[0074] "Amino acid modification" refers to amino acid substitution, deletion, addition, insertion, modification, etc., and can be performed by various methods known in the art. For example, but not limited to, amino acid substitution, addition, deletion, and insertion can be performed using any well-known PCR-based technique, and amino acid substitution can be performed by site-directed mutagenesis (see, for example, Zoller and Smith, Nucl. Acids Res 10:6487-6500 (1982); Kunkel, Proc. Natl. Acad. Sci USA, 82:488 (1985)). An example of an amino acid "modification" includes the addition or deletion of a glycosylation chain.

[0075] In one embodiment, the amino acid substitution(s) may be "conservative amino acid substitution(s)". A "conservative amino acid substitution" means that an amino acid residue is replaced by another amino acid residue having a side group with similar chemical properties (e.g., charge or hydrophobicity), and generally does not substantially change the functional properties of the protein. Examples of conservative amino acid substitutions are well known in the art and can be made, for example, by making substitutions within the classes of amino acids represented by one or more of the following: acidic residues: Asp, Glu; basic residues: Lys, Arg, His; hydrophilic uncharged residues: Ser, Thr, Asn, Gln; aliphatic uncharged residues: Gly, Ala, Val, Leu, Ile; non-polar uncharged residues: Cys, Met, Pro, and aromatic residues: Phe, Tyr, Trp.

[0076] The additional amino acid modification(s) can be at least one amino acid modification, for example, about 20 or less, about 10 or less, or about 1 to about 5 amino acid modifications. When the additional amino acid modification(s) is / are made to a wild-type sequence (e.g., a sequence of a wild-type Fc region), it is preferred that the sequence of the variant sequence (e.g., a variant Fc region) has at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the corresponding wild-type sequence. In one embodiment, the sequence of the variant sequence (e.g., a variant Fc region) has at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding wild-type sequence. Polypeptide sequence similarity or identity can be measured using known sequence analysis software (eg, GAP, BESTFIT, FASTA, or TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wis.)).

[0077] Examples of additional amino acid modifications include G236A, G236P, G236L, G236K, G236R, G236T, G236H, G236N, G237R, P238I, S239M, S239K, S239R, S239T, S239H, S239H, S239Q, V266I, V266L, S267P, S267K, H268A, H268V, H268F, H268P, H268M, H268I, H268L, H268Q, and the like. Included are amino acid substitutions such as 8K, H268R, H268T, H268Y, H268Q, H268W, E269A, E269V, E269D, E269K, E269R, E269S, E269T and E269H (all positions represented by EU numbering), which have been shown to increase the Tm of an antibody Fc over the parent polypeptide, as described in International Publication No. WO2013 / 118858.

[0078] In one embodiment, amino acid substitutions that further reduce Fcγ receptor binding activity may be used. Examples of additional amino acid substitutions that reduce Fcγ receptor binding activity include, for example, amino acid substitutions such as G237F / S239E / A327H, G237 / A327 / A330I, and S239E / S267E / H268D (all positions are represented by EU numbering) as shown in International Publication No. WO2011 / 120134.

[0079] In one embodiment, the additional amino acid modifications include substitutions that reduce susceptibility to proteolysis, substitutions that reduce susceptibility to oxidation, substitutions to change binding affinity to form protein complexes, substitutions that confer or alter other physicochemical or functional properties of such analogs, modifications that inhibit deamidation reactions, etc.

[0080] Preparation method The polypeptide of the present disclosure may be prepared by recombinant protein methods known in the art. Although not limited to the following methods, the polypeptide according to the present disclosure may be obtained by a method including the following steps: first, designing a DNA encoding a polypeptide comprising the above-mentioned variant Fc region, then chemically synthesizing the DNA and inserting the DNA into a vector or the like, followed by introducing the DNA / vector into a host cell, culturing the host cell in a medium under conditions suitable for the expression of the polypeptide, and recovering the polypeptide from the medium or the cell. A vector comprising a DNA encoding a polypeptide comprising a variant Fc region may be prepared, for example, by introducing an appropriate mutation into a polypeptide comprising a wild-type Fc region, for example, using a KOD-Plus-Mutagenesis kit (Toyobo Co., Ltd., Osaka, Japan) according to the manufacturer's instructions. If the polypeptide comprises other amino acid modifications of interest (e.g., additions, deletions, substitutions, etc.) other than those described above, a DNA sequence encoding a polypeptide comprising the specific amino acid substitutions of the above-mentioned variant Fc region and other amino acid modifications of interest (multiple possible) may be designed, and the polypeptide may be obtained in the same manner as described above. In addition, polypeptides of the present disclosure can be prepared by recombinant methods as well as peptide synthesis, chemical synthesis, in vitro translation, and the like.

[0081] Thus, in one embodiment, the disclosure relates to a nucleic acid comprising a nucleotide sequence encoding a polypeptide comprising the variant Fc region or a portion thereof, e.g., a heavy or light chain, as described above. In one embodiment, the disclosure relates to a vector comprising said nucleic acid. In one embodiment, the disclosure relates to a host cell comprising a nucleic acid encoding a polypeptide comprising the variant Fc region described above. In one embodiment, the disclosure relates to a method for producing a polypeptide described herein, e.g., comprising culturing a host cell comprising a nucleic acid encoding the variant Fc region or a polypeptide comprising same in a medium under conditions suitable for expression of the variant Fc region or a polypeptide comprising the variant Fc region, and recovering the variant Fc region or polypeptide from the medium or cell.

[0082] Embodiment Exemplary, non-limiting embodiments of the present disclosure include the following.

[0083] [1] A polypeptide comprising one or more variant IgG1 Fc regions or variant IgG4 Fc regions, each of said variant Fc regions comprising two CH2 domains, one or both of the CH2 domains in at least one of said variant Fc regions is variant CH2 domain(s), and the amino acid residues at positions 234, 235, and 265 (all positions represented by EU numbering) of the variant CH2 domain are Ala, Ala, and Gly, respectively.

[0084] [2] The polypeptide described in [1], wherein the amino acid residue at position 329 of the variant CH2 domain is Ala.

[0085] [3] A polypeptide comprising one or more variant IgG1 Fc regions or variant IgG4 Fc regions, each of said variant Fc regions comprising two CH2 domains, one or both of the CH2 domains in at least one of said variant Fc regions is variant CH2 domain(s), and the amino acid residues at positions 234, 235, and 265 (all positions represented by EU numbering) of the variant CH2 domain are Ala, Ala, and Asn, respectively.

[0086] [4] The polypeptide described in [1] or [2], wherein the thermal denaturation midpoint temperature (Tm) of the variant CH2 domain is at least 1°C higher than the Tm of the parent CH2 before the mutation is introduced.

[0087] [5] The polypeptide described in [4], wherein the thermal denaturation midpoint temperature (Tm) of the variant CH2 domain is 3°C or more higher than the Tm of the parent CH2.

[0088] [6] The polypeptide described in [3], wherein the thermal denaturation midpoint temperature (Tm) of the variant CH2 domain is not 1°C higher than the Tm of the parent CH2 before the mutation is introduced and not 1°C lower than the Tm of the parent CH2 before the mutation is introduced.

[0089] [7] A polypeptide described in any one of [1] to [6], which has a reduced binding affinity to at least one Fcγ receptor compared to a parent polypeptide containing the parent CH2 domain before the mutation is introduced.

[0090] [8] The polypeptide described in [7], which has a binding affinity to at least one Fcγ receptor that is reduced by 50% or more compared to the parent polypeptide.

[0091] [9] The polypeptide described in [7] or [8], wherein the at least one Fcγ receptor is at least one selected from a human Fcγ receptor and a cynomolgus monkey Fcγ receptor.

[0092]

[10] The polypeptide described in [9], wherein the Fcγ receptor is a human Fcγ receptor.

[0093]

[11] The polypeptide according to any one of [7] to

[10] , wherein the Fcγ receptor is FcγRI.

[0094]

[12] The polypeptide according to any one of [1] to

[11] , comprising one or more variant IgG1 Fc regions.

[0095]

[13] The polypeptide according to any one of [1] to

[12] , which is an antibody or a functional fragment thereof.

[0096]

[14] The polypeptide described in

[13] , wherein the antibody or functional fragment thereof binds to a tumor antigen.

[0097]

[15] A molecule comprising a polypeptide according to any one of [1] to

[14] .

[0098]

[16] The polypeptide according to

[15] , wherein the molecule is a fusion protein between a polypeptide according to any one of [1] to

[14] and a polypeptide other than the polypeptide.

[0099]

[17] A polypeptide-drug conjugate, in which the polypeptide according to any one of [1] to

[14] is conjugated to a drug.

[0100]

[18] A nucleic acid comprising a nucleotide sequence encoding a polypeptide or a part thereof according to any one of [1] to

[14] .

[0101]

[19] A host cell comprising the nucleic acid described in

[18] .

[0102]

[20] A pharmaceutical composition comprising a polypeptide according to any one of [1] to

[14] , a molecule according to

[15] or

[16] , or a polypeptide-drug conjugate according to

[17] , and a pharma- ceutically acceptable carrier.

[0103]

[21] A polypeptide according to any one of [1] to

[14] , a molecule according to

[15] or

[16] , or a polypeptide-drug conjugate according to

[17] , for use in treatment.

[0104]

[22] The polypeptide, molecule, or polypeptide-drug conjugate for use according to

[21] , wherein the treatment comprises treatment of cancer, an immune disease, an inflammatory disease or an infectious disease. EXAMPLES

[0105] The following examples are offered for illustrative purposes and are not intended to limit the scope of the present invention in any way.

[0106] Comparative Example 1: Evaluation of the binding activity of anti-EGFR antibodies containing known effectorless mutations to human FcγRI. The binding activity of anti-EGFR antibodies with known effector-reducing mutations introduced to human FcγRI (hFcγRI) was evaluated using Biacore™ T200 (Cytiva, Marlborough, MA). The Fc region of the anti-EGFR antibody panitumumab, which is originally a human IgG2 antibody, was replaced with the Fc region of human IgG1, and the resulting antibody was called IgG1 WT. The amino acid sequences of the heavy and light chains of IgG1 WT are represented by SEQ ID NOs: 13 and 14, respectively (see Figures 16A and 16B). IgG1 WT and the following variants were evaluated: a variant obtained by introducing L234A / L235A (LALA) mutations into the Fc region of IgG1 WT; a variant obtained by introducing L234A / L235A / D265A (LALA-DA) mutations into the Fc region of IgG1 WT; and a variant obtained by introducing L234A / L235A / P329G (LALA-PG described in International Publication No. WO2012 / 130831) mutations into the Fc region of IgG1 WT. The described mutations were introduced into both CH2 domains of the Fc region. These anti-EGFR antibodies (also referred to as "anti-EGFR antibody variants") were obtained by introducing a vector containing DNA encoding the amino acid sequence of the antibody into a host cell and culturing the host cell in a medium to cause the host cell to express the DNA in the cell. A His-tagged human FcγRI consisting of recombinant human Fc gamma RI / CD64 protein CF (R&D Systems, Minneapolis, MN, accession number: P12314), containing the Gln residue at position 16 of SEQ ID NO: 19 through the Pro residue at position 288 (see FIG. 19A) and with a 6×His tag added to the carboxyl terminus, was used as a ligand. The FcγRI ligand was captured on a sensor chip with immobilized anti-His antibodies. Various anti-EGFR antibodies were then added to the sensor chip as analytes, and the interaction between the ligand and the analyte was measured by surface plasmon resonance (SPR).

[0107] Briefly, anti-6X His tag antibody [AD1.1.10] (Abcam plc, Cambridge, UK) was immobilized on a sensor chip CM5 (Cytiva, Marlborough, MA) according to the manufacturer's instructions, and then recombinant human Fc gamma RI / CD64 protein (R&D Systems) prepared at 10 μg / mL in HBS-EP+ (GE Healthcare, Chicago, IL) was captured by contacting the sensor chip with the antibody at 10 μL / min for 60 seconds. Anti-EGFR antibodies prepared at each given concentration in HBS-EP+ were then added to the sensor chip at a flow rate of 30 μL / min for 120 seconds. Figure 2A shows a graph of the binding response (resonance units, RU) plotted against each test antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM). Table 1 below shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0108] [Table 1]

[0109] As shown in Table 1 above, all variants containing effectorless mutations had lower RU values, an index of binding to human FcγRI, than IgG1 WT. The RU values ​​of the variants were in the following order: LALA>LALA-PG>LALA-DA.

[0110] Comparative Example 2: Evaluation of the binding activity of anti-EGFR antibodies containing known effectorless mutations to various human FcγRs The binding activity of the anti-EGFR antibodies described in Comparative Example 1 to human FcγRIIa, FcγRIIa (H167), FcγRIIb / c, FcγRIIIa, FcγRIIIa (V176F), and FcγRIIIb was evaluated using Biacore™ T200. After human FcγR was directly immobilized on a sensor chip as a ligand, various anti-EGFR antibodies were added to the sensor chip as analytes, and the interaction between the ligand and analyte was measured by SPR. All antibodies contained either a 10×His tag or a 6×His tag at the carboxyl terminus and were R&D. The following FcγRs were obtained from Systems (Minneapolis, MN): recombinant human Fc gamma RIIA / CD32a (R167) protein (accession number: AAA35827, comprising an Ala residue at position 36 of SEQ ID NO:20 through an Ile residue at position 218 (see FIG. 19B) and including a 10×His tag), recombinant human Fc gamma RIIA / CD32a (H167) protein (accession number: P12318-1 (P12318.4), comprising an Ala residue at position 34 of SEQ ID NO:21 through an Ile residue at position 218 (see FIG. 19C) and including a 10×His tag), recombinant human Fc gamma RIIB / C (CD32b / c) protein (accession number: P31994, comprising an Ala residue at position 46 of SEQ ID NO:22 through an Ile residue at position 217 (see FIG. 19D) and including a 10×His tag). (see FIG. 19D) and containing a 10×His tag), recombinant human Fc gamma RIIIA / CD16a protein (accession number: AAH17865, containing a Gly residue at position 17 of SEQ ID NO: 23 to a Gln residue at position 208 (see FIG. 19E) and containing a 6×His tag), recombinant human Fc gamma RIIIA / CD16a (V176F) protein (accession number: P08637, containing a Gly residue at position 17 of SEQ ID NO: 31 to a Gln residue at position 208 (see FIG. 23) and containing a 6×His tag), and recombinant human Fc gamma RIIIB / CD16b protein (accession number: O75015, containing a Thr residue at position 20 of SEQ ID NO: 24 to a Gln residue at position 208 (see FIG. 19F) and containing a 10×His tag).

[0111] Briefly, each of the human FcγRs was immobilized on a sensor chip CM5 according to the manufacturer's instructions. Then, anti-EGFR antibodies prepared at each given concentration in HBS-EP+ were added to the sensor chip at a flow rate of 30 μL / min for 120 seconds. Figures 2B-2E show graphs plotting RU values ​​for each antibody concentration (0.9, 1.3, 2.0, 3.1, and 4.7 μM) when human FcγRIIa, FcγRIIb / c, FcγRIIIa, and FcγRIIIb were used as ligands. Table 1 shows the RU values ​​for various human FcγRs obtained at an antibody concentration of 4.7 μM.

[0112] As can be seen from Table 1, all variants containing effectorless mutations had lower RU values, which are an index of binding to each human FcγR, than IgG1 WT. The RU values ​​of the variants were in the order LALA>LALA-PG, and LALA-DA was essentially the same as LALA-PG.

[0113] Comparative Example 3: Evaluation of the binding activity of anti-EGFR antibodies containing known effectorless mutations to cynomolgus monkey FcγRI The binding activity of the anti-EGFR antibody described in Comparative Example 1 to cynomolgus monkey FcγRI (cFcγRI) was evaluated in the same manner as in Comparative Example 1. Cynomolgus monkey Fc gamma RI / CD64 protein (accession number: NP_001270969, containing the Val residue at position 11 of SEQ ID NO: 25 to the Pro residue at position 288 (see FIG. 20A), with a 6×His tag added to the carboxyl terminus: R&D Systems, Minneapolis, MN) was used as cynomolgus monkey FcγR. FIG. 3A shows a graph plotting the RU values ​​for each antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM). Table 2 below shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0114] [Table 2]

[0115] As shown in Table 2 above, all variants containing effectorless mutations had lower RU values ​​for binding to cynomolgus monkey FcγRI than IgG1 WT. The RU values ​​of the variants were in the following order: LALA>LALA-PG>LALA-DA.

[0116] Comparative Example 4: Evaluation of the binding activity of anti-EGFR antibodies containing known effectorless mutations to various cynomolgus monkey FcγRs The binding activity of the anti-EGFR antibody described in Comparative Example 1 to cynomolgus monkey FcγRIIa, FcγRIIb, and FcγRIII was evaluated in the same manner as in Comparative Example 2. The following cynomolgus FcγRs were used, each containing a 6×His tag at the carboxyl terminus, and all obtained from R&D Systems (Minneapolis, MN): recombinant cynomolgus Fc gamma RIIA / CD32a protein (accession number: NP_001270598, which contains from the Thr residue at position 29 of SEQ ID NO:26 through the Ile residue at position 211 (see FIG. 20B)), recombinant cynomolgus Fc gamma RIIB protein (accession number: NP_001271060, which contains from the Ala residue at position 46 of SEQ ID NO:27 through the Pro residue at position 217 (see FIG. 20C)), and recombinant cynomolgus Fc gamma RIII / CD16 protein (accession number: NP_001270121, which contains from the Gly residue at position 17 of SEQ ID NO:28 through the Gln residue at position 208 (see FIG. 20D)). Table 2 above shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0117] As shown in Table 2 above, all variants containing effectorless mutations had lower RU values ​​indicating binding to each cynomolgus monkey FcγR than IgG1 WT. The RU values ​​of the variants were in the following order: LALA>LALA-PG>LALA-DA.

[0118] Comparative Example 5: Evaluation of the thermostability of anti-EGFR antibodies containing known effectorless mutations The thermal stability of the anti-EGFR antibodies described in Comparative Example 1 was evaluated using a MicroCal VP-Capillary DSC System (Malvern Panalytical Ltd., Malvern, UK). Each anti-EGFR antibody was prepared at an antibody concentration of 0.5 mg / mL with 25 mM histidine and 5% w / v sorbitol (pH 6.0), and the heat capacity C p The change in heat capacity and peak top temperature (T m Of the two peaks observed, the peak at the lower temperature indicates thermal denaturation of the CH2 domain. The peak at the higher temperature is considered to indicate overlapping of the peaks of the Fab region and the CH3 domain. Table 3 below shows the T m values ​​and T between each variant and IgG1 WT m The difference in values ​​(ΔT m ) is shown.

[0119] [Table 3]

[0120] As can be seen from Table 3, the T m The T value was 0.6°C higher than that of IgG1 WT, but the T values ​​of the LALA-DA and LALA-PG variants were m The values ​​were 1.9°C and 1.0°C lower, respectively, than IgG1 WT.

[0121] Example 1: Evaluation of the binding activity of anti-EGFR antibodies containing L234A / L235A and D265 mutations to human FcγRI The binding activity of anti-EGFR antibodies containing the L234A / L235A and D265 mutations to human FcγRI was evaluated in the same manner as in Comparative Example 1. The Fc region of the anti-EGFR antibody panitumumab, which is originally a human IgG2 antibody, was replaced with the Fc region of human IgG1 (IgG1 WT). The variants used for evaluation were the LALA-DA variant of IgG1 WT; a variant obtained by introducing L234A / L235A / D265G (LALA-DG) mutations into the Fc region of IgG1 WT; a variant obtained by introducing L234A / L235A / D265N (LALA-DN) mutations into the Fc region of IgG1 WT; a variant obtained by introducing L234A / L235A / D265Q (LALA-DQ) mutations into the Fc region of IgG1 WT; and a variant obtained by introducing L234A / L235A / D265T (LALA-DT) mutations into the Fc region of IgG1 WT. The listed mutations were introduced into both CH2 domains of the Fc region. Figure 5 shows a graph plotting the RU values ​​for each antibody concentration tested (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM). Table 4 below shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0122] [Table 4]

[0123] As shown in Table 4, the RU values ​​of the LALA-DG and LALA-DN variants obtained at an antibody concentration of 4.7 μM for binding activity to human FcγRI were approximately 1 / 10 and 1 / 4 of that of the LALA-DA variant, respectively. The RU value of the LALA-DQ variant was approximately twice that of the LALA-DA variant.

[0124] Example 2: Evaluation of the binding activity of anti-EGFR antibodies containing L234A / L235A and D265 mutations to various human FcγRs The binding activity of the anti-EGFR antibody described in Example 1 to human FcγRIIa, FcγRIIa(H167), FcγRIIb / c, FcγRIIIa, FcγRIIIa(V176F), and FcγRIIIb was evaluated in the same manner as in Comparative Example 2. Table 4 above shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0125] As can be seen from Table 4, the RU values ​​for human FcγR obtained at an antibody concentration of 4.7 μM were almost the same for each variant, except for human FcγRIIa (H167). The RU value of the LALA-DG variant for human FcγRIIa (H167) was less than half that of the other variants.

[0126] Example 3: Evaluation of the binding activity of anti-EGFR antibodies containing L234A / L235A and D265 mutations to cynomolgus monkey FcγRI The binding activity of the anti-EGFR antibody described in Example 1 to cynomolgus monkey FcγRI was evaluated in the same manner as in Comparative Example 3. Figure 6 shows a graph plotting the RU value for each test antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM). Table 5 below shows the RU value at an antibody concentration of 4.7 μM.

[0127] [Table 5]

[0128] As can be seen from Table 5, the RU value of the LALA-DG variant obtained at an antibody concentration of 4.7 μM was about 1 / 10 that of the LALA-DA variant, and the RU value of the LALA-DN variant was about 1 / 4 that of the LALA-DA variant, while the RU values ​​of the LALA-DQ and LALA-DT variants were more than 1.4 times that of the LALA-DA variant.

[0129] Example 4: Evaluation of the binding activity of anti-EGFR antibodies containing L234A / L235A and D265 mutations to various cynomolgus monkey FcγRs The binding activity of the anti-EGFR antibody described in Example 1 to cynomolgus monkey FcγRIIa, FcγRIIb, and FcγRIII was evaluated in the same manner as in Comparative Example 4. Table 5 above shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0130] As can be seen from Table 5, the RU values ​​against cynomolgus monkey FcγRIIa, FcγRIIb, and FcγRIII obtained at an antibody concentration of 4.7 μM were almost the same for each variant.

[0131] Example 5: Evaluation of the thermostability of anti-EGFR antibodies containing the L234A / L235A and D265 mutations The thermal stability of each CH2 domain of the IgG1 WT and variants of the anti-EGFR antibody panitumumab described in Example 1 was evaluated in the same manner as in Comparative Example 5. Figures 7A to 7D show the changes in heat capacity and T m The following Table 6 shows the T values ​​of the CH2 domain of each test antibody. m values ​​and T between each variant and IgG1 WT m The difference in values ​​(ΔT m ) is shown.

[0132] [Table 6]

[0133] As shown in Table 6, the T of the LALA-DQ and LALA-DT variants m The T value for the LALA-DG variant was approximately 2°C lower than that for IgG1 WT, similar to that for the LALA-DA variant. m The T value of the LALA-DN variant was 4.5°C higher than that of the IgG1 WT. m The value was 0.3°C higher than IgG1 WT.

[0134] Example 6: Evaluation of antigen binding activity of LALA-DG variants of anti-EGFR antibodies The EGFR binding activity of the IgG1 WT and LALA and LALA-DG variants of the anti-EGFR antibody panitumumab was evaluated using Biacore™ T200. The anti-EGFR antibody was captured as a ligand on a sensor chip immobilized with an anti-human IgG Fc antibody, and EGFR was run as an analyte. The interaction between the ligand and the analyte was then measured by SPR.

[0135] Briefly, anti-human IgG Fc antibodies included in the Human Antibody Capture Kit (Cytiva, Marlborough, MA) were immobilized on a sensor chip CM5 according to the manufacturer's instructions, and then each anti-EGFR antibody prepared at 2 μg / mL in HBS-EP+ was captured by contacting it with the sensor chip at a flow rate of 10 μL / min for 30 seconds. Human EGFR protein (ACROBiosystems, Newark, DE) prepared at concentrations of 12, 37, 110, 330, and 1000 pM in HBS-EP+ was then added to the sensor chip at a flow rate of 30 μL / min for 120 seconds, and then allowed to dissociate for 1500 seconds. The dissociation constant K of each antibody against EGFR was D was calculated from the obtained sensorgram by single cycle kinetic analysis. The results are shown in Table 7 below.

[0136] [Table 7]

[0137] As can be seen from Table 7, the K D The values ​​were 27, 24, and 26 nM for IgG1 WT, LALA variant, and LALA-DG variant, respectively, and were nearly the same regardless of the presence or absence of mutation and its type.

[0138] Example 7: Evaluation of the binding activity of LALA-DG and LALA-DGPA variants of anti-EGFR antibodies to fetal Fc receptor (FcRn) The binding activity of the anti-EGFR antibody described in Example 6 to human FcRn (human fetal receptor large subunit p51 / accession number: P55899 / sequence number 29 / Figure 21) and beta2-microglobulin (accession number: NP_004039.1 / sequence number 30 / Figure 22) was evaluated using an Octet® RED384 system (Molecular Devices, LLC, San Jose, CA). Using biolayer interference, the binding reaction between the antibody and FcRn was measured when a Streptavidin Biosensor (Molecular Devices, LLC) capturing biotinylated FcRn was contacted with a solution of the anti-EGFR antibody, and the dissociation reaction between the antibody and FcRn was measured when the biosensor was transferred to another solution not containing the antibody. Both the binding reaction and the dissociation reaction were carried out in a buffer solution of pH 6.0, and the dissociation constant K at pH 6.0 was calculated from the sensorgrams of the obtained binding reaction and dissociation reaction. D In addition, the binding reaction was carried out in a buffer solution of pH 6.0, and the dissociation reaction was carried out in a buffer solution of pH 7.4. From the sensorgram of the dissociation reaction obtained, the dissociation rate constant k d The following Table 8 shows the calculated K for the anti-EGFR antibodies. D value and k d The values ​​are shown. The concentrations of the anti-EGFR antibody solutions were set at three points, namely, 1.7, 6.9, and 28 nM. A solution of 20 mM Bis-Tris, 150 mM NaCl, and 0.05% Surfactant P20 (pH 6.0 or 7.4) was used as a buffer solution.

[0139] [Table 8]

[0140] As can be seen from Table 8, the K values ​​at pH 6.0 for IgG1 WT, LALA variant, LALA-DG variant, and LALA-DGPA variant were all D In addition, the k value at pH 7.4 was 5-6 nM. dThe values ​​were nearly identical, i.e., 2.1 (1 / s) for IgG1 WT and 2.8, 2.2, 2.3 (1 / s) for the variants, respectively.

[0141] Example 8: Pharmacokinetic study of the LALA-DG variant of an anti-EGFR antibody in mice Pharmacokinetic studies of the anti-EGFR antibodies described in Example 6 were carried out in mice. Each anti-EGFR antibody was administered intravenously to mice at 3 mg / kg, and then blood was collected from the tail vein using heparinized hematocrit tubes under isoflurane anesthesia at 1, 6, 24, 72, 168, 336 and 360 hours later, followed by centrifugation at 4°C and 15000 rpm for 5 minutes to obtain plasma. The amount of anti-EGFR antibody in the obtained plasma was measured using a fully automated immunoassay platform Gyrolab™ xP Workstation (Gyros Protein Technologies AB, Uppsala, Sweden). Biotin-labeled anti-human IgG goat antibody (SouthernBiotech, Birmingham, AL) was used to capture the anti-EGFR antibody, and Alexa Fluor 647-labeled anti-human IgG goat antibody (SouthernBiotech) was used to detect it. Figure 8 shows a graph plotting the blood concentration of anti-EGFR antibody against time after administration.

[0142] As can be seen from FIG. 8, no significant differences were observed in the time course of blood concentrations of the anti-EGFR antibodies IgG1 WT, LALA, and LALA-DG variants in mice.

[0143] Example 9: Evaluation of the binding activity of anti-EGFR antibodies containing L234A / L235A / D265G / P329A mutations to human FcγRI The binding activity to human FcγRI of the LALA-DG variant of the anti-EGFR antibody panitumumab and the variant obtained by introducing L234A / L235A / D265G / P329A (LALA-DGPA) mutations into the Fc region of IgG1 WT was evaluated in the same manner as in Comparative Example 1. Figure 9A shows a graph plotting the RU values ​​for each test antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM) for the LALA-DG variant (see Example 1) and the LALA-DGPA variant of the anti-EGFR antibody.

[0144] As can be seen from Figure 9A, the LALA-DGPA variant showed a lower RU value against human FcγRI than the LALA-DG variant.

[0145] Example 10: Evaluation of the binding activity of anti-EGFR antibodies containing L234A / L235A / D265G / P329A mutations to cynomolgus monkey FcγRI The binding activity of the anti-EGFR antibody described in Example 9 to cynomolgus monkey FcγRI was evaluated in the same manner as in Comparative Example 3. Figure 9B shows a graph plotting the RU values ​​for each test antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM) for the LALA-DG variant (see Example 1) and the LALA-DGPA variant of the anti-EGFR antibody.

[0146] As can be seen from Figure 9B, the RU value of the LALA-DGPA variant against cynomolgus monkey FcγRI was lower than that of the LALA-DG variant.

[0147] Example 11: Evaluation of the thermal stability of anti-EGFR antibodies containing the L234A / L235A / D265G / P329A mutations The thermal stability of the anti-EGFR antibody described in Example 9 was evaluated in the same manner as in Comparative Example 5. Table 9 below shows the T of the CH2 domain of each of the LALA-DG variant and the LALA-DGPA variant of the anti-EGFR antibody. m The Tm values ​​of the CH2 domain of the LALA-DG variant are the same as in Table 6.

[0148] [Table 9]

[0149] As shown in Table 9, the T of the CH2 domain of the LALA-DGPA variant m The value was 1°C lower than the LALA-DG variant.

[0150] Example 12: Evaluation of the binding activity of LALA-DG variants of anti-CD70 antibodies to various human Fcγ receptors The binding activity of the LALA and LALA-DG variants of the anti-CD70 antibody borsetuzumab, an IgG1 antibody, to human FcγRI was obtained and evaluated in the same manner as in Comparative Example 1. The amino acid sequences of the light and heavy chains of the LALA variant of borsetuzumab are represented by SEQ ID NOs: 15 and 16, respectively (see Figures 17A and 17B). Figure 10A shows a graph plotting the RU values ​​for each test antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM). In addition, the binding activity of these antibodies to human FcγRIIa, FcγRIIb / c, FcγRIIIa, and FcγRIIIb was evaluated using the method described in Comparative Example 2. Table 10 shows the RU values ​​obtained at an antibody concentration of 4.7 μM.

[0151] [Table 10]

[0152] The RU value of the LALA-DG variant against human FcγRI obtained at an antibody concentration of 4.7 μM was approximately 1 / 200 of that of the LALA variant. In addition, the RU values ​​of the LALA-DG variant against other human FcγR obtained at an antibody concentration of 4.7 μM were lower than those of the LALA variant.

[0153] Example 13: Evaluation of the binding activity of LALA-DG variants of anti-LPS antibodies to human Fcγ receptors The binding activity of the LALA and LALA-DG variants of the anti-LPS antibody O11-1111, an IgG1 antibody, to human FcγRI was obtained and evaluated in the same manner as in Comparative Example 1. The amino acid sequences of the light and heavy chains of the 011-1111 antibody are represented by SEQ ID NOs: 17 and 18, respectively (see Figures 18A and 18B). Figure 10B shows a graph plotting the RU values ​​for each test antibody concentration (0.3, 0.4, 0.6, 0.9, 1.3, 2.0, 3.1, and 4.7 μM). In addition, the binding activity of these antibodies to human FcγRIIa, FcγRIIb / c, FcγRIIIa, and FcγRIIIb was evaluated using the method described in Comparative Example 2. Table 10 above shows the RU values ​​at an antibody concentration of 4.7 μM.

[0154] As can be seen from Table 10, the RU value of the LALA-DG variant against human FcγRI obtained at an antibody concentration of 4.7 μM was about 1 / 300 of that of the LALA variant. In addition, the RU values ​​of the LALA-DG variant against other human FcγR obtained at an antibody concentration of 4.7 μM were lower than that of the LALA variant.

[0155] Example 14: Evaluation of the thermostability of various antibodies containing the LALA-DG mutation The thermal stability of the following antibodies was evaluated in the same manner as in Comparative Example 5. The antibodies evaluated were the parent antibody (IgG1 WT) of the anti-LPS antibody O11-1111, and the parent antibody (IgG1 WT) of the anti-CD70 antibody, the LALA and LALA-DG variants of each of these antibodies, as well as antibodies derived from the variants lacking any Fab region. The LALA variant lacking Fab and the LALA-DG variant lacking Fab consist of amino acids 222 to 448 of SEQ ID NO: 16 and amino acids 104 to 330 of SEQ ID NO: 5, respectively (see Figures 17B and 12B). Table 11 shows the T of the CH2 domain of each test antibody. m Indicates the value.

[0156] [Table 11]

[0157] As shown in Table 11, for anti-CD70 antibodies, the T of the CH2 domain of the LALA-DG variant m The T value of the CH2 domain of the LALA-DG variant was 3.9°C higher than that of the LALA variant. m The values ​​were 3.7°C higher than those of the IgG1 WT and 3.3°C higher than those of the LALA variant. m The value was 3.6°C higher than the LALA variant.

Claims

1. 1. A polypeptide comprising one or more variant Fc regions, each of said variant Fc regions comprising two CH2 domains, wherein at least one CH2 domain in at least one of said variant Fc regions is a variant CH2 domain comprising amino acid substitutions at positions 234, 235, and 265 (all positions represented by EU numbering), wherein the amino acid residues at positions 234, 235, and 265 of said variant CH2 domain are Ala, Ala, and Gly, respectively, or Ala, Ala, and Asn, respectively, and wherein each of said one or more variant Fc regions is a variant IgG1 Fc region, a variant IgG4 Fc region, or a variant IgG1 / IgG4 Fc region. The polypeptide, which is an Fc region and has reduced binding affinity to an Fcγ receptor (FcγR) selected from the group consisting of FcγRI, FcγRII, and FcγRIII compared to a parent polypeptide comprising a parent CH2 domain that does not contain amino acid substitutions at positions 234, 235, and 265, and wherein the FcγR is a human or cynomolgus monkey FcγR.

2. 2. The polypeptide of claim 1, further comprising an amino acid substitution at position 329 of the variant CH2 domain, wherein the amino acid residue at position 329 of the variant CH2 domain is Ala.

3. The polypeptide of claim 1, which has a binding affinity to an Fcγ receptor (FcγR) that is reduced by 50% or more compared to a parent polypeptide comprising a parent CH2 domain that does not contain amino acid substitutions at positions 234, 235, and 265.

4. The polypeptide of claim 1, wherein the FcγR is a human FcγR.

5. The polypeptide of claim 1, wherein the FcγR is FcγRI.

6. The polypeptide of claim 1 , comprising one or more variant IgG1 Fc regions.

7. The polypeptide of claim 1 comprising one variant Fc region.

8. The polypeptide of claim 1 comprising one variant IgG1 Fc region.

9. 2. The polypeptide of claim 1, wherein the amino acid residues at positions 234, 235, and 265 of the variant CH2 domain are Ala, Ala, and Gly, respectively.

10. 2. The polypeptide of claim 1, wherein the amino acid residues at positions 234, 235, and 265 of the variant CH2 domain are Ala, Ala, and Asn, respectively.

11. 2. The polypeptide of claim 1, wherein the thermal denaturation midpoint (Tm) of the variant CH2 domain is within 1°C of the Tm of a parent CH2 domain that does not contain the amino acid substitutions at positions 234, 235, and 265.

12. the thermal denaturation midpoint (Tm) of the variant CH2 domain is higher than the Tm of a parent CH2 domain that does not contain the amino acid substitutions at positions 234, 235, and 265; (a) 1°C or more, or (b) 3℃ or more The polypeptide of claim 9, wherein the polypeptide has a high affinity.

13. The polypeptide of claim 1, which is an antibody or a functional fragment thereof.

14. The polypeptide of claim 13 , wherein the antibody or functional fragment thereof binds to a tumor antigen.

15. The polypeptide of claim 13, wherein the antibody is a bispecific or multispecific antibody.

16. A fusion protein comprising the polypeptide of claim 1 and a heterologous polypeptide.

17. A polypeptide-drug conjugate comprising the polypeptide of claim 1 conjugated to one or more drugs or non-proteinaceous polymers.

18. 18. The polypeptide-drug conjugate of claim 17, wherein the drug is a chemotherapeutic or cytotoxic agent.

19. A nucleic acid comprising a nucleotide sequence encoding a polypeptide or a part thereof according to any one of claims 1 to 15.

20. 20. A host cell comprising the nucleic acid of claim 19.

21. 21. A method for producing a polypeptide according to any one of claims 1 to 15, comprising culturing a host cell according to claim 20 in a medium under conditions suitable for expression of said polypeptide, and recovering said polypeptide from said medium or said host cell.

22. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 15, the fusion protein of claim 16, or the polypeptide-drug conjugate of claim 17, and a pharmaceutically acceptable carrier.

23. A pharmaceutical comprising a polypeptide described in any one of claims 1 to 15, a fusion protein described in claim 16, or a polypeptide-drug conjugate described in claim 17, for the treatment of cancer, an immune disease, an inflammatory disease, or an infectious disease.

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