JP2018138022A - Human igg1 fc region variants and uses thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Fc domain-containing polypeptides (for example, antibodies) that have increased complement-dependent cytotoxicity (CDC) and may also have other modified effector functions resulting from one or more amino acid modifications in the Fc-domain.SOLUTION: A mutation is introduced to the parent polypeptide in one or more amino acid residue(s) selected from the group corresponding to E430X, E345X, and S440W in the Fc region of a human IgG1 heavy chain. When the polypeptide or antibody binds a target thereof or antigen on the surface of a cell, the variant Fc domain causes a stabilized Fc:Fc interaction, therefore causing increased complement-dependent cytotoxicity (CDC).SELECTED DRAWING: Figure 32B

Description

The present invention relates to Fc with enhanced complement dependent cytotoxicity (CDC) and may also have other altered effector functions resulting from one or more amino acid modifications in the Fc domain. It relates to polypeptides containing domains (eg antibodies).

Background of the Invention Effector functions mediated by the Fc region of antibodies allow the destruction of foreign entities such as pathogen killing and antigen elimination and degradation. Antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP) are triggered by binding of the Fc region to cells bearing the Fc receptor (FcR), while complementing. Body-dependent cytotoxicity (CDC) is triggered by binding of the Fc region to C1q, which initiates the classical pathway of complement activation.

Each IgG antibody contains two binding sites for C1q, one in each heavy chain constant (Fc) region. However, a single molecule of IgG in solution does not activate complement (Sledge et al., 1973 J. Biol) because the affinity of monomeric IgG for C1q is very weak (K _d approximately 10 ^-4 M). Chem. 248,2818-13 (Non-Patent Document 1); Hughes-Jones et al., 1979 Mol. Immunol. 16,697-701 (Non-Patent Document 2)). Antigen-triggered IgG association can lead to much tighter binding of polyvalent C1q molecules (K _d ^{˜10 −8} M) and complement activation (Burton et al., 1990 Mol. Immunol. 22, 161-206 (Non-Patent Document 3)). In contrast, IgM exists in nature in the form of covalently linked pentamers or hexamers, and IgM pentamers and hexamers can efficiently induce CDC when bound to expressed or immobilized cell antigens. Antigen binding is a prerequisite for inducing conformational changes in IgM to expose the C1q binding site (Feinstein et al., 1986, Immunology Today, 169-174).

  It has also been suggested that IgG can also achieve complement activation through the formation of a hexamer ring structure through the interaction of the CH2 / CH3 domain of the Fc region (Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69 (Non-Patent Document 5)). Evidence supporting the existence of such hexameric IgG structures is based on two-dimensional crystals (Reidler et al., 1986 I Handbook of Experimental Immunology 4th edit. (Weir, DM ed.), Pp17.1-17.5. Blackwell, Edinburgh (non- 6); Pinic et al., 1971 Immunochem. 8, 1041-5 (Non-Patent Document 7)) and three-dimensional crystals, as well as IgG1, IgG2a and IgG4 and human Fc in solution. (Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115 (non-patent document 8)). Hexamer ring formation was also observed in the crystal structure of b12 human IgG1κ antibody (1HZH in PDB) targeting HIV-1 gp120 (Saphire et al., Science 2001 Aug 10; 293 (5532), 1155- 9 (Non-Patent Document 9)). In the b12 hexamer ring, six reachable C1q binding sites were presented on the hexamer surface, one from each of the six antibodies, with the remaining six binding sites facing down.

  C1q has a shape resembling a bunch of tulips, with six globular heads containing antibody binding regions tied to six collagen stalks (Perkins et al., 1985 Biochem J. 228, 13-26 (Non-patent document 10); Poon et al., 1983 J Mol Biol. 168, 563-77 (Non-patent document 11); Reid et al., 1983 Biochem Soc Trans 11, 1-12 (Non-patent document) 12); Weiss et al., 1986 J. Mol. Biol. 189, 573-81 (Non-patent Document 13)). C1q fits into the 1HZH crystalline b12 hexamer assembly from above, and as a result, each of the six globular heads has been found to contact one of the six C1q binding sites (Parren, FASEB Summer Research Conference, Snowmass, Co., 5-10 July 2010; "Crystal Structure of an intact human IgG: implications for HIV-1 neutralization and effector Function", Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, California. November 2000 (Non-patent Document 15)). Mutations in selected amino acids at the Fc interface observed between multiple b12 antibodies in symmetry within the crystal structure have been observed to reduce C1q binding, indicating that intermolecular Fc : Indicates the contribution of these amino acids to the Fc interaction.

  WO 2006/104989 (Patent Document 1) describes a modified antibody Fc region and its use.

  WO 2005/047327 describes a neonatal Fc receptor (FcRn) binding polypeptide variant, a dimeric Fc binding protein, and methods related thereto.

  WO 2010/106180 (Patent Document 3) describes an Fc variant with enhanced binding to the neonatal Fc receptor (FcRn).

  WO 2005/070963 (Patent Document 4) describes polypeptide Fc region variants and uses thereof.

  WO 2006/053301 (Patent Document 5) describes an Fc variant with a modified binding to FcRn.

  US 2011/0123440 describes a modified antibody Fc region and its use. The modified Fc region has one or more amino acid substitutions.

  US 2008/0089892 describes polypeptide Fc region variants and compositions comprising these Fc region variants.

  US 2010/0184959 describes a method for providing Fc polypeptide variants with altered Fc ligand recognition and / or effector function.

  US 2010/015133 (Patent Document 9) describes a method for producing a polypeptide by regulating the association of the polypeptides.

  US 2010/105873 describes an integrated approach to making multidomain protein therapeutics.

  US 6,737,056 (Patent Document 11) describes polypeptide variants with altered effector functions.

  Prior efforts have been made to identify antibody Fc variants with enhanced effector function or other altered properties. Such studies include, for example, exchanging segments between IgG isotypes to produce chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68 (10), 3863-72), or hinge regions (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138 (Non-patent Document 17)) or in or near the C1q binding site in the CH2 domain centered on residues D270, K322, P329 and P331 ( Idusogie et al., 2001 J Immunol 166, 2571-2575 (Non-Patent Document 18); Michaelsen et al., 2009 Scand J Immunol 70, 553-564 (Non-Patent Document 19) and WO 99/51642 (Patent Document 12) The aim was to generate amino acid substitutions in)). For example, Moore et al. (2010 mAbs 2 (2), 181-189) (Non-Patent Document 20) describes S267E, H268F, S324T, S239D, I332E, G236A related to enhancement of effector function via CDC or ADCC. And discusses various combinations of I332E. Binding to Fc receptors (WO 2006/105062 (patent document 13), WO 00/42072 (patent document 14), US Pat. No. 6,737,056 (patent document 11) and US Pat. No. 7,083,784 (patent document 15)) Alternatively, other Fc mutations affecting the physical properties of antibodies (WO 2007/005612 A1 (Patent Document 16)) have also been suggested.

  However, despite these and other advantages in the art, there remains a need for new and improved antibody-based therapeutics.

WO 2006/104989 WO 2005/047327 No. WO 2010/106180 WO 2005/070963 WO 2006/053301 US 2011/0123440 US 2008/0089892 US 2010/0184959 US 2010/015133 US 2010/105873 US 6,737,056 WO 99/51642 WO 2006/105062 WO 00/42072 U.S. Patent No. 7,083,784 WO 2007/005612 A1

Sledge et al., 1973 J. Biol. Chem. 248,2818-13 Hughes-Jones et al., 1979 Mol. Immunol. 16,697-701 Burton et al., 1990 Mol. Immunol. 22, 161-206 Feinstein et al., 1986, Immunology Today, 169-174 Burton et al., 1990 Trends in Biochem. Sci. 15, 64-69 Reidler et al., 1986 I Handbook of Experimental Immunology 4th edit. (Weir, D. M. ed.), Pp17.1-17.5. Blackwell, Edinburgh Pinteric et al., 1971 Immunochem. 8, 1041-5 Kuznetsov et al., 2000 J Struct. Biol. 131, 108-115 Saphire et al., Science 2001 Aug 10; 293 (5532), 1155-9 Perkins et al., 1985 Biochem J. 228, 13-26 Poon et al., 1983 J Mol Biol. 168, 563-77 Reid et al., 1983 Biochem Soc Trans 11, 1-12 Weiss et al., 1986 J. Mol. Biol. 189, 573-81 Parren, FASEB Summer Research Conference, Snowmass, Co., 5-10 July 2010 "Crystal Structure of an intact human IgG: implications for HIV-1 neutralization and effector Function", Thesis by Erica Ollmann Saphire, for the Scripps Research Institute, La Jolla, California. November 2000 Natsume et al., 2008 Cancer Res 68 (10), 3863-72 Dall'Acqua et al., 2006 J Immunol 177, 1129-1138 Idusogie et al., 2001 J Immunol 166, 2571-2575 Michaelsen et al., 2009 Scand J Immunol 70, 553-564 Moore et al. (2010 mAbs 2 (2), 181-189)

  The present invention provides polypeptide and antibody variants that have enhanced complement dependent cytotoxicity (CDC) and may have other effector functions compared to their parent polypeptide / antibody. Provide the body. Without being limited to theory, these variants allow for a more stable binding interaction between the Fc regions of two polypeptide / antibody molecules, thereby making them more enhanced or more specific It is thought to result in a more cohesive surface that leads to enhanced effector functions such as a robust CDC response. Certain variants are also characterized by improved ADCC response, ADCP response, and / or other enhanced effector functions. This tricky procedure of polypeptide / antibody manipulation can be applied, for example, to enhance the activity or specificity of antibody-based therapeutics, for example, as described herein.

  Accordingly, in one aspect, the invention provides a method for enhancing complement dependent cytotoxicity (CDC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein A method comprising introducing a mutation in one or more amino acid residues selected from the group corresponding to E430X, E345X and S440W in the Fc region of a human IgG1 heavy chain.

  In one further aspect, the invention provides a variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein the E430S, E430F, E430T, E345K, E345Q, E345R in the Fc region of a human IgG1 heavy chain. Including one or more mutations selected from the group corresponding to E345Y and S440W, provided that it does not contain additional mutations in the Fc domain that alter the binding of the mutant to the neonatal Fc receptor (FcRn) It is related with the variant.

  The present invention also enhances complement-dependent cytotoxicity (CDC) mediated by a polypeptide or antibody, eg, when bound to that antigen on the surface of a cell, cell membrane or virion expressing the antigen. The use of one or more such mutations is also provided.

  In one aspect, when referred to herein as a “single variant”, the variant has enhanced CDC and other effector functions compared to the parent polypeptide or antibody. Also good.

  In one aspect, when referred to herein as a “double mutant”, the mutant includes at least two mutations in the segment and includes only one of the at least two mutations. Compared with CDC, CDC may be improved and other effector functions may also be improved.

  In one aspect, when referred to herein as a “mixed variant”, the variant is combined with a second variant of the same or different polypeptide or antibody that contains a mutation at a different amino acid residue in the segment. When used in conjunction with one or more of the mutant, second mutant, and parent polypeptide or parent antibody alone, resulting in enhanced CDC and enhanced other effector functions. It may be.

  Typically, the mutation causes the parent amino acid residue to promote the formation of a new intermolecular Fc: Fc bond or to enhance the strength of an existing pair of interactions, size and / or physicochemical properties. Amino acid substitutions such as mutations that exchange for different ones. Exemplary amino acid residues for the mutations of the invention are shown in Table 1 and Tables 2A and 2B with exemplary amino acid substitutions. A non-limiting illustration of various aspects of the present invention is presented in FIG.

[Invention 1001]
A method for enhancing complement dependent cytotoxicity (CDC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the following steps:
Introducing into the parent polypeptide a mutation in one or more amino acid residues selected from the group corresponding to E430X, E345X and S440W in the Fc region of the human IgG1 heavy chain.
[Invention 1002]
The present invention 1001, wherein the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain the method of.
[Invention 1003]
The method of any of claims 1001 or 1002, wherein said mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.
[Invention 1004]
A method for enhancing CDC and antibody-dependent cell-mediated cytotoxicity (ADCC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the following steps:
Introducing a mutation in one or more amino acid residues corresponding to E430X, E345X and S440W in the Fc region of human IgG1 heavy chain into the parent polypeptide.
[Invention 1005]
The method of 1004 of this invention wherein said mutation in one or more amino acid residues corresponds to E345R, E430T and E430F in the Fc region of a human IgG1 heavy chain.
[Invention 1006]
The method according to any one of the inventions 1001 to 1005, wherein the parent polypeptide is a parent antibody comprising an Fc domain of an immunoglobulin and an antigen-binding region.
[Invention 1007]
The method of the present invention 1006, wherein the parent antibody is a monospecific antibody, a bispecific antibody or a multispecific antibody.
[Invention 1008]
A first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and a second polypeptide comprising an immunoglobulin second CH2-CH3 region and a second antigen-binding region; A method for enhancing the complement dependent cytotoxicity (CDC) of a parent antibody, which is a bispecific antibody comprising a peptide, comprising:
The first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens, and the method is directed against the first and / or second CH2-CH3 regions, Introducing a mutation in one or more amino acid residues selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the IgG1 heavy chain; and the first CH2-CH3 region comprises: A further amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; and the second CH2-CH3 region is human An additional amino acid mutation at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the IgG1 heavy chain; and the additional amino acid in the first CH2-CH3 region Mutations in the second CH2-CH3 region The method, which is different from an amino acid mutation.
[Invention 1009]
Wherein the mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain The method of invention 1008.
[Invention 1010]
The method of any of 1008 or 1009 of the invention, comprising introducing mutations into both the first and second polypeptides of the bispecific antibody.
[Invention 1011]
The further amino acid mutation of the first CH2-CH3 region is in a position corresponding to K409 in the Fc region of a human IgG1 heavy chain, for example K409R; and the further amino acid of the second CH2-CH3 region The method of any of the present invention 1008-1010, wherein the mutation is at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain, eg, F405L.
[Invention 1012]
Introducing a mutation other than S440Y and S440W at one or more positions, and (i) a mutation in each of the amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that S440 A mutation, provided that the mutation in is not S440Y or S440W,
(Ii) mutations in each of the amino acid residues corresponding to K447 and 448 in the Fc region of the human IgG1 heavy chain, for example K447K / R / H and 448E / D, preferably human IgG1 heavy chain in the Fc region of the human IgG1 heavy chain (Iii) mutations in each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of the human IgG1 heavy chain, eg, K447D / E, 448K in the Fc region of the human IgG1 heavy chain / R / H and 449P, preferably K447E, 448K and 449P in the Fc region of the human IgG1 heavy chain
The method according to any one of the inventions 1001 to 1011, further comprising the step of introducing
[Invention 1013]
Introducing a mutation other than S440Y and S440W at one or more positions, and further introducing a mutation into each of the amino acid residues corresponding to K439 and / or S440 in the Fc region of the human IgG1 heavy chain, However, the method of the present invention 1012 provided that the mutation in S440 is neither S440Y nor S440W.
[Invention 1014]
The mutation at a position corresponding to K439 in the Fc region of human IgG1 heavy chain is K439D / E, and / or the mutation at a position corresponding to S440 in the Fc region of human IgG1 heavy chain is S440K / R, The method of the invention 1013.
[Invention 1015]
A method of enhancing the complement dependent cytotoxicity (CDC) of a combination of at least a first and a second parent polypeptide, wherein the at least first and second parent polypeptides are each an immunoglobulin Fc domain and One or more selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain with respect to the at least first and / or second parent polypeptide. The method comprising introducing mutations in the amino acid residues of
[Invention 1016]
For said at least first and / or second parent polypeptide, selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain The method of the invention 1015 comprising the step of introducing mutations in one or more of the amino acid residues that are made.
[Invention 1017]
The method of the invention 1016 comprising the step of introducing mutations, which may be the same or different, into both the first and second parent polypeptides.
[Invention 1018]
(I) 1 selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide Introducing mutations in one or more amino acid residues;
(Ii) including mutations in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain Providing the second parent polypeptide,
The method of the present invention 1016, comprising:
[Invention 1019]
The mutation at one or more positions is different from S440Y and S440W, and the method comprises:
(I) introducing a second mutation into the amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide; and (ii) the second parent poly A step of introducing a second mutation into the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain with respect to the peptide, provided that the mutation is neither S440Y nor S440W Further comprising steps (i) and (ii)
(Iii) a step of introducing a second mutation into an amino acid residue corresponding to position S440 in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide, provided that the mutation is S440Y Stage, provided that it is not S440W;
(Iv) The present invention may include a step of introducing a second mutation into an amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain with respect to the second parent polypeptide. Either way.
[Invention 1020]
The mutation in the position corresponding to K439 in the Fc region of human IgG1 heavy chain is K439D / E and / or the mutation in the position corresponding to S440 in the Fc region of human IgG1 heavy chain is S440K / R The method of invention 1019.
[Invention 1021]
The method of any of the inventions 1015-1020, wherein the first and second parent polypeptides are first and second parent antibodies, each comprising an immunoglobulin Fc domain and an antigen binding region.
[Invention 1022]
The method of the present invention 1021, wherein the first and second parent antibodies are monospecific, bispecific or multispecific antibodies.
[Invention 1023]
The first and / or second parent antibody comprises a first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and a second CH2-CH3 region and a second A bispecific antibody comprising a second polypeptide comprising an antigen binding region, wherein the first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens, and The first CH2-CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; and Two CH2-CH3 regions contain additional amino acid mutations at positions selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain; and The additional amino acid mutation in the CH2-CH3 region of the second CH2-CH3 region The method of the present invention 1022, which differs from the amino acid mutation
[Invention 1024]
The first CH2-CH3 region comprises a further amino acid mutation, for example K409R, at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain, and the second CH2-CH3 region is a human IgG1 heavy chain The method of the invention 1023 comprising an additional amino acid mutation, eg F405L, in the position corresponding to F405 in the Fc region.
[Invention 1025]
The method of any one of the inventions 1001-1003 and 1006-1024, wherein the parent polypeptide or the antibody-dependent cell-mediated cytotoxicity (ADCC) of the parent antibody is not altered.
[Invention 1026]
The method of any of 1001 to 1025 of the invention, wherein the binding of the parent polypeptide or the parent antibody to the neonatal Fc receptor (FcRn) determined by the method disclosed in Example 34 is not altered.
[Invention 1027]
More than 30% of the binding of the parent polypeptide or the parent antibody to the neonatal Fc receptor (FcRn) as determined by the change in absorbance at OD405nm, as determined by the method disclosed in Example 34, for example The method of any of the inventions 1001-1025 which does not increase or decrease by more than 20%, 10% or 5%.
[Invention 1028]
Does not enhance the apparent affinity of the parent polypeptide or the parent antibody to the mouse neonatal Fc receptor (FcRn) by more than 0.5-fold, as determined by the method disclosed in Example 34, or the parent polypeptide or The method according to any of 1001 to 1025 of the invention, wherein the apparent affinity of the parent antibody for mouse FcRn is not reduced more than 2-fold.
[Invention 1029]
The method of any of the inventions 1001-1028, wherein the plasma clearance rate of the parent polypeptide or parent antibody determined by the method disclosed in Example 37 is not altered.
[Invention 1030]
Enhancing the plasma clearance rate of the parent polypeptide or antibody determined by the method disclosed in Example 37 by more than 3.0 times, for example, by 2.5 times, 2.0 times, 1.5 times or 1.2 times Or any of the methods of the invention 1001-1028.
[Invention 1031]
The method of any of the inventions 1001-1030, wherein the variant activation of the variant in the target-independent liquid phase determined by the method disclosed in Example 36 is not altered.
[Invention 1032]
The method of any one of 1001 to 1031 of the present invention, wherein the plasma half-life of the parent polypeptide or the parent antibody is not altered.
[Invention 1033]
A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain Said variant comprising one or more mutations selected from: provided that the variant does not contain further mutations in the Fc domain that alter binding of the variant to the neonatal Fc receptor (FcRn).
[Invention 1034]
A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain To the neonatal Fc receptor (FcRn) of the mutant as measured by the change in absorbance at OD405nm, as determined by the method disclosed in Example 34, comprising one or more mutations selected from Said mutant, provided that it does not contain further mutations in the Fc domain that increase or decrease binding by more than 30%, for example more than 20%, 10% or 5%.
[Invention 1035]
The variant of any of the invention 1033 or 1034, wherein the one or more of said mutations is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of a human IgG1 heavy chain.
[Invention 1036]
The variant of any of the present invention 1033-1035, wherein the variant does not comprise a further mutation in the Fc domain that alters antibody-dependent cell-mediated cytotoxicity (ADCC) of said variant.
[Invention 1037]
The variant of any of the present invention 1033-1036, comprising no additional mutations in the Fc domain that alter the plasma clearance rate of the variant as determined by the method disclosed in Example 37.
[Invention 1038]
Further increase in the plasma clearance rate of the mutant as determined by the method disclosed in Example 37 by more than 3.0 fold, for example 2.5 fold, 2.0 fold, 1.5 fold or 1.2 fold or more in the Fc domain The mutant of any one of the present invention 1033-1036, which does not contain a mutation.
[Invention 1039]
The variant of any of the present invention 1033-1036, which does not comprise a further mutation in the Fc domain that alters the serum half-life of the variant.
[Invention 1040]
Any of the invention 1033-1039, comprising no further mutations in the Fc domain that alter complement activation in the target-independent fluid phase of the mutant as determined by the method disclosed in Example 36 Mutants.
[Invention 1041]
The variant of any of the present invention 1033-1040, comprising no further mutations in the Fc domain.
[Invention 1042]
The variant of any of the present invention 1033-1041, comprising only one mutation.
[Invention 1043]
The invention 1033 comprising a combination of two mutations in the amino acid residues selected from the group corresponding to E345X / E430X, E345X / S440Y, E345X / S440W, E430X / S440Y and E430X / S440W in the Fc region of the human IgG1 heavy chain A variant of any of ~ 1042.
[Invention 1044]
A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising:
A first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain; and selected from the group corresponding to Said variant comprising a second mutation:
(I) amino acid residues corresponding to K439 and S440 in the Fc region of human IgG1 heavy chain, provided that the mutation in S440 is neither S440Y nor S440W, and the first mutation is S440Y or S440W 2 mutations are in the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain;
(Ii) amino acid residues corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of human IgG1 heavy chain; or (iii) K447D / in the Fc region of human IgG1 heavy chain Amino acid residues corresponding to E or corresponding to K447K / R / H and 448K / R / H and 449P.
[Invention 1045]
Corresponds to the first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y in the Fc region of human IgG1 heavy chain, and K439 and S440 in the Fc region of human IgG1 heavy chain The variant of the present invention 1044 comprising a second mutation in the amino acid residue to be obtained provided that the mutation in S440 is not S440Y or S440W.
[Invention 1046]
The variant of the present invention 1045, wherein the mutation in the amino acid residue corresponding to K439 is K439D / E, and the amino acid residue corresponding to S440 is S440K / R.
[Invention 1047]
The variant of any one of the present invention 1033 to 1046, wherein the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen-binding region.
[Invention 1048]
The variant of the present invention 1047, selected from monospecific, bispecific or multispecific antibodies.
[Invention 1049]
A first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and a second polypeptide comprising an immunoglobulin second CH2-CH3 region and a second antigen-binding region; A variant of a parent antibody that is a bispecific antibody comprising a peptide, wherein the first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens; and And / or the second CH2-CH3 region is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain or A further mutation in an amino acid residue comprising a plurality of mutations and wherein the first polypeptide is selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain And wherein the second polypeptide is a Fc region of a human IgG1 heavy chain Further mutations in amino acid residues selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409, and wherein the further mutation in the first polypeptide comprises the second poly Said variant different from said further mutation in a peptide.
[Invention 1050]
(I) the first polypeptide comprises a further mutation in the amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain, for example K409R; and (ii) the second polypeptide comprises human IgG1 heavy A further mutation in an amino acid residue corresponding to F405 in the Fc region of the chain, eg F405L; or alternatively (iii) the first polypeptide is an amino acid corresponding to F405 in the Fc region of a human IgG1 heavy chain A further mutation at a residue, for example F405L; and (iv) the second polypeptide comprises a further mutation at an amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain, for example K409R;
The variant of the present invention 1049.
[Invention 1051]
A variant of any of the invention 1033-1050 which is conjugated to a drug, toxin or radioactive label, eg conjugated to a toxin via a linker.
[Invention 1052]
The variant of any of the present invention 1033-1051, which is part of a fusion protein.
[Invention 1053]
The variant of any of the present invention 1033-1052, which is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally a human full length antibody, eg, a human full length IgG1 antibody.
[Invention 1054]
A composition comprising first and second variants of a parent polypeptide each comprising an immunoglobulin Fc domain and a binding region, wherein the first and / or second variant comprises a human IgG1 heavy chain The composition comprising one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of:
[Invention 1055]
The first and / or second variant is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain Or the composition of the present invention 1054 comprising a plurality of mutations.
[Invention 1056]
The composition of the present invention 1055 wherein both the first and second variants comprise one or more mutations which may be the same or different.
[Invention 1057]
Said first variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain And the second variant is in an amino acid residue selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain The composition of the present invention 1055 which does not contain one or more mutations.
[Invention 1058]
(I) the first mutant further comprises a mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and (ii) the second mutant is in the Fc region of the human IgG1 heavy chain. Further comprising a mutation at a position corresponding to S440, provided that the mutation is neither S440Y nor S440W; or (i) and (ii) alternatively
(Iii) the first variant further comprises a mutation at a position corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W; and (iv) the The second variant further comprises a mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain;
The composition of any of the present invention 1054-1057.
[Invention 1059]
The composition of the invention 1058 wherein the mutation at position K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at position S440 in the Fc region of a human IgG1 heavy chain is S440K / R.
[Invention 1060]
(I) the first variant further comprises a prodrug, and (ii) the second variant comprises an activator for the prodrug on the first variant; or (i) And (ii) is alternatively
(Iii) the second variant comprises a prodrug, and (iv) the first variant may comprise an activator for the prodrug on the second variant, The composition of any of the present invention 1054-1059.
[Invention 1061]
The composition of any of the present invention 1054-1060, wherein the first and second parent polypeptides are first and second parent antibodies, respectively, comprising an immunoglobulin Fc domain and an antigen binding region.
[Invention 1062]
Said first and second antibodies are each human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each human full length antibody, for example each human full length IgG1 antibody The composition of the present invention 1061.
[Invention 1063]
The composition of the present invention 1062 wherein said first and second antibodies are each selected from monospecific, bispecific or multispecific antibodies.
[Invention 1064]
The first and / or second parent antibody comprises a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin and a second CH2-CH3 region and a second, respectively. A bispecific antibody comprising a second polypeptide comprising a plurality of antigen-binding regions, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and The first CH2-CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; and Two CH2-CH3 regions contain additional amino acid mutations at positions corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain, and the first CH2-CH3 region Wherein said further amino acid mutation in said second CH2-CH3 region The composition of the present invention 1063 that differs from a mutation.
[Invention 1065]
The further amino acid mutation of the first CH2-CH3 region is in a position corresponding to K409 in the Fc region of a human IgG1 heavy chain, for example K409R; and the further amino acid of the second CH2-CH3 region The composition of the invention 1064 wherein the mutation is in a position corresponding to F405 in the Fc region of a human IgG1 heavy chain, such as F405L.
[Invention 1066]
The composition of any of the present invention 1054-1063, wherein said first and second variants of said composition bind to different epitopes on the same or different antigen.
[Invention 1067]
Any of the present invention 1054-1066, wherein one or both of said first and second variants are conjugated to a drug, toxin or radiolabel, eg, conjugated to a toxin via a linker Composition.
[Invention 1068]
The composition of any of the present invention 1054-1067, wherein one or both of the first and second variants is part of a fusion protein.
[Invention 1069]
The composition of any of the present invention 1054-1063 and 1066-1068, wherein the first and / or second variant of the composition comprises only one mutation.
[Invention 1070]
A composition comprising a variant of any of the inventions 1033-1053 or any of the compositions of the invention 1054-1069 and a pharmaceutically acceptable carrier.
[Invention 1071]
A kit of parts comprising a first variant and a second variant as defined in any of the inventions 1033-1053 for simultaneous, separate or sequential use in therapy.
[Invention 1072]
The variant, composition or kit of parts of any of the invention 1033-1071 for the treatment of diseases such as cancer.
[Invention 1073]
A method for the treatment of a disease in a human comprising administering a variant, composition or kit of parts of any of the invention 1033-1071.
[Invention 1074]
A method for the treatment of cancer in a human comprising administering a variant, composition or kit of parts of any of the invention 1033-1071.
[Invention 1075]
A variant, composition or kit-of-part of any of the invention 1033-1071 for use in imaging at least a portion of a human or other mammalian body.
[Invention 1076]
A method for imaging at least a part of a human or other mammalian body comprising administering a variant, composition or kit of parts of any of the invention 1033-1071.
These and other aspects of the invention, in particular various uses and therapeutic applications of polypeptide variants and antibody variants, are described in further detail below.

Schematic representation of IgG molecules in hexamer formation. Dotted circles illustrate two adjacent Fc: Fc interaction pairs of two adjacent IgG molecules. The arrows in the box indicate the orientation of the explanatory views in B, C, and D: rotate two adjacent Fc molecules 90 ° (in the plane of the drawing) from the Fab arm to the CH3 domain Looking for direction. Observational effect of oligomerization enhancing mutation on CDC. Schematic illustrating Fc: Fc interaction pairs with enhanced activity according to aspects of the single and double mutants of the invention. Observed effect of oligomerization-inhibiting mutation on CDC. At least two oligomerization-inhibiting mutations that compensate for each other, either grouped into one molecule (double mutant aspect) or separated into two molecules (mixed mutant aspect) Schematic illustrating how to restore or enhance the Fc: Fc interaction according to the double mutant and mixed mutant aspects of. Mixed mutants achieve specific effector function activation in response to the binding of both antibodies capable of recognizing multiple different targets. Theoretical effect of C1q binding inhibitory mutations on CDC. Since C1q cannot compensate for the defects introduced in the antibody, it illustrates that if the mutation inhibits C1q binding, they cannot be combined or mixed to restore CDC activity, Schematic representation of the Fc: C1q interaction. Using Clustal 2.1 software, human IgG1, IgG1f, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, corresponding to residues P247 to K447 in the IgG1 heavy chain, numbered according to the EU index described in Kabat Sequence alignment of IgE and IgM Fc segments. The sequences shown are human IgG1 heavy chain constant region (SEQ ID NO: 1; UniProt accession number P01857) and residues 130-330 (SEQ ID NO: 6 and SEQ ID NO of allotype variant IgG1m (f) : 7); residues 126-326 (SEQ ID NO: 8) of IgG2 heavy chain constant region (SEQ ID NO: 2; UniProt accession number P01859); and IgG3 heavy chain constant region (SEQ ID NO: 3; UniProt (Accession number P01860) residues 177-377 (SEQ ID NO: 9); and IgG4 heavy chain constant region (SEQ ID NO: 4; UniProt accession number P01861) residues 127-327 (SEQ ID NO: 10) ); And residues 225-428 (SEQ ID NO: 11) of the IgE constant region (Uniprot accession number P01854); and residues 133-353 (SEQ ID NO: 12) of the IgA1 constant region (Uniprot accession number P01876) ); And residues 120-340 (SEQ ID NO: 13) of the IgA2 constant region (Uniprot accession number P01877); and the IgM constant region (Uniprot Click session number P01871) residues 230-452 (SEQ ID NO: 14); and residues of IgD constant region (Uniprot accession number P01880) 176-384 (SEQ ID NO: represent 15). It is a figure which shows the continuation of FIGS. FIGS. 3A and B: Sequence alignment of anti-EGFr antibody 2F8 in IgG1 (SEQ ID NO: 16), IgG4 (SEQ ID NO: 18) and (partial) IgG3 (SEQ ID NO: 17) backbones. Amino acid numbering by Kabat and EU index is shown (both described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) . It is a figure which shows the continuation of FIG. 3A. Details of the K439 / S440 interaction between Fc of neighboring molecules (Fc and Fc ′, respectively) in a multimeric (eg hexamer) configuration, illustrating the interaction between wild-type unmodified Fc and Fc ′ molecules Figure. K439 / S440 between Fc of adjacent molecules (Fc and Fc ′, respectively) in a multimeric (eg hexamer) configuration illustrating the interaction between mutant Fc and Fc ′ molecules, including K439E and S440K mutations Detailed view of interaction. 7D8 Fc: C1q binding ELISA using Fc variants. The wells of the microtiter plate were coated with the indicated antibody concentration series and incubated with a fixed concentration of C1q. The efficiency of binding to C1q was comparable to wild type 7D8 for all of the coated mutants except I253D. Representative of at least 3 experiments are shown. CDC mediated by 7D8 mutant against CD20 positive Raji cells. To test CDC activity by measuring cell lysis, Raji cells were incubated with 7D8 mutant (K439E, S440K, K439E / S440K double mutant, K439E + S440K mixed) and C1q concentration series. A representative graph of repeated experiments is shown. CDC mediated by 7D8 mutant (7D8-WT, K439E, S440K, K439E / S440K double mutant, K439E + S440K mixed type) against CD20 positive Daudi cells. The concentration series of 7D8 mutants were examined for their activity to induce CDC. CDC mediated by a variant of the CD38 antibody HuMAb 005 against CD38 positive cells. CDC activity against Daudi cells by 005 mutant concentration series. Unpurified antibody samples isolated from those that had been transiently transfected were tested. As a negative control, the supernatant of mock-transfected cells was used. CDC mediated by a variant of the CD38 antibody HuMAb 005 against CD38 positive cells. CDC activity against Raji cells by HuMAb 005 mutant concentration series. Unpurified antibody samples isolated from those that had been transiently transfected were tested. As a negative control, the supernatant of mock-transfected cells was used. CDC mediated by a variant of the CD38 antibody HuMAb 005 against CD38 positive cells. CDC activity against Wien133 cells of E345R mutant of HuMAb 005 with either 20% or 50% NHS. Unpurified antibody samples isolated from those that had been transiently transfected were tested. As a negative control, the supernatant of mock-transfected cells was used. CDC mediated by a variant of the CD38 antibody HuMAb 005 against CD38 positive cells. CDC activity against Raji cells of E345R mutants of HuMAb 005 and 7D8 with either 20% or 50% NHS. Unpurified antibody samples isolated from those that had been transiently transfected were tested. As a negative control, the supernatant of mock-transfected cells was used. CDC with wild type and E345R mutants of CD38 antibody HuMAb 005 (A) and CD20 antibody HuMAb 7D8 (B) in competition experiments with Fc-binding peptides. Cell lysis after CDC was measured on Daudi cells opsonized with antibodies incubated with a concentration series of Fc-binding DCAWHLGELVWCT peptide (SEQ ID NO: 19). Unpurified antibody samples isolated from those that had been transiently transfected were used. As a negative control, mock-transfected cell supernatant was used. ADCC for Daudi cells expressing CD38 with wild-type CD38 antibody HuMAb 005 and mutant IgG1-005-E345R. ADCC of one donor's PBMC is shown and expressed as% dissolution. Binding of wild-type IgG1-7D8 and mutant IgG1-7D8-E345R to human, cynomolgus monkey and mouse FcRn as determined by ELISA at pH 6. Plasma concentrations of wild-type IgG1-7D8 and IgG1-7D8-E354R, IgG1-7D8-S440K and IgG1-7D8-K322A variants after intravenous injection in SCID mice. FIG. 14A: CDC against CD20 positive and CD38 positive Wien133 cells. FIG. 14B: CDC against CD20 positive and CD38 positive Wien133 cells. FIG. 14C: CDC against CD20 positive and CD38 positive Wien133 cells. FIG. 14D: CDC against CD20 positive and CD38 positive Wien133 cells. 15A and B: Evaluation of in vivo activity of IgG1-7D8-E345R in a subcutaneous xenograft model using Raji-luc # 2D1 cells. FIGS. 16A and B: Evaluation of in vivo activity of IgG1-005-E345R in a subcutaneous xenograft model using Raji-luc # 2D1 cells. CDC by CD38 / EGFR bispecific antibody having E345R mutation against CD38 positive and EGFR negative Wien133 cells. 18A and B: CDC against CD20 positive and CD38 negative Wienl33 cells or Raji cells with CD20 / CD38 bispecific antibodies with and without E345R mutation. CDC against EGFR positive A431 cells by EGFR antibody 2F8 having E345R mutation. Figures 20A and B: CDC mediated by E345R variant antibodies. Co-localization analysis of TF antibody (FITC) using lysosomal marker LAMP1 (APC). Figure 22A: In a study on various B cell lines, introduction of E345R resulted in enhanced CDC-mediated lethality compared to wild type rituximab. Figure 22B: In a study on various B cell lines, introduction of E345R resulted in enhanced CDC-mediated lethality compared to wild type rituximab. FIG. 22C: In a study on various B cell lines, introduction of E345R resulted in enhanced CDC-mediated lethality compared to wild type rituximab. FIG. 22D: In studies on various B cell lines, introduction of E345R resulted in enhanced CDC-mediated lethality compared to wild type rituximab. In various B cell lines with similar CD20 expression levels, introduction of E345R is independent of the expression levels of complement regulatory proteins CD46 (A), CD55 (B) or CD59 (C) compared to wild-type rituximab Resulting in enhanced maximum CDC-mediated lethality. CDC dynamics. E345R antibody results in faster and more substantial target cell lysis by CDC compared to wild type antibody. CDC dynamics. E345R antibody results in faster and more substantial target cell lysis by CDC compared to wild type antibody. CDC dynamics. E345R antibody results in faster and more substantial target cell lysis by CDC compared to wild type antibody. CDC dynamics. E345R antibody results in faster and more substantial target cell lysis by CDC compared to wild type antibody. CDC dynamics. Introduction of the E345R mutation into the bispecific CD38xCD20 antibody results in faster and more substantial CDC-mediated target cell lysis. Figures 25A-B: CDC dynamics. Introduction of the E345R mutation into the bispecific antibodies CD38 x EGFR (A) and CD20 x EGFR (B) that monovalently bind to EGFR-negative Raji cells results in faster and more substantial CDC-mediated target cell lysis . FIG. 26A: CDC against Wien133 cells with a combination of a wild type antibody and a mutant antibody containing E345R and Q386K. Since IgG1-b12 mutant does not bind to Wien133 cells, it was used as a negative control antibody. FIG. 26B: CDC on Wien133 cells with a combination of a wild type antibody and a mutant antibody containing E345R and Q386K. Since IgG1-b12 mutant does not bind to Wien133 cells, it was used as a negative control antibody. FIG. 26C: CDC against Wien133 cells with a combination of a wild type antibody and a mutant antibody containing E345R and Q386K. Since IgG1-b12 mutant does not bind to Wien133 cells, it was used as a negative control antibody. FIG. 26D: CDC against Wien133 cells with a combination of a wild type antibody and a mutant antibody containing E345R, E430G and Q386K. Since IgG1-b12 mutant does not bind to Wien133 cells, it was used as a negative control antibody. FIG. 26E: CDC against Wien133 cells with a combination of a wild type antibody and a mutant antibody containing E345R, E430G and Q386K. Since IgG1-b12 mutant does not bind to Wien133 cells, it was used as a negative control antibody. FIG. 26F: CDC against Wien133 cells with a combination of a wild type antibody and a mutant antibody containing E345R, E430G and Q386K. Since IgG1-b12 mutant does not bind to Wien133 cells, it was used as a negative control antibody. CDC activity of IgG1, IgG2, IgG3 and IgG4 isotype antibodies containing E345R mutation. Introduction of the Fc-Fc stabilizing E345R mutation into wild type CD38 antibody 005 results in enhanced lethality of primary CLL cells in the ex vivo CDC assay (mean ± standard error of the mean). FcRn binding to wild-type IgG1-005 and IgG1-005 mutant human, mouse and cynomolgus FcRn at pH 6.0 as determined by ELISA. CDC activity in 20% normal human serum of various rituximab mutants, wild type rituximab and an irrelevant negative control antibody IgG1-b12 in Ramos and SU-DHL-4 cell lines. Wild-type IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1-005-E430S and IgG1-005-S440Y as determined by Micro Vue C4d fragment ELISA , As well as C4d production in normal human serum by heat-aggregated IgG (HAG) (positive control). Wild-type IgG1-005 and antibody variants IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345R, IgG1-005-E345Y, IgG1-005-, IgG1-005- administered in SCID mice, as determined by total human IgG ELISA Plasma clearance rates of E430F, IgG1-005-E430G, IgG1-005-E430S, IgG1-005-E430T and IgG1-005-S440Y. Wild-type IgG1-005 administered in SCID mice and antibody variants IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345R, IgG1-005-E345Y, IgG1-005, as determined by human CD38 specific ELISA -Plasma clearance rates of E430F, IgG1-005-E430G, IgG1-005-E430S, IgG1-005-E430T and IgG1-005-S440Y.

Detailed Description of the Invention As explained herein, surprisingly, mutations in amino acids that are not directly involved in Fc: C1q binding can nevertheless enhance the CDC of an antibody, Furthermore, other Fc-mediated effector functions of the antibody can be improved. This supports the hypothesis that antibody molecules such as IgG1 antibodies can form oligomeric structures that are later bound by C1q. In addition, while certain mutations have been found to reduce CDC induction, certain combinations of such mutations in the same or different antibody molecules result in recovery of CDC induction and are directed against antibody oligomerization. Showed additional specificity, thereby promoting more specific CDC induction. Specific mutations that enhance the CDC response were also characterized by improved ADCC response, enhanced binding, enhanced intracellular translocation, and in vivo activity in mouse tumor model systems, as demonstrated in the Examples. . These discoveries allow new antibody-based therapeutics with enhanced CDC inducing ability, more selective CDC inducibility, and / or other improved effector functions.

  All of the polypeptide variants (including antibody variants) of the invention are full length or partial immunoglobulins containing one or more mutations in the binding region and the segment corresponding to amino acid residues E345-S440 in IgG1. Including Fc domain. Without being limited to theory, the identified mutations are schematically represented in FIG. 1, referred to herein as “single mutant”, “double mutant” and “mixed mutant”. Based on three different principles, it is believed to result in more effective and / or more specific CDC induction.

  The improved C1q and / or CDC effects of the variants of the present invention can only be detected mainly in assays that are capable of forming antibody oligomers, such as cell-based assays in which the antigen is not immobilized and is present in the flow membrane. . Furthermore, it can be verified according to the principle shown in FIG. 1C that these effects are caused by a more stable antibody oligomer and not by modification of the direct binding site of C1q.

Definitions The term "single mutant" shall be construed as variant of a parent polypeptide or antibody have been enhanced CDC is compared to and other effector functions present invention, which may be enhanced .

  The term “double mutant” refers to an improved CDC compared to a mutant that contains at least two mutations in the segment and contains only one of the at least two mutations, and other It shall be construed as a variant that may also have an enhanced effector function.

  The term “mixed variant” when used in combination with a second variant of the same or different polypeptide or antibody, which contains a mutation at a different amino acid residue in the segment, Variants and variants that result in improved CDC and optionally also enhance other effector functions compared to one or more of the parent polypeptide or antibody alone are to be construed.

  The term “polypeptide comprising an Fc domain of an immunoglobulin and a binding region” refers in the context of the present invention to any molecule such as an Fc domain of an immunoglobulin and a polypeptide present on the surface of a cell, bacteria or virion, for example. And a binding region that can bind to the polypeptide. An immunoglobulin Fc domain contains two CH2-CH3 regions of an immunoglobulin and a linking region (eg, a hinge region), typically occurring after digestion of an antibody with papain (known to those skilled in the art). Possible antibody fragments are defined. The antibody isotype (eg, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, or IgE) is defined by the constant domain of the antibody heavy chain. The Fc domain mediates the effector functions of antibodies along with cell surface receptors called Fc receptors and proteins of the complement system. The binding region may be a polypeptide sequence such as a protein, protein ligand, receptor, antigen binding region or ligand binding region that can bind to cells, bacteria, or virions. When the binding region is, for example, a receptor, a “polypeptide comprising an immunoglobulin Fc domain and a binding region” can be prepared as a fusion protein of the immunoglobulin Fc domain and the binding region. Where the binding region is an antigen binding region, a “polypeptide comprising an immunoglobulin Fc domain and binding region” refers to a chimeric, humanized, or human antibody or heavy chain only antibody, or ScFv-Fc fusion. An antibody such as A polypeptide comprising an immunoglobulin Fc domain and a binding region typically comprises a linking region (eg, a hinge region) and the two CH2-CH3 regions of an immunoglobulin heavy chain, and thus “immunoglobulin”. The “polypeptide comprising an Fc domain and a binding region” may be “a polypeptide comprising at least one Fc domain of an immunoglobulin and a binding region”. “Immunoglobulin Fc domain” means in the context of the present invention a linking region, eg a hinge, depending on the subtype of the antibody, eg human IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgM or IgE. As well as the CH2 and CH3 regions of an immunoglobulin. The polypeptide is not limited to being of human origin and may be of any origin (eg, mouse or cynomolgus monkey origin).

  The term “CH2 region” or “CH2 domain”, as used herein, is intended to refer to the CH2 region of an immunoglobulin. Thus, for example, the CH2 region of human IgG1 antibody corresponds to amino acids 228-340 according to the EU numbering system. However, the CH2 region may be any of the other subtypes as described herein.

  The term “CH3 region” or “CH3 domain”, as used herein, is intended to refer to the CH3 region of an immunoglobulin. Thus, for example, the CH3 region of human IgG1 antibody corresponds to amino acids 341 to 447 according to the EU numbering system. However, the CH3 region may be any of the other subtypes as described herein.

  The term "immunoglobulin" consists of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four of which are interconnected by disulfide bonds. Refers to a possible class of structurally related glycoproteins. The structure of the immunoglobulin has been characterized in detail. See, for example, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)). Briefly, each heavy chain is typically comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is typically comprised of three domains, CH1, CH2 and CH3. The heavy chains are interconnected via disulfide bonds in so-called “hinge regions”. Each light chain is typically comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is typically comprised of one domain, CL. Hypervariable regions (or hypervariable regions), also called complementarity determining regions (CDRs), interspersed between more conserved regions, called framework regions (FR), and VH and VL regions , Which may be hypervariable in terms of structurally defined loop arrangement and / or morphology). Each VH and VL is typically composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (Chothia and Lesk J. Mol. Biol. 196, 901 917 (1987)). Unless otherwise stated or contradicted by context, the constant region sequence amino acids are referred to herein by the EU index (Kabat, EA et al., Sequences of proteins of immunological interest. 5th Edition-US Department of Health and Human Services, NIH publication No. 91-3242, pp 662,680,689 (1991)).

The term “antibody” (Ab) in the context of the present invention, under typical physiological conditions, has a meaningful period of time, eg at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours. At least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or longer, about 48 hours or longer, about 3 days, 4 days, 5 days, 6 days, 7 days or more Such as long days, or any other reasonable functionally defined period of time (e.g., sufficient time to induce, promote, enhance and / or modulate a physiological response associated with antibody binding to an antigen, and / or An immunoglobulin molecule, fragment of an immunoglobulin molecule, or any group thereof that has the ability to specifically bind an antigen with a half-life of sufficient time for the antibody to drive effector activity) It refers to things. The antibody of the present invention comprises an Fc domain of an immunoglobulin and an antigen binding region. An antibody generally comprises two CH2-CH3 regions and one linking region (eg, a hinge region), eg, at least one Fc domain. Accordingly, the antibody of the present invention may comprise one Fc region and one antigen binding region. The variable regions of the heavy and light chains of an immunoglobulin molecule contain binding domains that interact with antigens. The constant region or “Fc” region of an antibody contains various cells of the immune system (eg, effector cells) and components of the complement system (eg, C1q, which is the first component in the classical pathway of complement activation). Or it may mediate the binding of an immunoglobulin to a factor. The antibody may also be a multispecific antibody such as a bispecific antibody or similar molecule. The term “bispecific antibody” refers to an antibody having specificity for at least two different, typically non-overlapping epitopes. Such epitopes may be on the same target or on different targets. Where the epitopes are on different targets, such targets may be on the same cell or on different cells or cell types. As noted above, unless otherwise stated or clearly contradicted by context, the term antibody herein includes at least a portion of the Fc region and retains the ability to specifically bind to an antigen. Antibody fragments. Such fragments can be obtained by any known technique such as enzymatic cleavage, peptide synthesis and recombinant expression techniques. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments that fall within the scope of the term “Ab” or “antibody” include monovalent antibodies (described in WO 007059782 by Genmab); consisting of only two heavy chains, eg natural in camelids Heavy chain antibodies (eg, Hamers-Casterman (1993) Nature 363: 446); ThioMab (Roche, WO2011069104), an asymmetric bispecific antibody-like molecule, the strand-exchange recombination domain (strand-exchange) engineered domain) (SEED or Seed-body) (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155: 219); FcΔAdp (Regeneron, WO2010151792), Azymetric Scaffold (Zymeworks / Merck, WO2012) No. 058768), mAb-Fv (Xencor, WO2011 / 028952), double variable domain immunoglobulin (Abbott, DVD-Ig, US Pat. No. 7,612,181); dual domain double-headed antibody (Unilever; Sanofi Aventis, WO20100226923) ), Di-diabody (ImClone / Eli Lilly, Knobs-into-holes antibody format (Genentech, WO9850431); DuoBody (Genmab, WO 2011/131746); Electrostatic steering antibody format (Amgen) , EP1870459 and WO 2009089004; Chugai, US201000155133; Oncomed, WO 2010129304A2); Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech) , Biclonic (Merus), dual targeting domain antibodies (GSK / Domantis), two-in-one antibodies that recognize two targets (Genentech, NovImmune), cross-linked Mabs (Cross-linked) Mab) (Karmanos Cancer Center), CovX-body (CovX / Pfizer), IgG-like bispecificity (ImClone / Eli Lilly, Shen, X, et al. J Immunol Methods, 2007. 318 (1-2): p 65-74), and DIG-body and PIG-body (Pharmabcine), and double parents Compatible retargeting molecules (Fc-DART or Ig-DART, by Macrogenics, WO / 2008/157379, WO / 2010/080538), Zybody (Zyngenia), common light chain (Crucell / Merus , US7262028) or an approach that uses a common heavy chain (κλBody by NovImmune), as well as scFv comprising a polypeptide sequence fused to an antibody fragment containing an Fc domain, such as BsAb by ZymoGenetics / BMS Fusion proteins such as fusions), HERCULES by Biogen Idec (US007951918), SCORPION by Emergent BioSolutions / Trubion, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009. 393 (3)): p. 672-92), scFv fusion by Novartis, scFv fusion by Changzhou Adam Biotech Inc (CN 102250246), TvAb by Roche (WO 2012025525, WO 2012025530), mAb ² by f-Star (WO2008 / 003116) No.) and double scFv fusions. In addition, unless the term antibody is specified otherwise, polyclonal antibodies, monoclonal antibodies (human monoclonal antibodies, etc.), antibody mixtures (recombinant polyclonal bodies), such as those produced by techniques developed by Symphonogen and Merus ( Oligoclonics), as well as antibody-like polypeptides, such as chimeric and humanized antibodies, should be understood. The antibody produced can potentially have any isotype.

  The term “full-length antibody” as used herein refers to an antibody (eg, a parental antibody) that includes all of the heavy and light chain constant and variable domains corresponding to those commonly found in wild-type antibodies of that isotype. Antibody or mutant antibody).

  The term “human antibody”, as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. A human antibody of the present invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations, insertions or insertions introduced by in vitro random or site-directed mutagenesis, or in vivo somatic mutation). Deletion). However, the term “human antibody”, as used herein, does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are joined to a human framework sequence. .

  The terms “monoclonal antibody”, “monoclonal Ab”, “monoclonal antibody composition”, “mAb” and the like as used herein refer to a preparation of Ab molecules of single molecular composition. A monoclonal antibody composition exhibits a single binding specificity and affinity for a particular epitope. Thus, the term “human monoclonal antibody” refers to Abs that exhibit a single binding specificity, having variable and constant regions derived from human germline immunoglobulin sequences. Human mAbs are obtained from transgenic or transchromosomal non-human animals, such as transgenic mice, having a genome comprising a human heavy chain transgene repertoire and a light chain transgene repertoire rearranged to produce functional human antibodies. The resulting B cells can be produced by hybridomas containing those fused with immortalized cells.

  As used herein, an “isotype” is an immunoglobulin class (eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA1, IgGA2, IgE or IgM, or any of them) encoded by a heavy chain constant region gene. Allotypes such as IgG1m (za) and IgG1m (f). Furthermore, each heavy chain isotype can be combined with either a kappa (κ) or lambda (λ) light chain.

  The term “monovalent antibody” in the context of the present invention allows an antibody molecule to bind an antigen in only one of the binding domains of the antibody, eg has a single antigen-antibody interaction, Therefore, it means that antigen crosslinking cannot be performed.

  As used herein, the term “target” refers to an antibody produced when, in the context of the present invention, the binding region of a polypeptide comprising an Fc domain and a binding region is used in the context of antibody binding. It should be understood as a molecule containing any antigen to which The terms “antigen” and “target” can be used interchangeably with respect to an antibody and have the same meaning and purpose with respect to any aspect or embodiment of the invention.

As used herein, the term “binding” in the context of antibody binding to a given antigen typically refers to surface plasmon resonance on a BIAcore 3000 instrument using, for example, an antigen as a ligand and an antibody as an analyte. as determined by (SPR) technology, K _D of about 10 ^-6 M or less, for example, 10 ^-7 M or less, for example about 10 ^-8 M or less, such as about 10 ^-9 M or greater Less than, about 10 ⁻¹⁰ M or less, or binding with an affinity corresponding to about 10 ⁻¹¹ M or less, etc., and non-specific antigens other than a given antigen or closely related antigen (Eg, BSA, casein) at least 10 times lower, eg, at least 100 times lower, eg, at least 1,000 times lower, eg, at least 10,000 times lower, eg, at least 100,000 times lower than its affinity for binding. With an affinity corresponding to a K _D of such times lower, it binds to a predetermined antigen. The amount of affinity is lower is dependent on K _D of an antibody, therefore, when the extremely low K _D of an antibody (i.e., high specificity of the antibody) is affinity affinity for antigen relative to non-specific antigen The degree of lower than gender may be at least 1 / 10,000. The term “K _D ” (M), as used herein, refers to the dissociation equilibrium constant for a particular antibody-antigen interaction.

別の保存的アミノ酸残基置換のクラス

アミノ酸残基の別の物理的および機能的な分類

A “variant” or “antibody variant” or “parent antibody variant” of the present invention is an antibody molecule comprising one or more mutations compared to a “parent antibody”. Different terms can be used interchangeably and have the same meaning and purpose with respect to any aspect or embodiment of the invention. Exemplary parent antibody formats include, but are not limited to, wild type antibodies, full length antibodies or antibody fragments containing Fc, bispecific antibodies, human antibodies, or any combination thereof. Similarly, a “variant” or “variant of a polypeptide comprising an immunoglobulin Fc domain and a binding region” or “variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region” of the present invention Is a “polypeptide comprising an immunoglobulin Fc domain and a binding region” comprising one or more mutations compared to a “parent polypeptide comprising an immunoglobulin Fc domain and a binding region”. These different terms can be used interchangeably and have the same meaning and purpose with respect to any aspect or embodiment of the invention. Exemplary mutations include amino acid deletions, insertions and substitutions of amino acids in the parent amino acid sequence. Amino acid substitutions may replace a natural amino acid with another natural amino acid or a non-natural amino acid derivative. Amino acid substitutions may be conservative or non-conservative. In the context of the present invention, conservative substitutions may be defined by substitutions within the class of amino acids represented in one or more of the following three tables:

Amino acid residue class for conservative substitutions

Another class of conservative amino acid residue substitutions

Alternative physical and functional classification of amino acid residues

In the context of the present invention, substitutions in the variant are indicated as follows:
Original amino acid-position-substituted amino acid;
The three letter code or the one letter code is used to indicate amino acid residues, including the codes Xaa and X. Thus, the notation "E345R" or "Glu345Arg" means that the variant contains a substitution of glutamic acid for arginine when aligned at the position of the variant amino acid corresponding to the amino acid at position 345 in the parent antibody. To do.

If such a position is not present in the antibody, the variant contains an amino acid insertion, eg:
Position-substituted amino acids; for example, the designation “448E” is used.

  Such notation is particularly relevant for modifications in a series of homologous polypeptides or antibodies.

Similarly, if the identity of the substituted amino acid residue is not important:
Original amino acid-position; or “E345”.

For modifications where the original amino acid and / or substituted amino acid includes multiple, but not all, amino acids, for the substitution of glutamic acid at position 345 with arginine, lysine or tryptophan:
"Glu345Arg, Lys, Trp" or "E345R, K, W" or "E345R / K / W" or "E345 to R, K or W (E345 to R, K or W)" in the context of the present invention Can be used interchangeably.

  Moreover, the term “substitution” encompasses substitution of any one of the other 19 natural amino acids, or substitution with other amino acids, such as unnatural amino acids. For example, the substitution of amino acid E at position 345 includes each of the following substitutions: 345A, 345C, 345D, 345G, 345H, 345F, 345I, 345K, 345L, 345M, 345N, 345Q, 345R, 345S, 345T , 345V, 345W, and 345Y. By the way, this is equivalent to the notation of 345X, where X represents any amino acid. These substitutions can also be expressed as E345A, E345C, other, or E345A, C, other, or E345A / C / other. The same is true for each and every position referred to herein, and any such substitutions are specifically included herein.

  An amino acid or segment in one sequence “corresponds” to an amino acid or segment in another sequence is (i) a standard sequence alignment program such as ALIGN, ClustalW or the like, typically with default settings. Used, aligned with other amino acids or segments, and (ii) having at least 50%, at least 80%, at least 90% or at least 95% sequence identity to SEQ ID NO: 1. For example, the sequence alignments shown in FIGS. 2 and 3 can be used to identify any amino acid in the IgG2, IgG3, or IgG4 Fc sequence that corresponds to a particular amino acid in the IgG1 Fc sequence.

  The present invention relates to variants, i.e. parent polypeptides and parent antibodies and / or variant polypeptides having some degree of identity to amino acids P247-K447 of SEQ ID No: 1, 2, 3, 4 and 5. Reference is made to variant antibodies, and such parental and / or variant antibodies are hereinafter referred to as “homologous antibodies”.

  For purposes of the present invention, the degree of identity between two amino acid sequences, as well as the degree of identity between two nucleotide sequences, is determined by the program “align”, a Needleman-Wunsch alignment (ie, global alignment). Is done. This program is used for alignment of polypeptide sequences as well as nucleotide sequences. The default scoring matrix BLOSUM50 is used for polypeptide alignment, the default identity matrix is used for nucleotide alignment, and the first residue penalty in the gap is -12 for the polypeptide. And -16 for nucleotides. The penalty for additional residues in the gap is -2 for polypeptides and -4 for nucleotides.

  "Align" is part of FASTA package version v20u6 (WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis", PNAS 85: 2444-2448, and WR Pearson (1990) "Rapid and Sensitive Sequence Comparaison with FASTP and FASTA ", Methods in Enzymology 183: 63-98). FASTA protein alignment uses the Smith-Waterman algorithm without limitation on gap size (see “Smith-Waterman algorithm”, T. F. Smith and M. S. Waterman (1981) J. Mol. Biolo. 147: 195-197).

  As used herein, the term “effector cells” refers to immune cells that are involved in the effector phase of the immune response, rather than the recognition and activation phases of the immune response. Exemplary immune cells include cells of myeloid or lymphoid origin, such as lymphocytes (such as B cells and T cells including cytolytic T cells (CTL)), killer cells, natural killer cells, macrophages, single cells. Spheres, eosinophils, polymorphonuclear cells such as neutrophils, granulocytes, mast cells and basophils are included. Certain effector cells express Fc receptors (FcR) or complement receptors and perform specific immune functions. In some embodiments, effector cells such as natural killer cells can induce ADCC. For example, monocytes, macrophages, neutrophils, dendritic cells and Kupffer cells that express FcR specifically kill target cells and present antigens to other components of the immune system, or Involved in binding to cells presenting antigen. In some embodiments, ADCC can further enhance classical complement activation triggered by antibodies that results in the deposition of activated C3 fragments on the target cell surface. C3 cleavage products are ligands for complement receptors (CR) such as CR3 that are expressed on myeloid cells. Recognition of complement fragments by CR on effector cells may facilitate enhancement of ADCC via Fc receptors. In some embodiments, antibody-triggered classical complement activation results in a C3 fragment on the target cell. These C3 cleavage products can promote direct complement dependent cytotoxicity (CDCC). In some embodiments, the effector cell can phagocytose the target antigen, target particle or target cell. Expression of specific FcR or complement receptors on effector cells can be regulated by humoral factors such as cytokines. For example, FcγRI expression has been found to be upregulated by interferon γ (IFNγ) and / or G-CSF. This enhanced expression enhances the cytotoxic activity of FcγRI-bearing cells against the target. Effector cells can phagocytose target antigens or phagocytose or lyse target cells. In some embodiments, antibody-triggered classical complement activation results in a C3 fragment on the target cell. These C3 cleavage products promote direct phagocytosis by effector cells or indirectly by enhancing antibody-mediated phagocytosis.

  The term “vector” as used herein refers to a nucleic acid molecule capable of inducing transcription of a nucleic acid segment linked into a vector. One type of vector is a “plasmid” in the form of a circular double stranded DNA loop. Another type of vector is a viral vector, in which a nucleic acid segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (such as non-episomal mammalian vectors), when introduced into a host cell, integrate into the host cell's genome and are thereby replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors, such as viral vectors (such as replication-defective retroviruses, adenoviruses and adeno-associated viruses) that perform equivalent functions.

  The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which an expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Even though such progeny may not actually be identical to the parental cell because certain modifications may occur in subsequent generations, either due to mutations or environmental influences, the present specification In the term “host cell”. Recombinant host cells include, for example, transfectomas, CHO cells, HEK-293 cells, PER.C6, NS0 cells and lymphocyte cells, and prokaryotic cells such as E. coli, and plant cells and Other eukaryotic hosts such as fungi are included.

  The term “transfectoma” as used herein refers to a recombinant eukaryotic host cell that expresses an Ab or target antigen, eg, CHO cells, PER.C6, NS0 cells, HEK-293 cells, plant cells. Or a fungus containing yeast cells.

  The term “preparation” enhances the ability to form oligomers when interacting with antigens bound to cells (eg, antigens expressed on the surface of cells), cell membranes, virions or other structures C1q binding, complement activation, CDC, ADCC, ADCP, other Fc-mediated effector functions, intracellular translocation, down-modulation, apoptosis, antibody-drug conjugate (ADC) uptake, binding Refers to a preparation of antibody variants and a mixture of different antibody variants that make it possible to enhance force, or any combination thereof. Exemplary assays include, for example, C1q binding strength (Example 4), CDC (Examples 5, 6 and 10, 16, 19, 22, 23, 24, 25, and 35); ADCC (Example 12), Regarding in vivo activity (Examples 20, 21), plasma clearance rate (Example 37), FcRn binding (Example 34), complement activation in a target-independent liquid phase (Example 36), etc. Presented in the examples. The variants of aspects herein, referred to as “single variants,” “double variants,” and “mixed variants,” along with exemplary processes for their preparation and methods of use, are described below. Further details will be described.

  As used herein, the term “affinity” refers to one molecule (eg, an antibody) and another molecule (eg, a target) at a single site, such as monovalent binding between an individual antigen binding site and an antigen of an antibody. Or the strength of binding to the antigen).

  As used herein, the term “binding force” is used between two structures, such as between multiple antigen binding sites of an antibody that interact with a target simultaneously, or between an antibody and C1q. Of composite strength for multiple binding sites. In the presence of multiple binding interactions, the two structures are considered to dissociate only when all of the binding sites are dissociated, and therefore the dissociation rate is lower than for individual binding sites, resulting in individual binding. It is believed to provide a greater effective total binding strength (binding force) compared to the strength of binding (affinity) at the site.

  As used herein, the term “oligomer” refers to a plurality, but a limited number of monomer units (eg, antibodies), as opposed to a polymer consisting at least in principle an infinite number of monomers. Refers to the molecule. Exemplary oligomers include dimers, trimers, tetramers, pentamers and hexamers. The Greek letter prefix is often used to represent the number of monomer units in the oligomer, eg tetramer is composed of 4 units and hexamer is composed of 6 units.

  The term “oligomerization” as used herein refers to the process of converting a monomer to a finite degree of polymerization. In the present specification, it has been observed that, after target binding by a polypeptide containing an Fc domain, such as an antibody, oligomerization of the Fc domain preferably occurs on the cell surface, but not limited thereto. Antibody oligomerization is described, for example, in cell surface C1q binding assays (as described in Examples 4 and 9), C1q activity assays (as described in Example 5), and in Examples 6, 10 and 19. The complement-dependent cytotoxicity can be evaluated.

  The term “C1q binding”, as used herein, is intended to refer to C1q binding in the context of C1q binding to an antibody bound to its antigen. An antibody bound to its antigen is to be interpreted as occurring both in vivo and in vitro in the context described herein. C1q binding can be achieved, for example, by using immobilized antibodies on an artificial surface (eg, plastic in an ELISA plate, as described in Example 3), or on the surface of a cell or virion (Example 4). And can be evaluated by use in combination with the predetermined antigen (as described in and 9). The binding of C1q to antibody oligomer is to be interpreted herein as a multivalent interaction that results in binding with high binding force.

  As used herein, the term “complement activation” refers to the activation of the classical complement pathway triggered by binding of complement component C1q to an antibody bound to its antigen. C1q is the first protein in the early events of the classical complement cascade and is involved in a series of cleavage reactions that result in the formation of an enzymatic activity called C3 convertase that cleaves complement component C3 into C3b and C3a . C3b covalently binds to C5 on the membrane to form C5b, which in turn complements the terminal complement components C5b, C6, C7, C8 and C9 into a membrane-damaging complex (MAC). Triggers late events for physical activation. The complement cascade results in pore formation, which causes cell lysis, also known as complement dependent cytotoxicity (CDC). Complement activation includes C1q activity (as described in Example 5), CDC kinetics (as described in Examples 28, 29 and 30), CDC assay (Examples 6, 10, 19, 25, 27 , 33 and 35) or by the cell deposition method of C3b and C4b as described in Beurskens et al April 1, 2012 vol. 188 no. 7 3532-3541.

  The term “complement dependent cytotoxicity” (“CDC”), as used herein, bound to its target on a cell or virion as a result of a pore in the membrane created by the MAC assembly. It is intended to refer to the process of antibody-mediated complement activation leading to antibody lysis. CDC is determined by in vitro assays such as the CDC assay using normal human serum as a complement source, as described in Examples 6, 10, 19, 25, 27, 33 and 35, or normal human serum limited to C1q. The C1q activity assay as described in Example 5 can be evaluated.

  The term “antibody-dependent cell-mediated cytotoxicity” (“ADCC”) as used herein is covered by an antibody by a cell expressing an Fc receptor that recognizes the constant region of the bound antibody. It is intended to refer to the mechanism of target cell or virion death. ADCC can be determined using methods such as, for example, the ADCC assay described in Example 12.

  The term “antibody-dependent cell phagocytosis” (“ADCP”), as used herein, refers to the mechanism of elimination of antibody-covered target cells or virions by intracellular translocation by phagocytic cells. Intended. The antibody-covered target cells or virions that have migrated intracellularly are housed in vesicles called phagosomes, which are subsequently fused with one or more lysosomes to form phagolysosomes. ADCP uses an in vitro cytotoxicity assay using macrophages as effector cells and video microscopy as described by van Bij et al. In Journal of Hepatology Volume 53, Issue 4, October 2010, Pages 677-685. Can be evaluated. Alternatively, for example, phagocytosis of S. aureus by PMN can be evaluated as described in Example 14.

  The term “complement dependent cytotoxicity” (“CDCC”), as used herein, refers to a complement third component (C3 that is covalently bound to a target cell or virion as a result of antibody-mediated complement activation. It is intended to refer to the mechanism of death of a target cell or virion by a cell expressing a complement receptor that recognizes the cleavage product. CDCC can be assessed in a manner similar to that described for ADCC.

  The term “plasma half-life”, as used herein, means that the concentration of a polypeptide in plasma is reduced to half its initial concentration during elimination (after the distribution phase). It points to this time. For antibodies, the distribution phase is typically 1-3 days, during which the plasma concentration is reduced by about 50% due to redistribution between plasma and tissue. Plasma half-life can be measured by methods well known in the art.

  The term “plasma clearance rate” as used herein refers to a quantitative measure of the rate at which a polypeptide is removed from the blood after administration to a living organism. The plasma clearance rate can be calculated as dose / AUC (mL / day / kg), where the AUC value (area under the curve) is determined from the concentration-time curve as in Example 37.

  The term “down-modulation” as used herein is intended to refer to a process that reduces the number of molecules such as antigens or receptors on the cell surface, eg, by binding of an antibody to the receptor. .

  The term “intracellular” as used herein refers to cells in which an antibody or Fc-containing polypeptide is transferred from the cell surface and / or surrounding medium, eg, via endocytosis, to a cell that expresses the target. It is intended to refer to any mechanism that undergoes inward migration. Antibody internalization can be assessed using a direct assay that measures the amount of antibody that has undergone internalization (eg, the lysosomal colocalization assay described in Example 26).

  The term “antibody-drug conjugate” as used herein refers to an antibody or Fc-containing polypeptide having specificity for at least one type of malignant cell, a drug, and a linker that links the drug to, for example, an antibody. Point to. The linker is cleavable or non-cleavable in the presence of malignant cells; antibody-drug conjugates kill malignant cells.

  The term “antibody-drug conjugate uptake” as used herein refers to antibody-drug conjugate binding to a target on a cell followed by uptake / embracing by the cell membrane, resulting in cellular It refers to the process of being drawn in. Incorporation of antibody-drug conjugates can be assessed as “antibody-mediated intracellular translocation and cell killing by anti-TF ADC in an in vitro lethal assay” as described in WO 2011/157741.

  The term “apoptosis” as used herein refers to the process of programmed cell death (PCD) that can occur in a cell. Biochemical events lead to characteristic cell changes (morphology) and cell death. These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation and chromosomal DNA fragmentation. Binding of antibodies to certain receptors can induce apoptosis.

  Fc receptor binding can also be measured indirectly as described in Example 12.

  The term “FcRn” as used herein is intended to refer to the neonatal Fc receptor, which is an Fc receptor. It was first discovered in rodents as a unique receptor capable of transporting IgG from maternal milk through the intestinal epithelium of the new rodent into the neonatal bloodstream. Further research has revealed similar receptors in humans. However, in humans, it has been found to help facilitate the transport of maternal IgG to the developing fetus in the placenta and has also been shown to play a role in monitoring IgG turnover. FcRn binds IgG at an acidic pH of 6.0-6.5 but does not bind at neutral or higher pH. Therefore, FcRn can bind to IgG from the intestinal lumen (inside the intestine) at a somewhat acidic pH, and the basolateral side (inside the body) where the pH is neutral to basic (pH 7.0-7.5) Efficient one-way transportation to can be ensured. This receptor also plays a role in IgG recycling in adults through its presence in the endocytic pathway in endothelial cells. The FcRn receptor in acidic endosomes binds to IgG that has been intracellularly moved by phagocytosis and recycles it to the cell surface, releasing it at the basic pH of blood, thereby undergoing lysosomal degradation prevent. This mechanism may explain the longer IgG half-life in blood compared to other isotypes. Examples 13 and 34 describe assays showing IgG binding to FcRn at pH 6.0 in an ELISA.

  The term “Protein A” as used herein is intended to refer to the 56 kDa MSCRAMM surface protein that was first found in the cell wall of the bacterium Staphylococcus aureus. It is encoded by the spa gene and its regulation is controlled by DNA topology, cell osmotic pressure, and a binary system called ArIS-ArIR. Because of its ability to bind immunoglobulins, it finds use in biochemical research. It is composed of five homologous Ig binding domains that fold into a three-helix bundle. Each domain can bind to a protein from many mammalian species, most notably IgG. It binds to the heavy chain Fc region of most immunoglobulins (which overlaps with the conserved binding site of the FcRn receptor) and also interacts with the Fab region of the human VH3 family. Through these interactions in serum, IgG molecules bind to bacteria not just through their Fab region, but also through their Fc region, which allows the bacteria to opsonize, complement activity Disruption and phagocytosis.

  The term “protein G” as used herein refers to an immunoglobulin-binding protein expressed in group C and group G streptococci that is very similar to protein A, except that it differs in specificity. Intended to point. This is a 65 kDa (G148 protein G) and 58 kDa (C40 protein G) cell surface protein that has been applied to purify antibodies through its binding to the Fc region.

Methods of Affecting CDC of Polypeptides All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody all comprise an immunoglobulin Fc domain and a binding region. It should be understood that this may also apply to other parent peptides, first parent peptides or second parent polypeptides.

  In one aspect, the invention relates to a method of enhancing complement dependent cytotoxicity (CDC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein It relates to a method comprising introducing mutations in one or more amino acid residues selected from the group corresponding to E430X, E345X and S440W in the Fc region of IgG1 heavy chain.

  In one embodiment, the parent polypeptide may be a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.

  Introduction of mutations into a parent polypeptide by the methods or uses of the present invention results in mutant polypeptides (which may also be referred to herein as “variants”). Thus, the methods of the invention can be performed to obtain any variant or variant polypeptide as described herein.

  Variant polypeptides obtained by the methods or uses of the invention have enhanced CDC compared to the parent polypeptide. Typically, the effect of a polypeptide on effector function can be determined by the EC50 value, which is the concentration of polypeptide required to obtain half the value of maximum lysis.

  Maximum lysis is the lysis obtained when a saturating amount of polypeptide is used, where saturation refers to the amount of polypeptide that all targets for the polypeptide are bound by the polypeptide. Intended.

  The terms “enhancing CDC”, “enhancing CDC”, “enhancing effector function” or “improving effector function” are used in the context of the present invention as compared to the parent polypeptide. It means that the EC50 value of the mutant polypeptide is decreased. The decrease in EC50 value may be, for example, at least or about one half, such as at least or about one third, or at least or about one fifth, or at least or about one tenth. Or “enhancing CDC”, “enhancing CDC”, “enhancing effector function” or “improving effector function” causes the parent polypeptide to lyse less than 100% of all cells. Under the conditions, the maximum amount of cells to be lysed (when the total amount of cells is set to 100%), for example from 10% to 100% of the total cells, for example about 10%, about 20%, about 30% , About 40%, about 50%, about 60%, about 70%, about 80%, about 90% and about 100%.

  Mutants were cloned into IgG1-005 or IgG1-7D8 heavy chain variable domains as described for Daudi (Example 6) and Wien (Example 10), and their activity was measured in a CDC assay. By testing, one can test for enhanced or improved effector function. When using IgG1-7D8 HC variable domains and Daudi cells, the enhancement is less than one-half lower than the EC50 of IgG1-7D8 under the test conditions, for example about one-half, about one-third, Defined by low EC50 values (concentration at which 50% lysis is observed) below about 1/5, about 1/10, or 1/10. When using IgG1-005 HC variable domains and Daudi cells, the enhancement is a lower EC50 that is less than one-half that of IgG1-005 under the test conditions, such as about one-half, about one-third, Defined by low EC50 values (concentration at which 50% lysis is observed) below about 1/5, about 1/10, or 1/10. When using IgG1-7D8 HC variable domains and Wien133 cells, the enhancement is a lower EC50 that is less than one-half that of IgG1-7D8 under the test conditions, e.g. about one-half, about one-third, Defined by low EC50 values (concentration at which 50% lysis is observed) below about 1/5, about 1/10, or 1/10. When using IgG1-005 HC variable domains and Wien133 cells, the enhancement ranges from 10% to 100% of total cells, eg, about 10%, about 20%, about 30%, about 40%, Defined by increases of about 50%, about 60%, about 70%, about 80%, about 90% and about 100%. It also enhances the activity of CDC with a low EC50 that is less than one-half that of IgG1-005 under the test conditions, e.g. about one-half, about one-third, about one-fifth, about It can also be defined by a low EC50 value below 1/10 or 1/10 (the concentration at which 50% lysis is observed under conditions where lysis of Wien133 cells is detectable).

  The inventors of the present invention surprisingly found that mutations at these specific positions introduced one or more mutations into the parent antibody according to the methods of the invention (eg, as shown in Example 19). It was found that the CDC of the mutant antibody obtained by doing so has an improved effect. Without being bound by theory, it is believed that substitution of one or more amino acids from the group of positions described above stimulates oligomerization. The antibody binds with higher avidity (illustrated by Example 2; direct labeling of IgG-7D8-E345R resulted in enhanced binding to Daudi cells compared to IgG-7D8-WT) , Which allows the antibody to bind to the cell for a longer period of time, thereby enabling various effector functions such as, for example, C1q binding, C1q activity CDC, ADCC, intracellular translocation, ADCP and / or enhanced in vivo activity . These effects are shown in Example 4 (C1q binding in cells), Example 5 (C1q activity in CDC assay), Examples 6, 7, 27, 28, 29 and 35 (CDC assay), Example 12 (ADCC) , Example 26 (intracellular translocation) and Examples 21 and 22 (in vivo activity), plasma clearance rate (Example 37), FcRn binding (Example 34), and complement in target-independent liquid phase Illustrated by activation (Example 36).

  Therefore, amino acid residues selected from those corresponding to E430X (eg, E430G, E430S, E430F or E430T), E345X (eg, E345K, E345Q, E345R or E345Y), S440Y and S440W in the Fc region of human IgG1 heavy chain Group mutations may also be referred to as “single variant” aspects or “CDC enhancing mutations” in the context of the present invention.

  Thus, in one embodiment, in the method of enhancing CDC, mutations in one or more amino acid residues are E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y in the Fc region of the human IgG1 heavy chain. And selected from the group corresponding to S440W.

  In one preferred embodiment, in the method of enhancing CDC, the mutation at one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

  In one embodiment, the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.

  In another aspect, the present invention also provides a method for enhancing CDC and antibody-dependent cell-mediated cytotoxicity (ADCC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region comprising the parent polypeptide In contrast, the method includes introducing a mutation in one or more amino acid residues corresponding to E430X, E345X and S440W in the Fc region of human IgG1 heavy chain, wherein X is any amino acid, such as a natural amino acid It also relates to the method.

  In one embodiment, the mutation at one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of the human IgG1 heavy chain .

  In one preferred embodiment, the mutation at one or more amino acid residues is selected from the group corresponding to positions E345R, E430T and E430F in the Fc region of the human IgG1 heavy chain.

  In one embodiment, at least one other effector function of the antibody, such as C1q binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc-γ receptor binding, protein A binding, protein G-binding, ADCP, complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, antibody-mediated opsonized antibody binding to complement receptors, antibody-mediated phagocytosis (ADCP), cells Also enhanced are internalization, apoptosis, and / or binding of opsonized antibodies to complement receptors, such as ADCC.

  In one embodiment, the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.

  In one embodiment, the CDC of the parent antibody is enhanced when the parent antibody binds to its antigen on an antigen-expressing cell, cell membrane or virion.

  In one embodiment, the parent antibody is a monospecific antibody, bispecific antibody or multispecific antibody.

In one further aspect, the invention provides a first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and an immunoglobulin second CH2-CH3 region and a second A method for enhancing complement-dependent cytotoxicity (CDC) of a parent antibody, which is a bispecific antibody comprising a second polypeptide comprising an antigen-binding region, wherein the first and second antigen-binding regions Binds to different epitopes on the same or different antigens and corresponds to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain relative to the first and / or second CH2-CH3 region Introducing a mutation in one or more amino acid residues selected from: and wherein the first CH2-CH3 region is K409, T366, L368, K370, D399, F405 in the Fc region of a human IgG1 heavy chain And additional amino acids at positions selected from those corresponding to Y407 The second CH2-CH3 region comprises a further amino acid mutation at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain; And the invention relates to a method wherein the further amino acid mutation in the first CH2-CH3 region is different from the further amino acid mutation in the second CH2-CH3 region.

  In one embodiment, the mutation at one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain Is done.

  In one preferred embodiment, the mutation at one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

  In one embodiment, the method includes introducing a mutation into only one of the first or second polypeptide of the bispecific antibody.

  In one embodiment, the method includes introducing mutations into both the first and second polypeptides of the bispecific antibody.

  In one preferred embodiment, the additional amino acid mutation of the first CH2-CH3 region is at a position corresponding to K409 in the Fc region of the human IgG1 heavy chain, such as K409R; and of the second CH2-CH3 region A further amino acid mutation is at a position corresponding to F405 in the Fc region of the human IgG1 heavy chain, for example F405L.

  The inventors of the present invention have also shown that introduction of mutations to the parent antibody in amino acid residues corresponding to either K439 or S440 in the Fc region of the human IgG1 heavy chain reduces the effector function of the parent antibody. (Examples 5, 6 and 10).

  As shown in Example 6, an amino acid substitution at position K439E or S440K as a “single variant” reduces CDC compared to any of the first mutations according to the method of the invention. I let you.

  The mutant antibody obtained by the above method for reducing the effector function has a reduced effector function compared to the parent antibody. Typically, the effect of an antibody on effector function can be measured by the EC50 value, which is the concentration of antibody required to obtain half maximal lysis.

  Maximum lysis is the lysis obtained when using a saturating amount of antibody, where saturating is intended to refer to the amount of antibody that all antigens to the antibody are bound by the antibody. .

  The term “reducing effector function” refers in the context of the present invention to an increase in the EC50 value of a variant antibody compared to the parent antibody. The increase in EC50 value may be, for example, at least or about 2-fold, such as at least or about 3-fold, or at least or about 5-fold, or at least or about 10-fold. Alternatively, “decreasing effector function” refers to the maximum amount of cells that are lysed under conditions where the parent antibody lyses less than 100% of all cells, eg, from 10% to 100% of all cells, For example, it also means that there is a decrease of about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and about 100%.

  Mutants were cloned as described for Daudi cells (Example 6) and Wien133 cells (Example 10) by cloning IgG1-005 or IgG1-7D8 heavy chain variable domains into their CDC activity. By testing in an assay, one can test for reduced effector function. When using IgG1-7D8 HC variable domains and Daudi cells, the decrease is less than one-half lower than the EC50 of IgG1-7D8 under the test conditions, for example about one-half, about one-third, Defined by low EC50 values (concentration at which 50% lysis is observed) below about 1/5, about 1/10, or 1/10. When using IgG1-005 HC variable domains and Daudi cells, the decrease is less than one-half lower than the EC50 of IgG1-005 under the test conditions, for example about one-half, about one-third, Defined by low EC50 values (concentration at which 50% lysis is observed) below about 1/5, about 1/10, or 1/10. When using IgG1-7D8 HC variable domains and Wien133 cells, the decrease is less than one-half lower than the EC50 of IgG1-7D8 under the test conditions, for example about one-half, about one-third, Defined by low EC50 values (concentration at which 50% lysis is observed) below about 1/5, about 1/10, or 1/10. When using IgG1-005 HC variable domains and Wien133 cells, the reduction ranges from 10% to 100% of total cells, eg, about 10%, about 20%, about 30%, about 40%, Defined by increases of about 50%, about 60%, about 70%, about 80%, about 90% and about 100%. Also, the decrease in CDC activity is lower than the EC50 of IgG1-005 under the test conditions by a lower EC50, such as about 1/2, about 1/3, about 1/5, about It can also be defined by a low EC50 value below 1/10 or 1/10 (the concentration at which 50% lysis is observed under conditions where lysis of Wien133 cells is detectable).

In one further aspect, the present invention is a method according to the present invention, as in the embodiments disclosed herein, comprising introducing mutations at one or more positions other than S440Y and S440W, and i) a mutation in each of the amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is neither S440Y nor S440W,
(Ii) mutations in each of the amino acid residues corresponding to K447 and 448 in the Fc region of the human IgG1 heavy chain, such as K447K / R / H and 448E / D, preferably K447K and 448E in the Fc region of the human IgG1 heavy chain, Or (iii) mutations in each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of the human IgG1 heavy chain, for example K447D / E, 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain, preferably Are K447E, 448K and 449P in the Fc region of human IgG1 heavy chain
The method further includes the step of introducing.

  Regarding embodiments in which further mutations are introduced as described in steps (ii) or (iii) above, it is noted that the lysine at position K447 is cleaved during antibody production in cells under normal circumstances There is a need. This can be prevented by protecting position K447 by adding one or more additional amino acid residues (such as 448 or 448/449). This is further described in WO 2013/004841 (Genmab A / S).

  In one embodiment, the method includes introducing mutations other than S440Y and S440W at one or more positions, and each of the amino acid residues corresponding to K439 and / or S440 in the Fc region of the human IgG1 heavy chain. Further introducing a mutation, provided that the mutation at S440 is not S440Y or S440W.

  In one preferred embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R It is.

  In one embodiment, the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.

  In one embodiment, the parent antibody is a monospecific antibody, bispecific antibody or multispecific antibody. Bispecific antibodies can be any one of the embodiments described herein.

  In addition, any of the mutations listed in Table 1 can be introduced into the bispecific antibody. Example 24 shows that the introduction of the E345R mutation to the bispecific CD20 × EGFR antibody enhances the activity of CDC. Examples 23, 29 and 30 also describe some of the various bispecific antibodies containing the mutations of the invention.

  Introduction of mutations to both amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain in the parent antibody, provided that the mutation in S440 is neither S440Y nor S440W Also referred to as the “heavy mutant” aspect. S440Y and S440W mutations were found to enhance CDC when introduced into the parent polypeptide, as described elsewhere.

  As also described elsewhere, the inventors of the present invention have shown that the introduction of the identified mutation into an amino acid residue corresponding to either K439 or S440 in the Fc region of the human IgG1 heavy chain is an effector function. (Examples 5, 6, and 10). However, when inhibitory mutations were introduced into both the amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, the effector function was restored, resulting in mutations at the K439 and S440 mutations. It is similar to the effector function of the parent antibody that does not have it. However, the presence of K439 and S440 mutations, although not bound by any theory, is thought to limit the induction of effector function on oligomeric complexes that correspond only to antibodies containing both K439 and S440 mutations. Thus, while the K439 and S440 mutations are included in therapeutic antibodies, and not bound by any theory, when such therapeutic antibodies are administered to a patient, induction of effector function is caused by the K439 / S440 mutation. Limited to oligomeric antibody conjugates that contain therapeutic antibodies, but not the K439 and S440 mutations, and do not contain the K439 and S440 mutations, which may be caused by the interaction of the therapeutic antibody with the patient's own antibody Certain side effects may be limited.

  Combining the mutation at position K439 and / or S440 with the first mutation results in enhanced CDC and increased CDC specificity. Similarly, CDC enhancement and specificity enhancement can also be obtained by introducing the mutations disclosed in embodiments (ii) and (iii) above.

  In another aspect, the present invention provides a method for enhancing complement dependent cytotoxicity (CDC) of a combination of at least a first and a second parent polypeptide, comprising at least a first and a second parent polypeptide. Each comprising an immunoglobulin Fc domain and a binding region, at least for the first and / or second parent polypeptide, from a group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain It relates to a method comprising introducing mutations in one or more selected amino acid residues.

  In one embodiment, the method comprises at least the first and / or second parent polypeptide E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y in the Fc region of the human IgG1 heavy chain. And introducing mutations in one or more amino acid residues selected from the group corresponding to S440W.

  In one preferred embodiment, the method is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain at least for the first and / or second parent polypeptide 1 Introducing a mutation in one or more amino acid residues.

  In one embodiment, the method comprises introducing a mutation that may be the same or different for both the first and second parent polypeptides.

In one further embodiment, the method comprises:
(I) one selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain relative to the first parent polypeptide Or introducing mutations in multiple amino acid residues,
(Ii) including mutations in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain Providing a second parent polypeptide.

  In one embodiment, the method comprises, relative to the first parent polypeptide, one or more amino acid residues selected from the group corresponding to E430G, E430S, E345K or E345Q in the Fc region of a human IgG1 heavy chain Introducing a mutation in

In one further embodiment, the mutation at one or more positions is separate from S440Y and S440W, and the method comprises:
(I) introducing a second mutation into the amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain relative to the first parent polypeptide; and (ii) into the second parent polypeptide In contrast, a step of introducing a second mutation into an amino acid residue corresponding to S440 in the Fc region of human IgG1 heavy chain, provided that the mutation is not S440Y or S440W; Or alternatively (i) and (ii)
(Iii) a step of introducing a second mutation into the amino acid residue corresponding to position S440 in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide, provided that the mutation is S440Y or S440W A stage subject to absence;
(Iv) introducing a second mutation into the amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain with respect to the second parent polypeptide.

  The second parent polypeptide can be any parent polypeptide that by itself does not result in a sufficient CDC response after binding to the target cell.

  Thus, without being bound by theory, providing a first variant polypeptide comprising a mutation in one or more amino acid residues according to the above list and thus having an enhanced CDC response, and It is believed that the above method of providing a second mutant polypeptide that does not contain such a mutation will result in a CDC response of the second parent polypeptide.

  A method of combining a first antibody comprising one of the aforementioned mutations capable of enhancing CDC with a second antibody that has not undergone modification according to the present invention was combined as shown in Example 31. Brings the enhancement of CDC by things. Thus, the method comprises, in one embodiment, a therapeutic antibody as a second antibody that has been demonstrated to be safe but not sufficiently efficient (or where increased efficiency is desirable) comprising a mutation, As a result, it can be used to combine with a first antibody that results in a combination that is effective.

  A suitable second antibody that does not contain mutations in amino acid residues selected from those corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain Examples of include, but are not limited to, any of the following: (90Y) crivutuzumab tetraxetane; (90Y) Takatuzumab tetraxetane; (99mTc) fanoresomab; (99mTc) nofetumo (99mTc) Pintumomab; 3F8; 8H9; Avagobomab; Abatacept; Absuximab; Actuxumab; Adalimumab; Adektumumab; Aferimomab; Alzumomab pentetate; Albirceptos dotox; Amatsu Shimabumab; AMG714 / HuMax-IL15; Anatumomabumafenatox; Anlucinzumab (= IMA-638); Apolizumab; Arcitsumumab; Acetizumab; Belatizumab; benlizumab betinumab; benlizumab betinumab; benlizumab Tanshin; Kantzuzumab tanshin; Caprazizumab zumab; Capromab; Capromabu pende Chid; Carlumumab; Katumaxomab; CC49; Cedelizumab; Sertolizumab Pegor; Cetuximab; Ch.14.18; Citazumab Bogatox; Dacetuzumab; daclizumab; darurizumab; edremomab; Rimomab Pegor; Enokizumab; Encituximab; Epitumomab; Epitumomab Shixetane; Fuzutumab; felitzumab; fetizumab; fetizumab; Ganitumab; gantenerumab; gabilimomab; gemtuzumab; gemtuzumab ozogamicin; geboxizumab; girentuximab; Anti-cMet antibodies as described above; anti-Her2 antibodies as disclosed in WO 2011/147986 or WO 2011/147982; WO 2004/058 Anti-IL8 antibody as disclosed in 797; anti-TAC antibody as disclosed in WO 2004/045512; anti-tissue factor as disclosed in WO 2010/066803 or WO 2011/157741 (TF) antibody; ivalizumab; ibritumomab tiuxetane; iculumab; Irituzumab; ixekizumab; keliximab; levetizumab; lebrikizumab; remaresomab; renercept; Muburisumumab; Metolisumumab; Metolizumab; Metolizumab; Neshitsumumabu; Nererimomabu; nimotuzumab; nivolumab; Nofetsumomabu; Merupentan; Obinitsuzumabu; Okaratsuzumabu; ocrelizumab; Odzurimomabu; ofatumumab; Oraratsumabu; Orokizumabu; omalizumab; Onarutsuzumabu; onercept; opportunistic Ruth's Mabu Mona Tok scan; oregovomab; Oterikishizumabu; Okiserumabu; Ozorarizumabu; Pagibakishimabu; palivizumab Panitumuma Panovazumab; paclizumab; paclitumumab; paclitumumab; patrizumab; petizumab; petizumab; petuzumab; HuMax-IGF1R; RG1512 / HuMax-p selectin; rilonacept; rirotumumab; rituximab; lovatumumab; loretuzumab; romozumab; Siplizumab; silk mab; solanezumab Sonetizumab; Sontuzumab; Sontuzumab; Sotatercept; Stamtelumab; TGN1412; ticilimumab (= tremelimumab); tigatuzumab; tretanizumab; tretanizumab; Ubrutuximab; Ulerumab; Urtoxazumab; Ustekinimab; Vapariximab; Tetlizumab; vedolizumab; veltuzumab; bepalimomab; besencumab; bicilizumab; borociximab;

  As shown in Example 10, the first and second variant antibodies are believed to have a preference for mutual oligomerization compared to any wild-type or native antibody.

  In one embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R. is there.

  Therefore, the enhancement of specificity relates to “induction of CDC”. Accordingly, the method is, in one embodiment, a method for increasing the specificity of induction of effector function by a combination of at least a first and a second parent polypeptide.

  A combination of the first variant and the second variant polypeptide by at least a combination of the first and second parent polypeptides by performing a method of increasing the specificity of the effector function or the induction of the effector function Is obtained.

  By introducing mutations into either the parent polypeptide, K439 or S440, the resulting mutant polypeptide has reduced effector function compared to the parent polypeptide. However, as also described elsewhere herein, mutations in K439 and S440 can complement each other or restore the effector function of a polypeptide containing both mutations. This ability of mutations in K439 and S440 to complement each other can be exploited in the two polypeptides as well. Thus, when the mutation at K439 is introduced into the first parent polypeptide and the mutation at S440 is introduced into the second parent polypeptide, or vice versa, the first and second mutant polypeptides are When used in combination, a decrease in effector function is no longer observed. The terms “enhancing specificity” or “enhancing specificity” in this context refer to a first mutant polypeptide comprising a mutation at K439 and a second mutant polypeptide comprising a mutation at S440. The effector response induced by the combination of is higher than the effector response induced by either the mutant polypeptide containing the first mutation at K439 or the second mutant polypeptide containing the mutation at S440 Point to.

  The introduction of both amino acid substitutions at K439 and S440 increases the specificity of oligomer formation.

  Combining the mutation at position K439 and / or S440 with the first mutation results in enhanced CDC and increased CDC specificity.

  In one embodiment, at least the first and second parent polypeptides bind to the same binding site or, for antibodies, bind to the same epitope.

  In one embodiment, at least the first and second parent polypeptides bind to different binding sites on the same target or, for antibodies, bind to different epitopes on the same antigen.

  In one embodiment, at least the first and second parent polypeptides bind to different epitopes on different targets.

  In one embodiment, the first and second parent polypeptides are first and second parent antibodies and have the same or different VL and VH sequences.

  In one embodiment, the combination of at least the first and second parent polypeptides comprises one first parent polypeptide and one second polypeptide.

  In one embodiment, the specificity is increased when the combination of the first and second parent polypeptides binds to its binding site or antigen on an antigen-expressing cell, cell membrane or virion.

Therefore, in another aspect, the invention also provides for the use of a mutation in two or more amino acid residues of a polypeptide when bound to that antigen on an antigen-expressing cell, cell membrane or virion. Of the polypeptide (eg, the specificity of CDC induced by the polypeptide), wherein the first mutation is an amino acid corresponding to K439 in the Fc region of the human IgG1 heavy chain In the residue;
The second mutation is in the amino acid residue corresponding to S440 in the Fc region of human IgG1 heavy chain.

  In one embodiment, the first and second parent polypeptides are first and second parent antibodies, each comprising an immunoglobulin Fc domain and an antigen binding region.

  In one embodiment, the first and second parent antibodies are monospecific antibodies, bispecific antibodies or multispecific antibodies.

  In one embodiment, the first and / or second parent antibody comprises a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin and a second CH2-CH3 region. A bispecific antibody comprising a second polypeptide comprising a second antigen-binding region, wherein the first and second antigen-binding regions bind to different epitopes on the same antigen or on different antigens, The first CH2-CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; The -CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain, and the first CH2-CH3 Additional amino acid mutations in the region may result in additional amino acids in the second CH2-CH3 region. Different from the acid mutations.

  In one preferred embodiment, the first CH2-CH3 region comprises an additional amino acid mutation (eg, K409R) at a position corresponding to K409 in the Fc region of the human IgG1 heavy chain; and the second CH2-CH3 region is Additional amino acid mutations, such as F405L, are included at positions corresponding to F405 in the Fc region of the human IgG1 heavy chain.

  By performing this method, a combination of at least the first and second mutant antibodies is obtained. At least the first and second variant antibodies obtained by this method have enhanced CDC when combined as compared to the combination of the first and second parent antibodies.

  The term “enhanced CDC” shall be construed as described below.

  The first and / or second parent antibody may be any parent antibody as described herein.

  A method of enhancing the CDC of a combination of a first and second antibody specifically includes first and / or second variant antibodies having any of the features of variant antibodies as described herein. Can be done to get.

  In one embodiment, at least the first and second parent antibody bind to the same epitope.

  In one embodiment, at least the first and second parent antibody bind to different epitopes on the same antigen.

  In one embodiment, at least the first and second parent antibodies bind to different epitopes on different targets.

  In one embodiment, the first and second parent antibodies have the same or different VL and VH sequences.

  In one embodiment, the combination of at least the first and second parent antibodies comprises one first parent antibody and one second antibody.

  In one embodiment, the combination of at least a first and second parent antibody comprises an additional parent antibody, such as a third, fourth or fifth parent antibody.

  In one embodiment, the first and second bispecific or multispecific parent antibodies are the same antibody or different antibodies. In one embodiment, the first and second bispecific or multispecific parent antibodies bind to different epitopes on the same or different antigens. Thus, in one embodiment, the at least first and second parent antibodies are bispecific or multispecific antibodies that bind to different epitopes on the same antigen or on different antigens.

  In one embodiment of the methods and / or uses of the present invention, the parent antibody affects effector function, whether it is a parent antibody, a first parent antibody or a second parent antibody. May contain other mutations other than the mutation of the present invention. Such other mutations may be introduced at the same time as the mutations of the invention that affect effector function, or they may be introduced sequentially, and the method or use of the invention may involve simultaneous introduction of mutations. It is not limited to any one of sequential introduction. The bispecific antibody can be any bispecific antibody since it is envisaged that various formats can be used, and the methods and uses of the present invention can be applied to any specific bispecific format. It is not limited.

  In one embodiment, the method does not alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the parent polypeptide or antibody.

  In one embodiment, the method does not alter the binding of the parent polypeptide or antibody to the neonatal Fc receptor (FcRn) as determined by the method disclosed in Example 34.

  In one embodiment, the method comprises binding of the parent polypeptide or parent antibody to the neonatal Fc receptor (FcRn) as measured by the change in absorbance at OD405 nm as determined by the method disclosed in Example 34. It does not increase or decrease more than 30%, for example more than 20%, 10% or 5%.

  In one embodiment, the method does not enhance the apparent affinity of the parent polypeptide or antibody to the mouse neonatal Fc receptor (FcRn) as determined by the method disclosed in Example 34 by more than 0.5 fold. Or does not reduce the apparent affinity of the parent polypeptide or antibody to mouse FcRn by more than 2-fold.

  In one embodiment, the method does not alter the plasma clearance rate of the parent polypeptide or antibody as determined by the method disclosed in Example 37.

  In one embodiment, the method provides a plasma clearance rate of the parent polypeptide or antibody determined by the method disclosed in Example 37 that is greater than 3.0 times, such as 2.5 times, 2.0 times, 1.5 times or 1.2 times. It does not increase or decrease more than twice.

  In one embodiment, the method does not alter complement activation in the target-independent fluid phase of the mutant as determined by the method disclosed in Example 36.

  In one embodiment, the method does not alter the plasma half-life of the parent polypeptide or antibody.

  Any of the mutations described herein or combinations thereof can be introduced according to the methods of the invention.

  Mutations selected from exemplary or preferred amino acid substitutions include oligomerization of antigen-bound antibodies, such as those described in the Examples, and C1q binding, complement activation, CDC, ADCC and / or cells It can be tested in a suitable assay that makes it possible to detect enhanced internalization. For example, C1q binding ability can be determined according to an assay similar to that described in Example 4 using cells expressing an antigen against the antibody variant. Exemplary CDC assays are presented in Examples 5, 6, 10, 16, 19, 22, 23, 24, 25, and 35. An exemplary ADCC assay is presented in Example 12. An exemplary intracellular translocation assay is presented in Example 26. Furthermore, in order to distinguish mutations in amino acid residues directly involved in C1q binding from mutations affecting oligomer formation, for example, C1q binding in an ELISA assay according to Example 3 is described in Example 4, for example. Compared to C1q binding in the cell-based assay followed, compared to plasma clearance rate according to the assay described in Example 37, compared to FcRn binding according to Example 34, and in a target-independent liquid phase Complement activation may be assessed according to the assay in Example 36.

  In one embodiment, the mutation at one or more amino acid residues may be an amino acid substitution, amino acid deletion or amino acid insertion.

  In one embodiment, the mutation at one or more amino acid residues is an amino acid deletion.

  In one embodiment, the mutation at one or more amino acid residues is an amino acid insertion.

  In one particular embodiment, the mutation at one or more amino acid residues is an amino acid substitution.

  In one embodiment, the mutation at one or more amino acid residues can be selected from any of the amino acid substitutions, amino acid deletions listed in Table 1.

  Thus, in one embodiment, E345X is E345R, Q, N, K, Y, A, C, D, F, G, H, I, L, M, P, S, T, V, W or Y; In particular E345A, D, G, H, K, N, Q, R, S, T, Y or W; or more particularly E345D, K, N, Q, R or W; or even more particularly E345R, Can be Q, N, K or Y. In a further preferred embodiment, E345X is E345K or E345Q.

  In another further embodiment, E430X is E430T, S, G, F, H, A, C, D, I, K, L, M, N, P, Q, R, V, W or Y; in particular E430T, It can be S, G, F or H. In a further preferred embodiment, E430X is E430G or E430S. In another embodiment, the mutation is optionally in C1q binding, as determined by comparing C1q binding in the ELISA assay according to Example 3 to C1q binding in the cell-based assay according to Example 4. There are no amino acid residues directly involved.

  In one embodiment, the one or more mutations are one mutation, i.e., no more than one mutation is introduced into the parent antibody.

  In another embodiment, the method or use of the invention introduces mutations into at least two of the amino acid residues in Table 1, such as 2, 3, 4, 5 or more amino acid residues. including.

  Any of the combinations of mutations described herein can be introduced according to the methods of the present invention.

  In one embodiment, the method comprises a plurality of mutations in an amino acid residue selected from the group corresponding to E345X, E430X, S440Y and S440W in the Fc region of a human IgG1 heavy chain relative to the parent polypeptide, for example two Introducing three, four or five, especially two or three mutations. For example, at least two or more amino acid residues corresponding to E345X, E430X, S440Y and S440W in the Fc region of human IgG1 heavy chain, for example, two or all of E345X, E430X, S440Y and S440W, optionally, Mutations can be combined with mutations in one or more other amino acids listed in Table 1. The at least two mutations may combine any amino acid residue substitution at position E345 with any amino acid residue substitution at position E430 or S440Y or S440W, or any amino acid substitution at position E430 at position S440Y or It may be combined with any amino acid residue of S440W. In one further embodiment, two or three mutations are introduced into the amino acid residues selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain relative to the parent antibody. The

  Such a combination of two mutations at amino acid residues is selected from the group corresponding to E345X / E430X, E345X / S440Y, E345X / S440W, E430X / S440Y and E430X / S440W in the Fc region of the human IgG1 heavy chain.

  In the method or application according to the invention, CDC is enhanced when the antibody binds to its antigen.

  While not being bound by any theory, CDC is believed to be enhanced when an antibody binds its antigen, where the antigen is on an antigen-expressing cell, on the cell membrane, or on a virion. In one embodiment, the Fc region of an IgG1 heavy chain comprises the sequence of residues 130-330 of SEQ ID NO: 1.

  The parent polypeptide or parent antibody may be any parent polypeptide or any parent antibody as described herein. Parent polypeptide and parent antibody in this context are also intended to be a first parent peptide and a second parent polypeptide and a first parent antibody and a second parent antibody.

  In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

  In one embodiment, the parent antibody is a human full length antibody, such as a human full length IgG1 antibody.

  In one embodiment, the parent antibody, the first parent antibody and the second parent antibody are human IgG1 antibodies, such as IgG1m (za) or IgG1m (f) allotypes, optionally comprising SEQ ID NO: 1 or 5 Includes Fc region.

  In one embodiment, the parent antibody is a human IgG2 antibody and optionally comprises an Fc region comprising SEQ ID NO: 2.

  In one embodiment, the parent antibody is a human IgG3 antibody and optionally comprises an Fc region comprising SEQ ID NO: 3.

  In one embodiment, the parent antibody is a human IgG4 antibody and optionally comprises an Fc region comprising SEQ ID NO: 4.

  In one embodiment, the parent antibody is a bispecific antibody.

In one embodiment, the parent antibody is any antibody as described herein, for example, an antibody fragment comprising at least a portion of the Fc region, a monovalent antibody (described in WO007059782 by Genmab) A heavy chain antibody consisting only of two heavy chains, eg naturally occurring in camelids (eg Hamers-Casterman (1993) Nature 363: 446); ThioMab (Roche, WO2011069104), asymmetric duplex Specific antibody-like molecules, chain exchange recombination domains (SEED or Seed-body) (Merck, WO2007110205); Triomab (Fresenius, Lindhofer et al. (1995 J Immunol 155: 219); FcΔAdp (Regeneron, WO2010151792) , Azymetric Scaffold (Zymeworks / Merck, WO2012 / 058768), mAb-Fv (Xencor, WO2011 / 028952), dual variable domain immunoglobulin (Abbott, DVD-Ig, US Pat. No. 7,612,181); Antibody (Unilever; Sanofi Aventis, WO20100226923), Jida Iabody (ImClone / Eli Lilly), Knob into hall antibody format (Genentech, WO9850431); Duobody (Genmab, WO 2011/131746); Electrostatic steering antibody format (Amgen, EP1870459 and WO 2009089004; Chugai , US201000155133; Oncomed, WO 2010129304A2); bispecific IgG1 and IgG2 (Rinat neurosciences Corporation, WO11143545), CrossMAb (Roche, WO2011117329), LUZ-Y (Genentech), Biclonic (Merus), dual target Directed domain antibody (GSK / Domantis), two-in-one antibody that recognizes two targets (Genentech, NovImmune), cross-linked Mab (Karmanos Cancer Center), CovX-body (CovX / Pfizer), IgG-like bispecific Sex (ImClone / Eli Lilly, Shen, X, et al. J Immunol Methods, 2007. 318 (1-2): p. 65-74), as well as DIG-body and PIG-body (Pharmabcine), and dual affinity Retargeting molecules (Fc-DART or Ig-DART, Macrogenic s approach using WO / 2008/157379, WO / 2010/080538), Zybody (Zyngenia), common light chain (Crucell / Merus, US7262028) or common heavy chain (κλbody according to NovImmune), Is a fusion protein such as a scFv fusion containing a polypeptide sequence fused to an antibody fragment containing an Fc domain, such as BsAb by ZymoGenetics / BMS), HERCULES by Biogen Idec (US007951918), Emergent SCORPION by BioSolutions / Trubion, Ts2Ab (MedImmune / AZ (Dimasi, N., et al. J Mol Biol, 2009. 393 (3): p. 672-92), scFv fusion by Novartis, scFv by Changzhou Adam Biotech Inc There are fusions (CN 102250246), TvAbs by Roche (WO 2012025525, WO 2012025530), mAb ² by f-Star (WO2008 / 003116) and double scFv fusions. Unless otherwise specified, the term antibody is produced by polyclonal antibodies, monoclonal antibodies (such as human monoclonal antibodies), antibody mixtures (recombinant polyclonal bodies), for example, techniques developed by Symphonogen and Merus (Oligoclonics). As well as antibody-like polypeptides, such as chimeric antibodies and humanized antibodies. The antibody produced can potentially have any isotype.

  In another embodiment, the antigen is expressed on the surface of the cell.

  In another embodiment, the cell is a human tumor cell.

  In one further embodiment, the antigen is erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, It is selected from the group consisting of Bigh3, SPARC, BCR, CD79, CD37, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3.

  In another embodiment, the antigen is bound to the cell membrane.

  In another embodiment, the antigen is bound to a virion, and optionally the antigen is contained within the protein coat or lipid envelope of the virion.

  In another embodiment, the antibody is a human antibody and optionally binds to at least one antigen selected from CD20 and CD38.

  In another embodiment, the antibody binds to the same epitope as at least one of 7D8 and 005, and optionally comprises at least one variable heavy and / or variable light chain region of 7D8 and 005.

  In any use of the disclosed invention, the antibody without any mutation of the invention may be any parent antibody. Accordingly, use herein results in any variant of such parent antibody.

  In one embodiment, the effector function is Fc receptor binding, including for example Fc-γ receptor binding.

  In one embodiment, the effector function is intracellular translocation of the Fc-containing polypeptide.

  In one embodiment, the effector function is a combination of complement dependent cytotoxicity (CDC) and antibody dependent cell mediated cytotoxicity (ADCC).

  As used herein, the term “C1q binding” refers to variants to a host tissue or factor, including various cells of the immune system (such as effector cells) when used in the context of a parent antibody variant or antibody. Or includes any mechanism of the first component to the classical pathway of complement activation mediated by antibody binding. C1q binding of the antibody can be assessed using an ELISA (eg, the C1q binding ELISA used in Examples 3 and 4), or C1q activity is assessed using a CDC assay (eg, the CDC used in Example 5). Assay). In one further embodiment, the C1q binding strength of the antibody is determined according to the assay described in Example 4.

  In all disclosed methods of the invention, the antibody without any mutation of the invention may be any parent antibody. Accordingly, the methods herein result in any variant of such parent antibody.

  The parent antibody, first parent antibody, second parent antibody or variant thereof obtained by the methods and / or uses of the present invention may bind to any target as described herein.

  Examples of antigens or targets that can be addressed by the present invention include: 5T4; ADAM-10; ADAM-12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CCR11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD6; CD15; CD18; CD19; CD20; CD22; CD25; CD29; CD37; CD38; CD40; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD74; CD79a; CD79b; CD80; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; claudin 4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FcγRI; FCER2; FGFR3; fibrin II β chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; GDF2; GLP1R; glypican-3; GPNMB; HBV (hepatitis B virus); HCMV (human cytomegalovirus); Heat shock Protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1 IIIB gp120 V3 loop; HLA-DRB (HLA-DRβ); human respiratory syncytial virus; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE junction region; IL12B; IL13; IL15; IL17A; IL1A; IL1RA; IL4; IL5; IL5RA; IL6; IL6R; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; Lewis-y; lipid A, lipopolysaccharide LPS domain; LTA; MET; MMP14; MMp15; MST1R MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin 4; NGF; NRP; NY-ESO-1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine PTK-7; Pseudomonas aeruginosa serotype IATS O11; RSV (human respiratory syncytial virus, sugar tongue) ROR1; RTN4; SELL; SELP; STEAP1; Shiga toxin-like toxin II B subunit [Escherichia coli]; SLAM7; SLC44A4; SOST; Staphylococcus epidermidis lipoteichoic acid; T cell reception TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2; TYRP1; VAP-1; and vimentin.

In a principal aspect, the present invention is a method for inducing CDC on a cell, cell membrane or virion expressing a target to which a parent polypeptide comprising an immunoglobulin Fc domain and a binding region binds, comprising:
(I) providing one parent polypeptide or a combination of at least a first parent polypeptide and a second parent polypeptide mutated according to any one of the embodiments disclosed herein; ii) preparing a mutated parent polypeptide of step (i) or a combination of at least a first parent polypeptide and a second parent polypeptide mutated of step (i) of human complement or effector cells; In the presence, it relates to a method comprising the step of contacting with an antigen-expressing cell, cell membrane or virion.

  In one embodiment, any or all of the parent polypeptide, the first parent polypeptide and the second parent polypeptide may be an antibody.

  In another embodiment, the method is mediated by ADCC, Fc-γ receptor binding, protein A binding, protein G binding, ADCP, complement dependent cytotoxicity (CDCC), complement enhanced cytotoxicity, antibody Enhances one additional effector function selected from the binding of the opsonized antibody to the complement receptor and any combination thereof.

  In further embodiments, the present invention also induces antibody-dependent cell-mediated cytotoxicity (ADCC).

  In still further embodiments, the present invention also induces intracellular translocation of Fc-containing polypeptides.

  In one embodiment, the cell is a human tumor cell or a bacterial cell.

  In another embodiment, the IgG1 parent antibody is a human IgG1 antibody.

  In another embodiment, the first and second antigens are erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, It is independently selected from the group consisting of CXCR5, c-Met, HERV-envelope protein, periostin, Big3, SPARC, BCR, CD79, EGFrvIII, IGFr, L1-CAM, AXL, tissue factor (TF), EpCAM and MRP3.

  In another embodiment, the first and second parent antibodies are fully human antibodies, and optionally the first and second parent antibodies bind to an antigen that is separately selected from CD20 and CD38.

  In one further embodiment, the first and second parent antibodies are separately selected from 7D8 and 005.

  In one further embodiment, the cell is a bacterial cell.

  In another embodiment, the bacterial cell is S. aureus, S. Epidermidis, S. pneumonia, Bacillus anthracis, Pseudomonas aeruginosa , Chlamydia, E. coli, Salmonella, Shigella, Yersinia, S. typhimurium, Neisseria meningitides and Mycobacterium tuberculosis).

  In another embodiment, the first and / or second antigen is lipoteichoic acid (LTA), and optionally at least one of the first and second parent antibodies is pagibaximab.

  In another embodiment, the antigen is expressed on virions.

  In another embodiment, the first and second antibodies bind to the same antigen.

  In another embodiment, the first and second antibodies comprise the same VH sequence, VL sequence, or both VH and VL sequences.

  For the purposes of the present invention, a target cell that expresses or otherwise entails an antigen can be any prokaryotic or eukaryotic cell. Exemplary antigen-expressing cells include, but are not limited to, mammalian cells, particularly human cells such as human cancer cells; and unicellular organisms such as bacteria, protozoa and unicellular fungi such as yeast cells. Cell membranes that contain or otherwise accompany antigens include partially and / or disrupted cell membranes derived from antigen-expressing cells. Antigens that bind to virion particles or virus particles may be contained within or otherwise bound within the virion protein coat and / or lipid envelope.

  The target cell can be, for example, a human tumor cell. Suitable tumor antigens include any target or antigen described herein, but erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD25, CD32, CD37, CD38, CD74, CD138, CXCR5, c-Met, HERV-envelope protein, periostin, Big3, SPARC, BCR, CD79, EGFrvIII, IGFR, L1-CAM, AXL, tissue factor (TF), EpCAM and MRP3 It is not limited to. Preferred antigens include CD20, CD38, HER2, EGFR, IGFR, CD25, CD74 and CD32. Exemplary antibodies include anti-CD20 antibody 7D8 as disclosed in WO 2004/035607, anti-CD38 antibody 005 as disclosed in WO 06/099875, disclosed in WO 2004/035607. Anti-CD20 antibody 11B8, anti-CD38 antibody 003 as disclosed in WO 06/099875, anti-EGFr antibody 2F8 as disclosed in WO 02/100348. Other detailed antibody examples are presented herein.

  Or target cells are bacterial cells such as Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia, Escherichia coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, Meningococcus It may be. An exemplary antigen includes lipoteichoic acid (LTA), and an exemplary antibody includes pagibaximab.

  Or the target is West Nile virus, dengue virus, hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papilloma virus, Epstein-Barr virus, herpes virus, pox virus, avian influenza virus, RVS, Aspergillus ), Viruses such as Candida albicans, Cryptococcus and Histoplasma, or on the surface of fungal cells or other particles.

  In one embodiment, the contacting step (ii) is performed in vitro.

  In one embodiment, the contacting step (ii) is performed in vivo.

  In another embodiment, step (ii) comprises administering the variant to the subject.

  In one further embodiment, the subject is suffering from a cancer, bacterial infection or viral infection. The contacting step (ii) of the embodiment described above can be performed in vitro or in vivo. In the latter case, step (ii) may further comprise administering one or more preparations to the subject, optionally to a subject suffering from a cancer or bacterial infection. Further details regarding therapeutic applications are presented below.

  The first and second antibodies comprise an antigen binding region that can bind to the same epitope or can bind to different epitopes. Such epitopes may be on the same target or on different targets.

  In one embodiment, the first and second antibodies bind to different epitopes on different targets. Such targets may be expressed on the same cell or cell type, or may be expressed on different cells or cell types. In one such embodiment, the enhanced effector function is directed only to cells or cell types that express both targets, thereby reducing the risk of incidental damage to cells or cell types that are not the cause of the disease being treated. Reduce.

  Without being bound by any theory, the first and second antibodies bind to an epitope found on the same cell, thereby utilizing the complex expression of the target to improve enhanced selectivity of CDC induction If possible, enhancement of CDC could be limited to target cells that simultaneously express two specific targets / antigens.

  If the target is expressed on different cells or cell types, but not bound by theory, administration of the first and second antibodies in any order improves CDC enhancement, and The “mobilization” of the second cell or cell type that expresses the second target will probably also improve other effector functions.

  In one embodiment using a combination of the first and second antibodies, step (ii) comprises in the presence of human complement and / or effector cells, the cells are mutated to the first and second parent antibodies. It may be done by contacting them separately or sequentially.

  The present invention also provides a method of inducing CDC or other effector responses (eg ADCC) to target cells, cell membranes, virions or other particles to which an antigen to which an IgG1 or IgG3 antibody binds binds, comprising: Providing a variant of an antibody comprising a mutation in K439, which is K439E in the Fc region, and a mutation in S440, which is S440K or S440R; and (ii) a preparation of the variant is prepared for human complement and / or effector cells. Contacting with a cell in the presence is also provided.

  The present invention also provides a CDC or other effector response (eg, ADCC) to a target cell, cell membrane or virion that expresses a first antigen to which the first IgG1 antibody binds and a second antigen to which the second antibody binds. (I) providing a first variant that is a first antibody comprising a K439E mutation and a second variant that is a second antibody comprising a S440K or S440R mutation; and ii) contacting the cell with the first and second variant preparations simultaneously, separately or sequentially in the presence of human complement or effector cells.

  In separate and specific embodiments, the first and second antibodies comprise (i) different antigens; (ii) different epitopes on the same antigen, (iii) the same epitope on one antigen, and (iv) 1 It binds to the same epitope on two antigens and contains the same VH and / or VL sequences.

In another major aspect of another method , the present invention is a method for identifying a mutation in an antibody that enhances the effector function of the antibody that binds C1q, comprising:
(I) preparing at least one antibody comprising a mutation in one or more amino acids selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain;
(Ii) evaluating the C1q activity of the antibody when bound to the surface of an antigen-expressing cell compared to the parent antibody; and (iii) selecting a mutation of any mutant with enhanced C1q binding ability. ,
Relates to a method comprising:

  In one embodiment, the at least one antibody is one or more selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain. Amino acid substitutions such as E430G, E430S, E345K and E345Q.

In yet another major aspect, the present invention is a method for identifying a mutation in a parent antibody that enhances the antibody's ability to induce a CDC response, comprising:
(I) preparing at least one variant of a parent antibody comprising a mutation in one or more amino acids selected from the group corresponding to E430X, E345X, S440Y or S440W in the Fc region of a human IgG1 heavy chain;
(Ii) assessing the CDC response induced by the variant when bound to the surface of an antigen-expressing cell in the presence of effector cells or complement compared to the parent antibody; and (iii) enhancing the CDC response. Selecting a mutation of any given mutant,
Relates to a method comprising:

  In one embodiment, the at least one antibody is one or more selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain. Amino acid substitutions such as E430G, E430S, E345K and E345Q in the Fc region of human IgG1 heavy chain.

Polypeptide of the present invention
Parent Polypeptides As described herein, the present invention relates to variants of a parent polypeptide that include one or more mutations in the CH3 region of an immunoglobulin, eg, the heavy chain of an antibody, among others. A “parent polypeptide” may be a “parent antibody”. A “parent” antibody, which may be a wild-type antibody, to be used as a starting material of the invention prior to modification is a hybridoma originally described, for example, by Kohler et al., Nature 256, 495 (1975). It may be made by a method or may be made by a recombinant DNA method. Monoclonal antibodies can also be obtained using the techniques described in, for example, Clackson et al., Nature 352, 624 628 (1991) and Marks et al., J. Mol. Biol. 222, 581 597 (1991). It can also be isolated from an antibody library. Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse splenic B cells obtained from mice immunized with the antigen of interest, eg, in the form of cells expressing the antigen on the surface, or of interest It can be obtained in the form of a nucleic acid encoding the antigen. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of an immunized human or non-human mammal (eg, rabbit, rat, dog, primate, etc.).

  The parent antibody may be, for example, a chimeric antibody or a humanized antibody. In another embodiment, the antibody is a human antibody. Human monoclonal antibodies can be produced using transgenic mice or transchromosomal mice, eg, HuMAb mice, that possess part of the human immune system rather than the mouse system. HuMAb mice target the human immunoglobulin gene minilocus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, targeting mutations that inactivate endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856 859 (1994)). Therefore, this mouse shows a decrease in the expression of mouse IgM or κ, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation in response to immunization, and high affinity human IgG, κ Generate monoclonal antibodies (Lonberg, N. et al. (1994), supra; Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536 546 (1995)). HuMAb mice were prepared by Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996). ing. US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, See also WO 92/22645, WO 92/03918 and WO 01/09187. Using splenocytes from these transgenic mice, hybridomas that secrete human monoclonal antibodies can be produced according to well-known techniques.

  In addition, human antibodies of the present invention, or antibodies of the present invention from other species, are not limited to phage display, retroviral display, ribosome display, mammalian display, yeast display and other techniques known in the art. And the resulting molecule can be subjected to further maturation, such as affinity maturation, as is well known in the art. The specific strategy described in Example 17 can be applied to any antibody to prepare and obtain mutants of the invention using phage-display.

  The parent antibody is not limited to naturally occurring, eg, antibodies having a human Fc domain, as it affects other mutations other than those of the invention, eg, glycosylation, or the antibody is bispecific. It may be an antibody having a mutation that makes it possible to be an antibody. The term “native antibody” means any antibody that does not contain any genetically introduced mutations. Antibodies containing naturally occurring modifications, such as various allotypes, should therefore be construed as “natural antibodies” within the meaning of the present invention and as a result, can be interpreted as parent antibodies. Such an antibody can serve as a template for one or more mutations of the present invention, thereby obtaining a mutant antibody of the present invention. An example of a parent antibody containing other mutations other than those of the present invention utilizes reducing conditions to facilitate half-molecule exchange of two antibodies containing an IgG4-like CH3 region, thereby concomitant formation of aggregates. Bispecific antibodies as described in WO 2011/131746 (Genmab) that form bispecific antibodies without accompanying. Other examples of parent antibodies include heterodimeric bispecifics: Triomab (Fresenius) ;; Bispecific IgG1 and IgG2 (Rinat neurosciences Corporation); FcΔAdp (Regeneron); Knob Into Hall (Genentech); Bispecific antibodies such as, but not limited to, electrostatic steering (Amgen, Chugai, Oncomed); SEEDbody (Merck); Azymetric scaffold (Zymeworks); mAb-Fv (Xencor); and LUZ-Y (Genentech) . Other exemplary parent antibody formats include, but are not limited to, wild type antibodies, full length antibodies or antibody fragments containing Fc, human antibodies, or any combination thereof.

  The parent antibody can bind to any target, and examples of such targets or antigens of the present invention may include, but are not limited to: 5T4; ADAM-10; ADAM- 12; ADAM17; AFP; AXL; ANGPT2 anthrax antigen; BSG; CAIX; CAXII; CA 72-4; cancer-associated antigen CTAA16.88; CCL11; CCL2; CCR4; CCR5; CCR6; CD2; CD3E; CD4; CD5; CD15; CD18; CD19; CD20; CD22; CD24; CD25; CD29; CD30; CD32B; CD33; CD37; CD38; CD40LG; CD44; CD47; CD52; CD56; CD66E; CD72; CD86; CD98; CD137; CD147; CD138; CD168; CD200; CD248; CD254; CD257; CDH3; CEA; CEACAM5; CEACAM6; CEACAM8; claudin4; CS-1; CSF2RA; CSPG-4; CTLA4; Cripto; DLL4; ED-B; EFNA2; EGFR; endothelin B receptor; ENPP3; EPCAM; ERBB2; ERBB3; FAPα; FcγRI; FCER2; FGFR3; fibrin II β chain; FLT1; FOLH1; FOLR1; FRP-1; GD3 ganglioside; Gripi -3; GPNM B; HBV (hepatitis B virus); HCMV (human cytomegalovirus); heat shock protein 90 homolog [Candida albicans]; herpes simplex virus gD glycoprotein; HGF; HIV-1; HIV-1IIIB HLA-DRB (HLA-DRβ); human respiratory syncytial virus, glycoprotein F; ICAM1; IFNA1; IFNA1; IFNB1 bispecific; IgE Fc; IGF1R; IGHE junction region; IL12B; IL13; IL15; IL1A; IL1B; IL2RA; IL4; IL5; IL5RA; IL6; IL6R; IL9; interleukin-2 receptor β subunit; ITGA2; ITGA2B ITGB3; ITGA4 ITGB7; ITGA5; ITGAL; ITGAV_ITGB3; ITGB2; KDR; L1CAM; y; Lipid A, Lipopolysaccharide LPS domain; LTA; MET; MMP14; MMp15; MST1R; MSTN; MUC1; MUC4; MUC16; MUC5AC; NCA-90 granulocyte cell antigen; Nectin4; NGF; NRP; 1; OX40L; PLAC-1; PLGF; PDGFRA; PD1; PDL1; PSCA; phosphatidylserine; PTK-7; Pseudomonas aeruginosa serotype IA TS O11; RSV (human respiratory syncytial virus, glycoprotein F); ROR1; RTN4; SELL; SELP; STEAP1; Shiga toxin-like toxin IIB subunit [E. coli]; SLAM7; SLC44A4; SOST; S. epidermidis lipoteichoic acid; TF; TGFB1; TGFB2; TMEFF2; TNC; TNF; TNFRSF10A; TNFRSF10B; TNFRSF12A; TNFSF13; TNFSF14; TNFSF2; TNFSF7; TRAILR2; TROP2;

  The parent antibody may be any human antibody such as IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgE, IgM and IgD, and optionally a human full-length IgG1 antibody. It may be a long antibody. The parent antibody may comprise a sequence according to any of SEQ ID NOs: 1, 2, 3, 4 and 5.

  Monoclonal antibodies, such as parent antibodies and / or variants, for use in the present invention may be generated by the hybridoma method first described by, for example, Kohler et al., Nature 256, 495 (1975) or It may be produced by a replacement DNA method. Monoclonal antibodies can also be obtained using techniques described in, for example, Clackson et al., Nature 352, 624-628 (1991) and Marks et al., J. Mol. Biol. 222, 581-597 (1991). It can also be isolated from a phage antibody library. Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas prepared from mouse splenic B cells obtained from mice immunized with the antigen of interest, eg, in the form of cells expressing the antigen on the surface, or of interest It can be obtained in the form of a nucleic acid encoding the antigen. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of an immunized human or non-human mammal (eg, rat, dog, primate, etc.).

  In one embodiment, the antibody is a human antibody. Human monoclonal antibodies directed to any antigen can be produced using transgenic mice or transchromosomal mice carrying part of the human immune system rather than the mouse system. Such transgenic mice or transchromosomal mice include mice referred to herein as HuMAb® mice and KM mice, which are collectively referred to herein as “transgenic mice”. Is done.

  HuMAb® mice inactivate the human immunoglobulin gene minilocus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, and the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). As such, this mouse exhibits reduced expression of mouse IgM or κ, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation in response to immunization, resulting in high affinity human IgG, κ Generate monoclonal antibodies (Lonberg, N. et al. (1994), supra; Lonberg, N. Handbook of Experimental Pharmacology 113, 49 101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 13 65 93 (1995) and Harding, F. and Lonberg, N. Ann. NY Acad. Sci 764 536 546 (1995)). Preparation of HuMAb® mice is described in Taylor, L. et al., Nucleic Acids Research 20, 6287 6295 (1992), Chen, J. et al., International Immunology 5, 647 656 (1993), Tuaillon et al. ., J. Immunol. 152, 2912 2920 (1994), Taylor, L. et al., International Immunology 6, 579 591 (1994), Fishwild, D. et al., Nature Biotechnology 14, 845 851 (1996) It is described in detail. US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, See also WO 92/22645, WO 92/03918 and WO 01/09187.

  HCo7, HCo12, HCo17 and HCo20 mice have JKD disruption in their endogenous light chain (κ) genes (as described in Chen et al., EMBO J. 12, 821-830 (1993)), their CMD disruption in the endogenous heavy chain gene (as described in Example 1 of WO 01/14424) and the KCo5 human kappa light chain transgene (Fishwild et al., Nature Biotechnology 14, 845-851 (1996) As described in). In addition, Hco7 mice have an HCo7 human heavy chain transgene (as described in US 5,770,429), and HCo12 mice have an HCo12 human heavy chain transgene (described in Example 2 of WO 01/14424). HCo17 mice have an HCo17 human heavy chain transgene (as described in Example 2 of WO 01/09187) and HCo20 mice have an HCo20 human heavy chain transgene. The resulting mice express human immunoglobulin heavy chain and kappa light chain transgenes in a background in which the endogenous mouse heavy chain and kappa light chain loci are homozygously disrupted.

  In the KM mouse strain, the endogenous mouse κ light chain gene is homozygously disrupted as described in Chen et al., EMBO J. 12, 811-820 (1993), and WO 01/09187 As described in Example 1, the endogenous mouse heavy chain gene has been homozygously disrupted. This mouse strain has the human kappa light chain transgene KCo5 as described in Fishwild et al., Nature Biotechnology 14, 845-851 (1996). This mouse strain also has a human heavy chain transchromosome composed of chromosome 14 fragment hCF (SC20) as described in WO 02/43478. HCo12-Balb / C mice can be generated by crossing HCo12 with KCo5 [J / K] (Balb) as described in WO / 2009/097006. Using splenocytes from these transgenic mice, hybridomas that secrete human monoclonal antibodies can be produced according to well-known techniques.

  In addition, any antigen binding region may be identified as a human antibody or other identified by display-type techniques including, but not limited to, phage display, retroviral display, ribosome display, and other techniques using techniques well known in the art. And the resulting molecules can be subjected to further maturation, such as affinity maturation, as is well known in the art (eg, Hoogenboom et al ., J. Mol. Biol. 227, 381 (1991) (phage display), Vaughan et al., Nature Biotech 14, 309 (1996) (phage display), Hanes and Plucthau, PNAS USA 94, 4937-4942 (1997) ) (Ribosome display), Parmley and Smith, Gene 73, 305-318 (1988) (phage display), Scott TIBS 17, 241-245 (1992), Cwirla et al., PNAS USA 87, 6378-6382 (1990) , Russel et al., Nucl.Acids Research 21, 1081-1085 (1993), Hogenboom et al., Immunol. Reviews 130, 43-68 (1992), Chiswell and McCafferty TIBTECH 10, 80-84 (1992), and (See US 5,733,743). When producing non-human antibodies using display technology, such antibodies can also be humanized.

  The mutation of the present invention may be, but is not limited to, a deletion, insertion or substitution of one or more amino acids. Such substitution of amino acids may be by any natural or unnatural amino acid.

"Single mutant"
All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody include other parent polypeptides comprising the Fc domain of an immunoglobulin and a binding region, the first It should be understood that it can also be applied to a parent polypeptide or a second parent polypeptide.

  The antibody or polypeptide variant of the “single variant” aspect of the present invention replaces each amino acid residue numbered according to the EU index in the human IgG1 antibody with the amino acid at the corresponding position in the IgG2, IgG3 and IgG4 parent antibodies. As well as mutations in one or more of the amino acid residues shown in Table 1, listed with “exemplary” and “preferred” amino acid substitutions. The segment of IgG2 corresponding to residues 126-326 of IgG1, the segment of IgG3 corresponding to residues 177-377 of IgG1, and the segment of IgG4 corresponding to residues 127-327 of IgG1 are shown in FIG. Yes.

TABLE 1 Exemplary mutation sites and amino acid substitutions for the “single variant” aspect

  As seen in Table 1, amino acid substitutions that resulted in enhanced cytolysis of Wien133 cells were included as “preferred substitutions” in Example 19.

  In one aspect, the invention relates to a variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein E430G, E430S, E430F, E430T, E345K, E345Q in the Fc region of a human IgG1 heavy chain. Contains one or more mutations selected from the group corresponding to E345R, E345Y and S440W, but does not contain additional mutations in the Fc domain that alter the binding of the mutant to the neonatal Fc receptor (FcRn) It relates to a mutant as a condition. (FcRn) can be determined by the method disclosed in Example 34.

  In another aspect, the invention provides a variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein E430G, E430S, E430F, E430T, E345K, E345Q in the Fc region of a human IgG1 heavy chain. A newborn of a mutant comprising one or more mutations selected from the group corresponding to E345R, E345Y and S440W, but measured by the change in absorbance at OD405nm as determined by the method disclosed in Example 34 It relates to variants that do not contain further mutations in the Fc domain that increase or decrease binding to the Fc receptor (FcRn) by more than 30%, for example more than 20%, 10% or 5%.

  In another aspect, the invention provides a variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein E430G, E430S, E430F, E430T, E345K, E345Q in the Fc region of a human IgG1 heavy chain. Mouse neonatal Fc of a parent polypeptide or parent antibody comprising one or more mutations selected from the group corresponding to E345R, E345Y and S440W, provided that the variant is determined by the method disclosed in Example 34 Does not contain additional mutations in the Fc domain that enhance the apparent affinity for the receptor (FcRn) by more than 0.5-fold or reduces the apparent affinity of the parent polypeptide or antibody to mouse FcRn by more than 2-fold It is related with the mutant on condition that it is not allowed.

  In one embodiment, the one or more mutations are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

  In one embodiment, the variant does not include additional mutations in the Fc domain that alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the variant.

  In one embodiment, the variant does not contain additional mutations in the Fc domain that alter the plasma clearance rate of the variant as determined by the method disclosed in Example 37.

  In another embodiment, the variant has a plasma clearance rate determined by the method disclosed in Example 37 that is greater than 3.0 times, such as greater than 2.5 times, 2.0 times, 1.5 times, or 1.2 times. Does not include additional mutations in the Fc domain that increase or decrease.

  In one embodiment, the variant does not include additional mutations in the Fc domain that alter the serum half-life of the variant.

  In one embodiment, the variant does not contain additional mutations in the Fc domain that alter the complement activation in the target-independent fluid phase of the variant as determined by the method disclosed in Example 36. .

  In one embodiment, the variant does not contain further mutations in the Fc domain.

  In one embodiment, the variant contains only one mutation.

  In one embodiment, the variant polypeptide may be a variant antibody comprising an immunoglobulin Fc domain and an antigen binding region.

  In one specific embodiment, the amino acid substitution is E345R.

  As shown in the examples, the CD38 antibodies HuMab-005 and -003 (as described in WO WO 2006/099875) and / or the CD20 antibody HuMab- containing one of these amino acid substitutions are included. Mutants of 7D8 and -11B8 (as described in WO 2004/035607) and rituximab and / or EGFR antibody HuMab-2F8 (as described in WO 2002/100348) are each wild type Has higher C1q binding, complement activation and / or CDC than HuMab 005 and 7D8.

  It should be understood that a variant may contain only one of the “exemplary substitution” mutations listed in Table 1. A variant may also include multiple mutations of any mutation listed in Table 1, eg 2, 3, 4, 5, or 6.

  In addition to the designated mutation, the variant may have any of the characteristics described for the parent antibody. In particular, it may be a human antibody. The variant may be of any IgG subtype in addition to the mutation.

  When bound to that antigen on the surface of an antigen-expressing cell, on the cell membrane, on a virion, or on another particle, or when the antigen is bound to a virion, optionally in the protein envelope or lipid envelope of the virion Such antibody variants, compared to the parent antibody, include (i) antibody-mediated CDC, (ii) antibody-mediated complement activation, (iii) C1q binding, It may have at least one of (iv) oligomer formation, (v) oligomer stability, or any combination of (i)-(v). In one embodiment of (iv) or (v), the oligomer is a hexamer. In one embodiment, the variant also has ADCC enhancement compared to the parent polypeptide or parent antibody. In one further embodiment, the variant retains the same or similar plasma clearance rate as compared to the parent polypeptide or parent antibody. In one further embodiment, the variant has a plasma clearance rate determined by the method disclosed in Example 37 when compared to the parent polypeptide or antibody of greater than 3.0 fold, for example 2.5 fold, 2.0 fold. Does not increase or decrease more than double, 1.5 times or 1.2 times.

  Without being limited to any particular theory, an effect caused by substituting an amino acid at a specified position with an amino acid residue of the present invention can be caused by, for example, the effect itself, another molecule Can be directly involved in contact with one Fc domain, or can be mutated to interact with another Fc domain that directly or indirectly affects intermolecular Fc: Fc interactions. Good. Thus, the substitution is not bound by theory, but directly or indirectly enhances the binding strength between antibody molecules in oligomeric form, resulting in oligomeric structures such as hexamer, pentamer, tetramer, trimer or dimer structures. It is thought to enhance stability. For example, amino acid substitutions promote or enhance the formation of new intermolecular Fc: Fc bonds, such as but not limited to van der Waals interactions, hydrogen bonds, charge-charge interactions, Alternatively, it may be an aromatic stacking interaction or the like, or one that promotes entropy increase by release of water molecules during Fc: Fc interaction. Further, with reference to Table 1, “exemplary substitutions” can be selected based on size and physicochemical properties that involve or promote intermolecular Fc: Fc or intramolecular interactions. “Preferred substitutions” can be selected based on optimal size and physicochemical properties to involve or stimulate intermolecular Fc: Fc or intramolecular interactions.

  In one embodiment, the variant may comprise a further mutation selected from Table 1.

  In one embodiment, the variant comprises a combination of two mutations in amino acid residues selected from the group corresponding to E345X / E430X, E345X / S440Y, E345X / S440W, E430X / S440Y and E430X / S440W.

  In any embodiment where such a mutation in at least two amino acids is included in the variant, it may be present in each of the heavy chains of the variant, or each one of the two is included in one of the heavy chains The other may be included in the other heavy chain, or vice versa.

  In one embodiment, the mutation at two amino acid residues is a deletion, insertion or substitution. Such substitution of amino acids may be by any natural or artificial amino acid.

  Each mutation according to the invention may be, but is not limited to, a deletion, insertion or substitution of one or more amino acid residues. Such substitution of amino acids may be by any natural or artificial amino acid.

  Thus, in one embodiment, the mutation at at least one amino acid residue is a deletion.

  In another embodiment, the mutation at at least one amino acid residue is an insertion.

  In another embodiment, the mutation at at least one amino acid residue is a substitution.

  Exemplary specific combinations of mutations at two amino acid residues include E345R / E430T, E345R / S440Y, E345R / S440W, E345R / E430G, E345Q / E430T, E345Q / S440Y, E345Q / S440W, E430T / S440Y, And E430T / S440W.

  In addition to mutations in one or more amino acids according to embodiments of the invention, the IgG heavy chain may contain additional mutations known in the art, such as mutations that further improve effector function. Such additional mutations include known mutations that enhance CDC, Fc-γ receptor binding or FcRn binding and / or improve Fc-γ receptor-mediated effector function.

  In one embodiment, the variants of the present invention are known CDC-enhancing modifications, such as exchanging segments between IgG isotypes to create chimeric IgG molecules (Natsume et al., 2008 Cancer Res 68 (10 ), 3863-72); one or more amino acid substitutions in the hinge region (Dall'Acqua et al., 2006 J Immunol 177, 1129-1138) and / or around residues D270, K322, P329 and P331 One or more amino acid substitutions within or near the C1q binding site within the CH2 domain (Idusogie et al., 2001 J Immunol 166, 2571-2575; Michaelsen et al., 2009 Scand J Immunol 70, 553-564 and WO 99/51642) and the like. For example, in one embodiment, a variant of the invention further comprises any combination of amino acid substitutions S267E, H268F, S324T, S239D, G236A and I332E that results in enhanced effector function via CDC or ADCC (Moore et al., 2010 mAbs 2 (2), 181-189)). Also, binding to Fc receptors (described in WO 2006/105062, WO 00/42072, US Pat. No. 6,737,056 and US Pat. No. 7,083,784) or antibody physical properties (described in WO 2007/005612 A1) Other Fc mutations that affect can also be used in the mutants of the invention.

  In one embodiment, the variants of the invention further comprise modifications that enhance Fc-γ receptor binding and / or Fc-γ receptor-mediated effector function. Such modifications include (i) reducing the amount of fucose in CH2-linked glycosylation (glycan engineering) (Umana P, et al., Nat Biotechnol 1999; 17: 176-80; Niwa R, et al., Clin Cancer Res 2004; 10: 6248-55.)), and (ii) site-directed mutagenesis (protein engineering) of amino acids in the hinge or CH2 region of the antibody (Lazar GA, et al., Proc Natl Acad Sci USA 2006; 103: 4005-10).

  In one embodiment, the variants of the invention are further engineered at the FcRn binding site, eg, to increase the half-life (t1 / 2) of IgG antibodies. Such modifications include (i) N434A and T307A / E380A / N434A mutations (Petcova et al. Int Immunol. 2006 Dec; 18 (12): 1759); (ii) Pro238, Thr256, Thr307, Gln311, Asp312, Substitution of one or more of Glu380, Glu382 and Asn434 to an alanine residue that improves FcRn binding (Shields RL, et al. J. Biol. Chem. 2001; 276: 6591); and (iii) against FcRn Increase affinity, IgG1 M252Y / S254T / T256E, M252W, M252Y, M252Y / T256Q, M252F / T256D, V308T / L309P / Q311S, G385D / Q386P / N389S, G385R / Q386T / P387R / N389P, H433K / N434F / Amino acid substitution or combination of amino acid substitutions selected from Y436H, N434F / Y436H, H433R / N434Y / Y436H, M252Y / S254T / T256E-H433K / N434F / Y436H or M252Y / S254T / T256E-G385R / Q386T / P387R / N389P 'Acqua et al., Supra).

"Double mutant"
All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody include other parent polypeptides comprising the Fc domain of an immunoglobulin and a binding region, the first It should be understood that it may also be applicable to a parent polypeptide or a second parent polypeptide.

  As described above and further described below, the present invention also provides a “double mutant, where each of the two mutations individually reduces effector function, but when combined, restores effector function to the level of the parent antibody. It also relates to the aspect. When used together, the specificity of the mutant is enhanced. Antibody variants of the “double variant” aspect are two mutations (typically amino acid substitutions) in specific amino acid residue interaction pairs K439 and S440, K447 and 448, or K447, 448 and 449. including.

Accordingly, in one aspect, the invention provides a variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, wherein E430G, E430S, E430F, E430T, E345K in the Fc region of a human IgG1 heavy chain, Includes a first mutation selected from the group corresponding to E345Q, E345R, E345Y, S440Y and S440W (eg, E430G, E430S, E345K or E345Q, etc.) and a second mutation selected from the group corresponding to Regarding variants:
(I) the amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that the second mutation is S440Y or S440W and the first mutation is S440Y or S440W The mutation is at an amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain,
(Ii) amino acid residues corresponding to K447D / E in the Fc region of human IgG1 heavy chain or corresponding to K447K / R / H and 448P; or (iii) K447D / E in the Fc region of human IgG1 heavy chain Amino acid residues corresponding to or corresponding to K447K / R / H and 448K / R / H and 449P. Tables 2A and B show “exemplary” and “preferred substitutions” for the “double mutant” aspect (Table A) and the “mixed mutant” aspect (Table 2B).

Table 2A: Exemplary mutation sites and amino acid substitutions for the “double mutant” aspect

Table 2B: Exemplary mutation sites and amino acid substitutions for the “mixed mutant” aspect (Ab1 + Ab2)

  In one embodiment, the variant is a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, and E345Y, and K439 and S440 in the Fc region of the human IgG1 heavy chain. A second mutation in the amino acid residue corresponding to (provided that the mutation in S440 is neither S440W nor S440Y).

  It is contemplated by the present invention that a variant may contain only one of the amino acid residue substitutions, e.g. either K439E or S440K, e.g. the variant comprises one mutation in K439 and optionally S440 Has no mutation in

  In one embodiment, the invention relates to a variant wherein the mutation in K439 is an amino acid substitution (eg, K439E) with an amino acid selected from E and D.

  In another embodiment, the mutant comprises a mutation at S440 and optionally has no mutation at K439.

  In one embodiment, the invention relates to a variant wherein the mutation in S440 is an amino acid substitution (eg, S440K) with an amino acid selected from K and R.

  In one embodiment, the mutant comprises a mutation in both K439 and S440.

  In another embodiment, the mutation at K439 is selected from a K439 to D, E or R mutation (eg, K439D / E) and the mutation at S440 is S440 to D, E, K and R (eg, S440K / R). Selected from mutations to

  In another embodiment, the mutation at K439 is selected from K439D and K439E, and the mutation at S440 is selected from S440K and S440R.

  In another embodiment, the variant comprises the K439E and S440K mutations.

  In one embodiment, the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.

As described in Examples 4-6, the antibody variant comprising only one of the K439E and S440K mutation have a dramatic increase in the K _D for CIq, which complement activation and / Or reflects the decline in CDC capacity. Surprisingly, antibody variants of HuMAb 7D8 or 005 containing both mutations were found to recover or enhance C1q binding or CDC. Without being bound by any particular theory, the underlying mechanism may probably be explained by the steric compensation of each mutation as shown in Figures 4 and 5 it is conceivable that.

  In one embodiment, the parent polypeptide (and also variants thereof) may be an antibody comprising an immunoglobulin Fc domain and an antigen binding region.

  In another embodiment, a variant comprising a mutation at both positions K439 and S440 as described herein is compared to a parent antibody or an antibody variant comprising a mutation in only one of K439 and S440. Complement-dependent cytotoxicity (CDC), C1q binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC), Fc receptor binding, including Fc-γ receptor binding, protein A binding , Protein G binding, antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDCC), complement-enhanced cytotoxicity, opsonization, intracellular translocation of Fc-containing polypeptides, target down-modulation, Has enhanced Fc-mediated effector function selected from ADC uptake, induction of apoptosis, cell death, cell cycle arrest and any combination thereof.

  The present invention also includes (i) an antibody-mediated CDC, (ii) an antibody compared to a parent antibody, which can be, for example, a wild-type antibody, or an antibody variant comprising only one of the K439E or S440K mutations. Restores one or more of mediated complement activation, (iii) C1q binding power, (iv) oligomer formation, (v) oligomer stability, or any combination of (i)-(v) For use of the K439E and S440K mutations in antibodies. In one embodiment of (iv) or (v), the oligomer is a hexamer.

  In one embodiment, the variant is selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

All embodiments described herein with reference to a mixed variant parent antibody, first parent antibody or second parent antibody include other parent polypeptides comprising an Fc domain of an immunoglobulin and a binding region; It should be understood that it may also be applicable to the first parent polypeptide or the second parent polypeptide.

  As noted above, the inventors of the present invention alone reduced effector function when used together, such as mutations at positions K439 and S440 in the Fc region of the human IgG1 heavy chain, but when used together, We also found a mutation that restores function. That is, this concept can be used to reliably pair two different antibodies by introducing K439 into one antibody and S440 into the other. Thus, an antibody variant of the “mixed variant” aspect contains one mutation, which typically results in a reduced or significant reduction in Fc: Fc interactions between identical Fc molecules. However, because “mixed variant” antibody variants of the invention can pair with each other; thereby, for example, specific for each variant alone or compared to a mixture of one or more parent antibodies. For some antibody variant pairs, CDC, C1q binding, complement activation, oligomerization, and / or oligomer stability can be restored or even enhanced. In one embodiment of the invention, the oligomer is a hexamer. In one embodiment, antibody variant pairs may additionally or alternatively have other effector functions that are retained or improved, such as C1q binding, complement activation, antibody-dependent cell-mediated cytotoxicity (ADCC). ), FcRn binding, Fc receptor binding including Fc-γ receptor binding, protein A binding, protein G binding, antibody dependent cellular phagocytosis (ADCP), complement dependent cytotoxicity (CDCC), complement enhancement It has sex cytotoxicity, opsonization, intracellular translocation of Fc-containing polypeptide, target down-modulation, ADC uptake, induction of apoptosis, cell death, cell cycle arrest and any combination thereof. This aspect of the invention provides a number of uses that can modulate selectivity as well as the strength of C1q binding, complement activation, CDC or other effector functions.

  Exemplary mutation sites for each antibody variant in the “mixed variant” pair are shown in Table 2B. Specifically, the present invention relates to a variant of an antibody comprising an antigen-binding region and an immunoglobulin Fc domain, the residue corresponding to one of K439 and S440 in the Fc region of human IgG1 heavy chain. Variants containing mutations are provided.

  In one embodiment, the mutation is at K439, which is an amino acid substitution to an amino acid selected from E or D, such as K439E. In one embodiment, the mutation is in S440, which is an amino acid substitution to an amino acid selected from K or R, such as S440K.

  In one embodiment, the variant comprises an amino acid mutation only at the position corresponding to K439 in the Fc region of the IgG1 heavy chain and not at the position corresponding to position S440.

  In one embodiment, the variant comprises an amino acid mutation only at a position corresponding to S440, provided that the mutation at S440 is not S440Y or S440W, at a position corresponding to K439 in the Fc region of the IgG1 heavy chain. Does not contain amino acid mutations.

  Thus, in one embodiment, the invention also provides a first selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of a human IgG1 heavy chain. Mutations; as well as variants comprising a second mutation in the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain.

  In another embodiment, the present invention also provides a first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y in the Fc region of a human IgG1 heavy chain; and human IgG1 heavy It also includes a second mutation at the amino acid residue corresponding to S440 in the Fc region of the chain, provided that the second mutation is not S440Y or S440W.

  In one embodiment, the above two embodiments can also be combined in a “mixed variant” pair aspect according to the present invention.

  Each variant in the “mixed variant” pair may further comprise a mutation in an amino acid listed in Table 1.

In one embodiment of the invention, the “mixed variant” pair comprises a first variant of the parent antibody and a second variant of the parent antibody, wherein the first variant is the first of the immunoglobulin. An Fc domain and an antigen binding region, wherein the first variant is (i) selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of a human IgG1 heavy chain, other than a mutation at K439, Position corresponding to the first mutation in one or more amino acid residues, e.g. E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, and K439 in the Fc region of the human IgG1 heavy chain And wherein the second variant comprises an immunoglobulin second Fc domain and an antigen binding region, wherein the second variant is (i) in the Fc region of a human IgG1 heavy chain One other than the mutation at S440, selected from the group corresponding to E430X and E345X Or a first mutation at a plurality of amino acid residues, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y, and (ii) a second at a position corresponding to S440 in the Fc region of the IgG1 heavy chain Mutations, provided that the mutation at S440 is not S440Y or S440W.

  Other exemplary “mixed variant” pairs may further include, but are not limited to, any of the following pairs; a first variant comprising mutation K447E and a first variant comprising mutation K447 / P448. 2 mutants; a first mutant comprising mutation K447E and a second mutant comprising mutation K447 / K448 / P449.

  In one embodiment, the mutation is a deletion, insertion or substitution. Such substitution of amino acids may be by any natural or unnatural amino acid.

  In one embodiment, the mutation is a deletion.

  In another embodiment, the mutation is an insertion.

  In another embodiment, the mutation is an amino acid substitution.

  In one particular embodiment, the first variant and / or the second variant is one or more selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain. Includes mutations at amino acid residues.

  For example, in one embodiment, one variant in a “mixed variant” pair comprises one of E430G, E430S, E345K or E345Q in combination with the K439E mutation, while the other variant is E430G, E430S, E345K. Or one of E345Q in combination with the S440K mutation, thereby enhancing and more specific C1q binding power, complement activation, CDC, oligomer formation, oligomer stability, and / or other Effector-related functions such as ADCC, Fc-γ receptor binding, protein A binding, protein G binding, ADCP, CDCC, complement-enhanced cytotoxicity, antibody-mediated phagocytosis, intracellular translocation, apoptosis, opsonized antibody This results in binding to complement receptors, and / or combinations thereof.

  The “mixed variant” aspect also includes, for example, a first variant comprising the mutation S440K / K447E and a second variant comprising the mutation K439E / K447 / P448; for example, a first variant comprising the mutation K439E / K447E and Two mutants each comprising a plurality of mutations listed in Table 2A in the Fc region of the human IgG1 heavy chain may be included, such as a second mutant comprising mutation S440K / K447 / P448.

  Variants in “mixed variant” pairs as described herein may be derived from the same parent antibody or from different parent antibodies. Furthermore, the “mixed variant” aspect can also be used for bispecific or asymmetric antibodies. Furthermore, the first, second and third antibodies may bind to different epitopes on the same or different targets.

  Furthermore, the “mixed variant” aspect utilizes a first antibody against the first antigen having the K439E mutation and a second antibody against the second antigen having the S440K or S440R mutation, to CDC or other effector responses can be generated that more specifically target tumor cells that express one specific tumor antigen. By utilizing a “mixed variant” aspect comprising three variants, optionally bispecific antibodies, at least 2, eg 2, 3, 4, 5 or 6 specific tumors A CDC or other effector response that more specifically targets tumor cells expressing the antigen may result.

  In one embodiment of any of the “single variant”, “double variant” and “mixed variant” aspects, the variant is a monospecific antibody, bispecific antibody or multispecific Selected from antibodies.

  In any embodiment of the “mixed variant” aspect, the first, second and / or third variants may comprise the same or different mutations of any of the amino acid substitutions listed in Table 1.

All embodiments described herein with reference to a multispecific antibody parent antibody, first parent antibody or second parent antibody are other parent polypeptides comprising an Fc domain of an immunoglobulin and a binding region. It should be understood that this may also apply to the first parent polypeptide or the second parent polypeptide.

  Any embodiment of the “single variant”, “double variant” and “mixed variant” aspects described herein may be used in the multispecific antibody aspect described below. Should be understood.

  Thus, in one embodiment, the variant is an antibody selected from monospecific antibodies, bispecific antibodies or multispecific antibodies.

  In one particular embodiment, the bispecific antibody has the format described in WO 2011/131746.

In one major aspect, the present invention provides a first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region, and an immunoglobulin second CH2-CH3 region. A variant of a parent antibody that is a bispecific antibody comprising a second polypeptide comprising a second antigen-binding region, wherein the first and second antigen-binding regions are on the same antigen or on different antigens The first and / or second CH2-CH3 regions correspond to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain Selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain. A further mutation in the amino acid residue to be selected; and a second polypeptide Contains a further mutation in an amino acid residue selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain, and further in the first polypeptide It relates to a variant, wherein the mutation is different from further mutations in the second polypeptide.

  In one embodiment, the mutation is a deletion, insertion or substitution. Such substitution of amino acids may be any natural or non-natural acid substitution.

  The bispecific antibodies of the present invention are not limited to a particular format and may be those described above and any described herein.

In one particular embodiment of the invention, (i) the first polypeptide comprises a further mutation in the amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain, for example K409R; and (ii) the second The polypeptide comprises a further mutation in the amino acid residue corresponding to F405 in the Fc region of the human IgG1 heavy chain, eg, F405L; or alternatively (iii) the first polypeptide is the Fc of the human IgG1 heavy chain Further mutations in the amino acid residues corresponding to F405 in the region, eg F405L; and (iv) the second polypeptide is further mutations in amino acid residues corresponding to K409 in the Fc region of the human IgG1 heavy chain, eg K409R including.

  In one particular embodiment, the mutation at one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

  Such bispecific antibodies of the invention can be made as described in Example 22. Moreover, the effect of the generated heterodimeric protein on CDC lethality can be tested by an assay as used in Example 23.

  Bispecific antibodies can include, for example, amino acid substitutions in one or more amino acids listed in Tables 1 and / or 2A / B, as well as the antigen binding region of the CD20 antibody and the antigen binding region of the CD38 antibody. Exemplary CD20 binding regions include those of ofatumumab (2F2), 7D8 and 11B8, and rituximab (WO 2005/103081) described in WO 2004/035607, which is incorporated herein by reference in its entirety. included. Exemplary CD38 binding regions include those of 003 and daratumumab (005) described in WO 2006/099875, which is incorporated herein by reference in its entirety.

  In one embodiment, bispecific antibodies bind to different epitopes on the same or different targets.

  In another embodiment, the first mutation in the first and second polypeptides can be the same or different.

  In one embodiment of the “single variant”, “double variant”, “mixed variant” and multispecific antibody aspects, the variant is a human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally a human full length antibody such as a human full length IgG1 antibody.

  In any “single variant”, “double variant”, “mixed variant” aspect and multispecific antibody aspect, the C1q binding of the antibody is determined according to the assay described in Example 4, and the CDC is Determined according to the assay described in Example 5, 6 or 10, the mutation is not in the amino acid residues directly involved in C1q binding, which optionally c1q binding in the ELISA assay according to Example 3 in Example 4. The ADCC is determined according to the assay described in Example 12 and determined by comparison with C1q binding in the cell-based assay followed.

  Furthermore, the present invention provides preparations of variants of any of the “single variants”, “double variants”, “mixed variants” and multispecific antibody aspects or embodiments described above. The invention also provides compositions, eg, pharmaceutical compositions, comprising variants of any of the “double variants” aspects and embodiments described above. The invention also provides the use of any such variant, preparation or composition as a medicament.

The above “single variant”, “double variant”, “mixed variant” and multispecific antibody aspects of the invention are directed to the human IgG1 heavy chain constant region (UniProt accession number P01857; SEQ ID NO: 1) It is particularly applicable to human antibody molecules having an IgG1 heavy chain comprising the corresponding segment P247-K447 corresponding to underlined residues 130-330.

The present invention can also be applied to antibody molecules having a human IgG2 heavy chain portion. The amino acid residues P247-K447 of the IgG1 heavy chain correspond to the underlined residues 126-326 of the IgG2 heavy chain constant region (Accession No. P01859; SEQ ID NO: 2).

The present invention can also be applied to antibody molecules having a human IgG3 heavy chain portion. Amino acid residues P247-K447 of the IgG1 heavy chain correspond to residues 177-377 of the IgG3 heavy chain constant region (UniProt accession number P01860, SEQ ID NO: 3), underlined below.

The present invention can also be applied to antibody molecules having a human IgG4 heavy chain portion. Amino acid residues P247 to K447 of the IgG1 heavy chain correspond to the underlined residues 127 to 327 of the IgG4 heavy chain constant region (Accession No. P01859, SEQ ID NO: 4).

The present invention can also be applied to an antibody having a human IgG1m (f) allotype heavy chain moiety. IgG1m (f) allotype amino acid sequence (CH3 sequence is underlined)-SEQ ID NO: 5

  The alignment of each segment of the IgG1, IgG2, IgG3, IgG4 and IgG1m (f) constant regions is shown in FIG. Thus, any mutation in the amino acid listed in Table 1 or Tables 2A and B is introduced at its equivalent position in IgG2, IgG3, IgG4 and / or IgG1m (f) as defined by the alignment, and the mutation of the invention You can get a body.

  In one embodiment, the present invention provides variants of full-length IgG1, IgG2, IgG3 or IgG4 antibodies comprising one or more amino acid substitutions of any of the above aspects.

  In any “single variant”, “double variant”, “mixed variant” aspect, and multispecific antibody, the Fc region of the IgG1 heavy chain comprises residues 130-330 of SEQ ID NO: 1, It may comprise the sequence of residues 126-326 of SEQ ID NO: 2, residues 177-377 of SEQ ID NO: 3, or residues 127-327 of SEQ ID NO: 4.

  In one embodiment, the parent antibody is a sequence selected from SEQ ID No .: 1-5, such as SEQ ID No .: 1, SEQ ID No .: 2, SEQ ID No .: 3, SEQ ID No .: 4 or SEQ ID No .: 5 is included.

  In one embodiment, the Fc region of an IgG1 heavy chain comprises the sequence of residues 130-330 of SEQ ID NO: 1.

  The parent antibody may be any parent antibody as described herein. A parent antibody in this context is also intended to be a first parent antibody and a second parent antibody.

  In one embodiment, the parent antibody is a human IgG1, IgG2, IgG3 or IgG4, IgA1, IgA2, IgD, IgM or IgE antibody.

  In one embodiment, the parent antibody is a human full length antibody, such as a human full length IgG1 antibody.

  In one embodiment, the parent antibody, the first parent antibody and the second parent antibody are human IgG1 antibodies, such as IgG1m (za) or IgG1m (f) allotypes, optionally comprising SEQ ID NO: 1 or 5 Includes Fc region.

  In one embodiment, the parent antibody is a human IgG2 antibody and optionally comprises an Fc region comprising SEQ ID NO: 2.

  In one embodiment, the parent antibody is a human IgG3 antibody and optionally comprises an Fc region comprising SEQ ID NO: 3.

  In one embodiment, the parent antibody is a human IgG4 antibody and optionally comprises an Fc region comprising SEQ ID NO: 4.

  In certain embodiments of any of the “single variants”, “double variants”, “mixed variants” and any of the multispecific antibody aspects, the variants are SEQ ID NOs, except for mutations introduced according to the present invention. ID No: at least 70%, 72%, 74%, 76%, 78%, 80%, 81%, 82%, 83%, 84% for amino acids P247-K447 of 1, 2, 3, 4 and 5 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% identity An amino acid sequence having a degree of

  Thus, a variant, except for any mutation defined herein, is a sequence according to SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 3, SEQ ID No: 4 or SEQ ID No: 5. May be included.

  Any of the above “single variants”, “double variants”, “mixed variants” and multispecific aspects of the invention can be construed to include the following embodiments.

In one embodiment, the first and / or second parent antibody is an antibody fragment, which is optionally a monovalent antibody, heavy chain antibody, chain exchange recombination domain (SEED), triomab, double variable domain immunity Globulin (DVD-Ig), knob-in-hole antibody, mini-antibody, dual affinity retargeting molecule (Fc-DART or Ig-DART); LUZ-Y antibody, Biclonic antibody, dual-targeting ( DT) -Ig antibody, two-in-one antibody, cross-linked Mab, mAb ² , CovX-body, IgG-like bispecific antibody, Ts2Ab, BsAb, HERCULES antibody, TvAb, ScFv / Fc Fusion antibody, SCORPION, Fc domain Selected from a scFv fragment fused to and a double scFv fragment fused to an Fc domain.

  In one further embodiment, both the first and second parent antibodies bind to an antigen expressed on the surface of a human tumor cell.

  In one further embodiment, the antigens for the first and second parent antibodies are erbB1 (EGFR), erbB2 (HER2), erbB3, erbB4, MUC-1, CD4, CD19, CD20, CD38, CD138, CXCR5, c- It is selected separately from the group consisting of Met, HERV-envelope protein, periostin, Big3, SPARC, BCR, CD79, CD37, EGFrvIII, L1-CAM, AXL, tissue factor (TF), CD74, EpCAM and MRP3.

  In one further embodiment, the first and second parent antibodies are fully human antibodies.

  In one further embodiment, the antigens for the first and second parent antibodies are selected from CD20 and CD38, in any order, and optionally the first and second parent antibodies are in any order, 7D8 and 005 Selected from.

  In one further embodiment, both the first antibody and the second antibody bind to an antigen expressed on the surface of a bacterial cell or virion.

  In another embodiment, the bacterial cell is S. aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Bacillus anthracis, Pseudomonas aeruginosa, Chlamydia trachomatis, E. coli, Salmonella, Shigella, Yersinia, Salmonella typhimurium, meninges Selected from the group consisting of Streptococcus and Mycobacterium tuberculosis.

  In one further embodiment, the first and second parent antibodies bind to the same antigen.

  In another embodiment, the first and second parent antibodies are the same antibody.

  In another embodiment, the parent antibody is selected from 7D8 and 005.

All embodiments described herein with reference to a composition parent antibody, a first parent antibody or a second parent antibody include other parent polypeptides comprising an immunoglobulin Fc domain and a binding region, It should be understood that it can also be applied to one parent polypeptide or a second parent polypeptide.

  The invention also relates to compositions comprising a variant and parent antibody, which may be any variant and parent antibody as described herein. Specific aspects and embodiments will be described below. Moreover, such mutants can be obtained by any method described herein.

  In one aspect, the present invention is a composition comprising first and second variants of a parent polypeptide, each comprising an immunoglobulin Fc domain and a binding region, wherein the first and / or second The composition relates to a composition comprising one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain.

  In one embodiment, the first and / or second variant is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain Containing one or more mutations.

  In one preferred embodiment, the first and / or second variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.

  In one embodiment, both the first and second variants comprise one or more mutations that may be the same or different.

In another embodiment, the first variant is E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain, e.g., E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W. An amino acid residue selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain, comprising one or more mutations selected from the corresponding group Does not include one or more mutations such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W.

  In one embodiment, the composition comprises at least one molecule comprising at least a CH2-CH3 domain of an immunoglobulin and a variant according to the invention, wherein the molecule comprises E430X, E345X in the Fc region of a human IgG1 heavy chain. , Mutations in one or more amino acid residues selected from the group corresponding to S440Y and S440W, such as E430G, E430S, E345K and E345Q.

  The molecules described in this embodiment can be referred to as “Fc-only molecules” and may further include, for example, a hinge region. However, such a hinge region may not be included.

  A composition comprising an Fc-only molecule and any variant according to the invention is applied for use in diagnostic imaging or to modulate the binding power of the variant once bound to the cell surface. Can do.

  The Fc-only molecule may further include additional mutations in amino acid residues corresponding to K439 and / or S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation in S440 is not S440Y or S440W, and the first mutation If is S440Y or S440W, further mutations are provided at the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain.

In another embodiment, (i) the first variant further comprises a mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain, and (ii) the second variant is a human IgG1 heavy chain Further comprising a mutation at a position corresponding to S440 in the Fc region, provided that the mutation is not S440Y or S440W; or alternatively (i) and (ii)
(Iii) the first variant further comprises a mutation at a position corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is neither S440Y nor S440W; and (iv) the second The variant may further comprise a mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain.

  In one embodiment, the mutation at position K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at position S440 in the Fc region of the human IgG1 heavy chain is S440K / R.

In one further embodiment, the present invention is a composition as defined herein comprising:
(I) the first variant further comprises a prodrug, and (ii) the second variant comprises an activator for the prodrug on the first variant; or alternatively (i) and ( ii) (iii) the second variant comprises a prodrug, and (iv) the first variant comprises an activator for the prodrug on the second variant,
It may relate to a composition.

  The term “prodrug” is used as a relatively non-cytotoxic drug precursor that must undergo chemical transformation, eg, by metabolic processes, to become an active pharmacological (anticancer) substance. It needs to be understood according to the invention. Examples of prodrugs and methods for preparing them are well known in the art. One example is the binding of an antibody conjugated to a prodrug to an antigen target present on the same cell, and the binding of an antibody conjugated to an activator to the prodrug, resulting in drug delivery An antibody compound containing a prodrug. This allows the prodrug and its activator to be in close proximity, thereby releasing the drug locally, for example to penetrate surrounding cells and kill these cells (Senter and Springer, 2001 Adv Drug Deliv Rev. 2001 Dec 31; 53 (3): 247-64, Senter, 1994 FASEB J. 1990 Feb 1; 4 (2): 188-93).

  The term “prodrug activator” is to be understood according to the invention as a molecule capable of converting a prodrug into an active drug. Examples of prodrug activators and methods of preparing them are well known in the art. An example of an activator may be an enzyme that acts as a catalyst for the conversion of a prodrug to an active drug (Senter and Springer, 2001 Adv Drug Deliv Rev. 2001 Dec 31; 53 (3): 247-64, Senter , 1994 FASEB J. 1990 Feb 1; 4 (2): 188-93).

  In one embodiment, the first and / or second parent polypeptide is a first and second parent antibody, each comprising an immunoglobulin Fc domain and an antigen binding region.

  In one embodiment, the first and second antibodies are human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibodies, respectively, optionally each human full length antibody, e.g. each human full length antibody. IgG1 antibody.

  In one embodiment, the first and second antibodies are each selected from a monospecific antibody, a bispecific antibody, or a multispecific antibody.

  In one further embodiment, the first and / or second parent antibody each comprises a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin and a second CH2-. A bispecific antibody comprising a second polypeptide comprising a CH3 region and a second antigen binding region, wherein the first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens. And the first CH2-CH3 region comprises a further amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; And the second CH2-CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain, and Additional amino acid mutations in the CH2-CH3 region of 1 Different from further amino acid mutations in the three regions.

  In one preferred embodiment, the additional amino acid mutation of the first CH2-CH3 region is at a position corresponding to K409 in the Fc region of the human IgG1 heavy chain, such as K409R; and of the second CH2-CH3 region A further amino acid mutation is at a position corresponding to F405 in the Fc region of the human IgG1 heavy chain, for example F405L.

  In one embodiment, the first and second variants of the composition bind to different epitopes on the same antigen or on different antigens.

  In one embodiment, one or both of the first and second variants is a drug, toxin, such that one or both of the first and second variants are conjugated to the toxin via a linker. Or conjugated with a radioactive label.

  In one embodiment, one or both of the first and second variants are part of a fusion protein.

  In one particular embodiment, the first and / or second variant of the composition comprises only one mutation.

  In embodiments where the second variant does not include any of the listed mutations described herein, such second variant is listed above in connection with methods for enhancing CDC. Any of the examples of suitable second antibodies can be included.

  In one embodiment, at least one first mutation in the first and second mutants is different.

  In one embodiment, the first variant and the second variant are human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibodies, respectively, optionally human full length IgG1 antibodies, for example Each is a human full-length antibody.

  In one embodiment, the first variant and the second variant are each selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

  In one further embodiment, the first and second variants bind to different epitopes on the same antigen or on different antigens. Thus, in embodiments where the first and second antibodies are bispecific, each antibody may bind to two different epitopes. The at least two bispecific antibodies may be the same or different. Thus, where bispecific antibodies are different, the composition includes targeting up to four different epitopes on the same or different targets.

  In another aspect, the invention relates to a composition comprising any variant, any bispecific antibody or any composition described herein and a pharmaceutically acceptable carrier.

  It is also contemplated that any of the embodiments of the “mixed variant” aspect can be included in any of the composition embodiments.

  In one embodiment, the variants of the first and second parent antibody bind to an antigen expressed on the same cell.

  In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 with an amino acid selected from E and D.

  In another embodiment, the amino acid substitution in the variant of the first parent antibody is K439E.

  In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 with an amino acid selected from K and R.

  In another embodiment, the amino acid substitution in the variant of the second parent antibody is S440K.

  In another aspect, the present invention relates to a pharmaceutical composition comprising a first parent polypeptide or parent antibody variant and a second parent polypeptide or parent antibody variant of any of the embodiments listed above. .

  The pharmaceutical composition can be formulated according to conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995. . The pharmaceutical composition of the present invention can be, for example, a diluent, a bulking agent, a salt, a buffer, a surfactant (eg, a nonionic surfactant such as Tween-20 or Tween-80), a stabilizer (eg, , Sugar or protein-free amino acids), preservatives, isotonic agents, antioxidants, tissue fixatives, solubilizing agents, and / or other materials suitable for inclusion in pharmaceutical compositions. Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyol (eg, glycerol, propylene glycol, polyethylene glycol). ) Is included.

  The pharmaceutical composition can be administered by any suitable route and mode. In one embodiment, the pharmaceutical composition of the present invention is administered parenterally. As used herein, the term “administered parenterally” refers to modes of administration other than enteral and topical administration, usually by injection, including the epidermis, intravenous, intramuscular, intraarterial. Intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendon, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, spinal cord, intracranial, intrathoracic, dura mater Includes injections and infusions outside and into the sternum.

All embodiments described herein with reference to a kit-of-part parent antibody, first parent antibody or second parent antibody include other parental polypeptides comprising an immunoglobulin Fc domain and a binding region. It should be understood that it may also apply to a peptide, a first parent polypeptide or a second parent polypeptide.

  The present invention also relates to a kit of parts for use simultaneously, separately or sequentially in a therapy comprising a parent polypeptide and a variant of the parent antibody, wherein any of the parent polypeptide and parent antibody. The variant may be as described herein. Specific aspects and embodiments will be described below. Moreover, such variants can be obtained according to any method described herein.

In one aspect, the invention is a kit of parts for use simultaneously, separately or sequentially in a therapy comprising a first variant of a parent polypeptide and a second variant of a parent polypeptide. Wherein the first variant is one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain, such as E430G, E430S, E430F, E430T, E345K , E345Q, E345R, E345Y, S440Y and S440W, etc., provided that the variant does not contain additional mutations in the Fc domain that alter the binding of the variant to the neonatal Fc receptor (FcRn), and (I) the first mutant contains a mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain, and the second mutant corresponds to S440 in the Fc region of a human IgG1 heavy chain Including mutations in position And, with the proviso that not even S440W mutation in S440 even S440Y,
(Ii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and the second variant comprises K447K / in the Fc region of a human IgG1 heavy chain Or (iii) the first variant comprises a mutation at a position corresponding to K447D / E in the Fc region of a human IgG1 heavy chain; and The variant comprises mutations at positions corresponding to K447K / R / H, 448K / R / H and 449P in the Fc region of the human IgG1 heavy chain,
It relates to kits of parts.

  In one embodiment, the first of the variants of the parent polypeptide, or both the first of the variants of the parent polypeptide and the second variant of the parent polypeptide are immunoglobulin Fc domains. And an antigen-binding region.

  In one embodiment, the mutation at the position corresponding to K439 in the Fc region of the human IgG1 heavy chain is K439D / E and / or the mutation at the position corresponding to S440 in the Fc region of the human IgG1 heavy chain is S440K / R. is there.

In another aspect, the invention provides a first variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region and a second variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region. From the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain for simultaneous, separate or sequential use in therapy, comprising One or more selected mutations, such as E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W, provided that the mutant is a mutant neonatal Fc receptor (FcRn) The second variant is selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain, provided that it does not contain further mutations in the Fc domain that alter binding to Mutations in amino acid residues, not including e.g. E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, and S440Y and S440W,
It relates to kits of parts.

  In embodiments where the second variant does not include any of the listed mutations described herein, such second variant is suitable as listed above in connection with the method of effector function. Any of the examples of the second antibody.

  In one embodiment, at least one first mutation in the first and second mutants is different.

  In one embodiment, the first variant and the second variant are human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibodies, respectively, optionally human full length IgG1 antibodies, for example Each is a human full-length antibody.

  In one embodiment, the first variant and the second variant are each selected from a monospecific antibody, a bispecific antibody or a multispecific antibody.

  In one further embodiment, the first and second variants bind to different epitopes on the same antigen or on different antigens. Thus, in embodiments where the first and second antibodies are bispecific, each antibody may bind to two different epitopes. The at least two bispecific antibodies may be the same or different. Thus, when bispecific antibodies are different, kits of parts for simultaneous, separate or sequential use in therapy can target up to four different epitopes on the same or different targets. Including.

  In one further embodiment, one or both of the first variant and the second variant is conjugated to a drug, a toxin, a radioactive label, eg, of the first variant and the second variant. One or both are conjugated to the toxin via a linker.

  In one further embodiment, one or both of the first variant and the second variant is part of a fusion protein.

  It is also contemplated that any of the embodiments of the “mixed variant” aspect can be included in any of the kit-of-part embodiments for simultaneous, separate or sequential use in therapy. ing.

  In one embodiment, the variants of the first and second parent antibody bind to an antigen expressed on the same cell.

  In another embodiment, the variant of the first parent antibody comprises an amino acid substitution of K439 with an amino acid selected from E and D.

  In another embodiment, the amino acid substitution in the variant of the first parent antibody is K439E.

  In another embodiment, the variant of the second parent antibody comprises an amino acid substitution of S440 with an amino acid selected from K and R.

  In another embodiment, the amino acid substitution in the variant of the second parent antibody is S440K.

  In another aspect, the present invention relates to a therapeutic method comprising any one first parent polypeptide or parent antibody variant and second parent polypeptide or parent antibody variant of any of the embodiments listed above. It relates to a pharmaceutical kit of parts for simultaneous, separate or sequential use.

  Pharmaceutical kits of parts for simultaneous, separate or sequential use in therapy may be administered by any suitable route and mode. In one embodiment, the pharmaceutical kit of parts of the present invention for simultaneous, separate or sequential use in therapy is administered parenterally. As used herein, the term “administered parenterally” refers to modes of administration other than enteral and topical administration, usually by injection, including the epidermis, intravenous, intramuscular, intraarterial. Intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendon, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, spinal cord, intracranial, intrathoracic, dura mater Includes injections and infusions outside and into the sternum.

Combinations Further, the present invention also provides variant preparations of any “single variant” aspect or embodiment described above, ie, preparations comprising multiple copies of the variant. The present invention also provides compositions, eg, pharmaceutical compositions, comprising variants of any “single variant” aspect and embodiment described above. The invention also provides the use of any such “single variant” variant, preparation or composition as a medicament.

  The invention also provides a combination of variants, wherein one variant comprises at least one variant according to the invention and one variant comprises at least one other variant according to the invention, as well as of such variants Combination preparations and pharmaceutical compositions and their use as pharmaceuticals are also provided. Preferably, the two variants bind to the same antigen or to different antigens that are typically expressed on the surface of the same cell, cell membrane, virion and / or other particle.

All embodiments described herein with reference to a conjugated parent antibody, a first parent antibody or a second parent antibody include other parent polypeptides comprising an immunoglobulin Fc domain and a binding region, It should be understood that it may also be applicable to one parent polypeptide or a second parent polypeptide.

  In one aspect, the invention relates to variants conjugated to drugs, toxins or radiolabels, for example, variants conjugated to toxins via a linker.

  In one embodiment, the variant is part of a fusion protein.

  In another aspect, the variants of the invention are not conjugated at the C-terminus with another molecule such as a toxin or label. In one embodiment, the variant is conjugated to another molecule at another site, typically one that does not interfere with oligomer formation. For example, the antibody variant is linked at the other site with a compound selected from the group consisting of toxins (including radioisotopes), prodrugs or drugs. Such compounds can make target cell killing more effective, for example, in cancer therapy. Accordingly, the resulting variant is an immunoconjugate.

  Accordingly, in one further aspect, the present invention provides an antibody conjugated or conjugated with one or more therapeutic moieties such as cytotoxins, chemotherapeutic agents, cytokines, immunosuppressive agents and / or radioisotopes. provide. Such conjugates are referred to herein as “immunoconjugates” or “drug conjugates”. Immunoconjugates that contain one or more cytotoxins are referred to as “immunotoxins”.

  A cytotoxin or cytotoxic agent includes any agent that is detrimental to (eg, kills) cells. Suitable therapeutic agents for forming the immunoconjugates of the invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy Endiyne antitumor antibiotics, anthracyclines, mitoxan, including anthracindione, maytansine or analogs or derivatives thereof, neocardinostatin, calicheamicins, esperamicins, dynemicins, lidamycin, kedarcidin or analogs or derivatives thereof Throne, mitramycin, actinomycin D, 1-dehydro-testosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, And puromycin, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, etc.), alkylating agents (eg, mechlorethamine, Thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin; And duocarmycin A, duocarmycin SA, CC-1065 (also known as rachelmycin) or an analogue or derivative of CC-1065), dolastatin, pyrrolo [2 , 1-c] [1,4] benzodiacepines (PDB) or their analogs, antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, daunorubicin (formerly daunomycin), doxorubicin, idarubicin, mitra Mycin, mitomycin, mitoxantrone, pricamycin, anthramycin (AMC), etc.), mitotic inhibitors (eg, tubulin inhibitors) such as monomethyl auristatin E, monomethyl auristatin F, or other dolastatin 10 Analogues or derivatives; histone deacetylase inhibitors such as trichostatin A hydroxamic acid, vorinostat (SAHA), belinostat, LAQ824, and panobinostat and benzamides, entinostat, CI994, mosetinostat, and fatty acids Compounds, such as phenylbutyrate and valproic acid, proteasome inhibitors such as danoprevir, bortezomib, amatoxins such as α-amantine, diphtheria toxins and related molecules (eg, diphtheria A chain and its active fragments, and hybrid molecules) ); Ricin toxin (eg, ricin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga toxin-like toxin (SLT-I, SLT-II, SLT-IIV), LT toxin, C3 toxin, Shiga toxin, Pertussis toxin, tetanus toxin, soybean Bowman-Birk protease inhibitor, Pseudomonas aeruginosa exotoxin, alloline, saporin, modesin, geranin, abrin A chain, modesin A chain, α-sarcin, Aleurites fordii protein, Giantin protein, Phytolacca americana protein (PA PI, PAPII and PAP-S), momordica charantia inhibitors, crucine, clotine, sapaonaria officinalis inhibitors, gelonin, mitogenin, restrictocin, phenomycin, and enomycin toxin. Other suitable conjugate molecules include antibacterial / lytic peptides such as CLIP, magainin 2, melittin, cecropin and P18; ribonuclease (RNase), DNase I, staphylococcal enterotoxin-A, pokeweed ( pokeweed) antiviral proteins, diphthelin toxin, and Pseudomonas aeruginosa endotoxin. See, for example, Pastan et al., Cell 47, 641 (1986) and Goldenberg, Calif. A Cancer Journal for Clinicians 44, 43 (1994). Therapeutic agents that can be administered in combination with the antibodies of the invention as described elsewhere herein, such as anti-cancer cytokines or chemokines, are also useful therapeutic moieties for conjugation with the antibodies of the invention. Is a candidate.

  In one embodiment, a drug conjugate of the invention comprises an antibody disclosed herein conjugated with auristatins or auristatin peptide analogs and derivatives (US5635483; US5780588). Auristatin interferes with microtubule dynamics, GTP hydrolysis and nuclear and cell division (Woyke et al (2001) Antimicrob. Agents and Chemother. 45 (12): 3580-3584), and anticancer activity (US5663149) And antifungal activity (Pettit et al., (1998) Antimicrob. Agents and Chemother. 42: 2961-2965). The auristatin drug moiety can also be linked to the antibody via the N (amino) terminus or C (terminal) of the peptide drug moiety by a linker.

  Exemplary auristatin embodiments are disclosed in Senter et al., Proceedings of the American Association for Cancer Research.Volume 45, abstract number 623, published March 28, 2004, and described in US 2005/0238649. N-terminally linked monomethyl auristatin drug moieties DE and DF are included.

  One exemplary auristatin embodiment is MMAE (monomethyl auristatin E). Another exemplary auristatin embodiment is MMAF (monomethyl auristatin F).

  In one embodiment, the antibody of the invention comprises a conjugated nucleic acid or nucleic acid binding molecule. In one such embodiment, the conjugated nucleic acid is a cytotoxic ribonuclease, antisense nucleic acid, inhibitory RNA molecule (eg, siRNA molecule) or immunostimulatory nucleic acid (eg, DNA containing an immunostimulatory CpG motif) Molecule). In another embodiment, the antibody of the invention is conjugated to an aptamer or ribozyme.

  In one embodiment, an antibody is provided that comprises one or more radiolabeled amino acids. Radiolabeled variants can be used for both diagnostic and therapeutic purposes (conjugation with radiolabeled molecules is another possible feature). Non-limiting examples of labels for polypeptides include 3H, 14C, 15N, 35S, 90Y, 99Tc and 125I, 131I and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, eg, Junghans et al., In Cancer Chemotherapy and Biotherapy 655-686 (2nd Ed., Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE 35,500), US 5,648,471 and US 5,697,902). For example, radioisotopes can be conjugated by the chloramine-T method.

In one embodiment, the variants of the invention are conjugated to a radioisotope or a chelate containing a radioisotope. For example, the variant may be conjugated with a chelator linker that allows the antibody to be complexed with a radioisotope, such as DOTA, DTPA or thixetane. Variants may additionally or alternatively include or be conjugated with one or more radiolabeled amino acids or other radiolabeled molecules. Radiolabeled variants can be used for both diagnostic and therapeutic purposes. In one embodiment, the variants of the invention are conjugated with an alpha emitter. Non-limiting examples of radioisotopes include ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹²⁵ I, ¹¹¹ In, ¹³¹ I, ¹⁸⁶ Re, ²¹³ Bs, ²²⁵ Ac and ²²⁷ Th Is included.

  In one embodiment, the variants of the invention are IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL- Select from the group consisting of 23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα, IFNβ, IFNγ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ancestim and TNFα Can be conjugated to the cytokine.

  The variants of the invention can also be chemically modified by covalent conjugates with polymers, for example, to increase their blood half-life. Exemplary polymers and methods for linking them to peptides are described, for example, in US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Other polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (eg, PEG having a molecular weight of about 1,000 to about 40,000, such as about 2,000 to about 20,000).

Hunter et al., Nature 144 , 945 (1962), David et al., Biochemistry 13 , 1014 (1974), Pain et al., J. Immunol. Meth. 40 , 219 (1981) and Nygren, J. Histochem. and Cytochem. 30 , 407 (1982) are used to conjugate the variants of the present invention to conjugated molecules such as those described above using any method known in the art. be able to. Such variants are made by chemically conjugating the other part to the N-terminus or C-terminus of the variant or fragment thereof (eg, antibody H chain or L chain). (See, for example, Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such conjugated mutant derivatives can also be made by conjugation with internal residues or sugars as appropriate.

  These agents can be coupled directly or indirectly to the variants of the invention. An example of indirect coupling of a second agent is coupling through a spacer or linker moiety to a cysteine or lysine residue in a bispecific antibody. In one embodiment, the variant is conjugated via a spacer or linker with a prodrug molecule that can be activated in vivo to become a therapeutic drug. In some embodiments, the linker becomes cleavable under intracellular conditions such that cleavage of the linker releases the drug unit from the antibody in the intracellular environment. In some embodiments, the linker is cleavable by a cleavable substance present in the intracellular environment (eg, inside a lysosome or endosome or caveolae). For example, a spacer or linker can be cleavable by enzymes associated with tumor cells or other tumor-specific conditions, thereby forming an active drug. Examples of such prodrug techniques and linkers are described by Syntarga BV, et al. In WO02083180, WO 2004043493, WO2007018431, WO2007089149, WO2009017394 and WO201062171. Suitable antibody-prodrug technology and duocarmycin analogs are also described in US Pat. No. 6,989,452 (Medarex), incorporated herein by reference. The linker may additionally or alternatively be a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme including, for example, but not limited to lysosomal or endosomal proteases. In some embodiments, the peptidyl linker is at least 2 amino acids long or at least 3 amino acids long. Cleavage substances include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives resulting in the release of active drugs in target cells (eg, Dubowchik and Walker, 1999, Pharm. Therapeutics 83: 67-123). In one specific embodiment, the peptidyl linker cleavable by an intracellular protease is a Val-Cit (valine-citrulline) linker or a Phe-Lys (phenylalanine-lysine) linker (eg, of doxorubicin with a Val-Cit linker). See US6214345 which describes synthesis and various examples of Phe-Lys linkers). Examples of structures of Val-Cit linker and Phe-Lys linker include, but are not limited to, MC-vc-PAB, MC-vc-GABA, MC-Phe-Lys-PAB or MC-Phe-Lys-GABA described below. Where MC is an abbreviation for maleimidocaproyl, vc is an abbreviation for Val-Cit, PAB is an abbreviation for p-aminobenzylcarbamate, and GABA is an abbreviation for γ-aminobutyric acid. One advantage of using intracellular proteolytic release of a therapeutic agent is that the drug is typically attenuated when conjugated and the serum stability of the conjugate is typically high. is there.

  In yet another embodiment, the linker unit is non-cleavable and the drug is released by antibody degradation (see US 2005/0238649). Typically, such linkers are not substantially sensitive to the extracellular environment. As used herein, “not substantially sensitive to the extracellular environment” in the context of a linker means that the variant antibody drug conjugate conjugate is present in the extracellular environment (eg, in plasma). In some cases, 20% or less, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less of the linker in a sample of a variant antibody drug conjugate mixture. Means that only about 3% or less or about 1% or less is cut. Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating the mutant antibody drug conjugate conjugate with plasma for a predetermined period of time (eg, 2, 4, 8, 16 or 24 hours). And subsequently can be determined by quantifying the amount of free drug present in the plasma. An exemplary embodiment comprising MMAE or MMAF and various linker components has the following structure (where Ab means antibody, drug load (or cell division inhibitor or cytotoxicity per antibody molecule) P representing the average number of drugs) is from 1 to about 8, for example, p may be 4-6, such as 3-5, or p is 1, 2, 3, 4, 5, 6, 7 or 8).

  Examples of combining cleavable linkers with auristatin include MC-vc-PAB-MMAF (also referred to as vcMMAF) and MC-vc-PAB-MMAF (also referred to as vcMMAE), where MC is maleimidocaproyl Abbreviation, vc is an abbreviation for a linker based on Val-Cit (valine-citrulline), and PAB is an abbreviation for p-aminobenzylcarbamate.

  Other examples include combinations of auristatin and non-cleavable linkers, such as mcMMAF (where mc (MC is the same as mc in this context) is an abbreviation for maleimidocaproyl).

  In one embodiment, the drug linker moiety is vcMMAE. vcMMAE drug linker moieties and methods of conjugation are disclosed in WO2004010957, US7659241, US7829531, US7851437 and US11 / 833,028 (Seattle Genetics, Inc.), which are incorporated herein by reference. The vcMMAE drug linker moiety is attached to the antibody at the cysteine site using methods similar to those disclosed therein.

  In one embodiment, the drug linker moiety is mcMMAF. mcMMAF drug linker moieties and methods of conjugation are disclosed in US7498298, US 11 / 833,954 and WO2005081711 (Seattle Genetics, Inc.), which are incorporated herein by reference, and include mcMMAF drug linkers The moieties are attached to the variant at the cysteine using methods similar to those disclosed therein.

  In one embodiment, the variants of the invention are coupled with a chelator linker, such as thixetane, that allows the bispecific antibody to be conjugated to a radioisotope.

  In one embodiment, each arm (or Fab arm) of the variant is coupled directly or indirectly to the same one or more therapeutic moieties.

  In one embodiment, only one arm of the variant is coupled directly or indirectly to one or more therapeutic moieties.

  In one embodiment, each arm of the variant is coupled directly or indirectly to a plurality of different therapeutic moieties. For example, as described herein, the variant is a bispecific antibody, prepared by controlled Fab arm exchange of two different monospecific antibodies, e.g., first and second antibodies In certain embodiments, such bispecific antibodies can be obtained by using monospecific antibodies that are conjugated or associated with different therapeutic moieties.

Further uses All embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody include other parent polypeptides comprising an immunoglobulin Fc domain and a binding region, It should be understood that this should also be interpreted as an embodiment relating to one parent polypeptide or a second parent polypeptide.

  In one further aspect, the present invention is for use as a medicament, in particular CDC-mediated of target cells (eg tumor cells, bacterial cells or fungal cells) or target organisms (eg viruses) or cells infected with bacteria or viruses. It relates to a variant of the invention as described herein for use as a medicament for the treatment of a disease or disorder in which sexual lethality is desired. Examples of such diseases and disorders include, but are not limited to, cancer and bacterial infections, viral infections or fungal infections.

  In another aspect, the invention relates to variants, bispecific antibodies, compositions and kits of parts described herein for the treatment of diseases such as cancer.

  In another aspect, the invention relates to a method for the treatment of humans comprising the administration of the variants, compositions or kits of parts described herein.

  In another aspect, the invention relates to a method for the treatment of cancer in humans comprising the administration of a variant, composition or kit of parts.

  “Treatment” refers to the administration of an effective amount of a therapeutically active compound of the present invention for the purpose of alleviating, ameliorating, suppressing, or eradicating (curing) symptoms or disease states.

  “Effective amount” or “therapeutically effective amount” refers to an amount effective to achieve the desired therapeutic result at the required dosage and duration. A therapeutically effective amount of the antibody can vary depending on factors such as the disease state, age, sex and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. A therapeutically effective amount is also an amount that provides a therapeutically beneficial effect over the toxic or adverse effects of the antibody or antibody portion.

  In another aspect, the invention relates to the use of a variant, composition or kit of parts according to any of the embodiments described herein for use in a diagnostic method.

  In another aspect, the present invention provides a variant, composition or kit of parts according to any of the embodiments described herein to at least a part of a human or other mammalian body. It relates to a diagnostic method comprising the step of administering.

  In another aspect, the present invention provides a variant, composition or kit of parts according to any of the embodiments described herein in the imaging of at least a part of a human or other mammalian body. About the use of.

  In another aspect, the present invention relates to at least a human or other mammalian body comprising administering a variant, composition or kit of parts according to any of the embodiments described herein. It relates to a method for imaging a part.

  Without being bound by theory, an effective amount of a therapeutically active compound decreases when any “single variant” aspect or embodiment of the invention is introduced into such therapeutically active compound. there's a possibility that.

  Suitable antigens for cancer antibodies may be the same as those described herein. Examples 15-18 describe specific applications for producing enhanced and / or more specific complement activation or CDC of tumor cells. For example, anti-tumor antibodies of the “single variant” aspect, including E345R mutations, can result in enhanced CDC or ADCC, ADCP responses of tumor cells. In addition, in variants of this method, mutations of the “single variant” aspect, such as E345R, E430, or S440S / W or any other mutation as listed in Table 1, are added to each antibody. Thus, it may also provide an enhanced CDC and / or ADCC response specifically directed to tumor cells that express at least two antigens.

  Suitable antibodies against bacterial infections target S. aureus, for example lipoteichoic acid (LTA) embedded in the cell wall of staphylococci, both of which are incorporated by reference in their entirety Chimeric monoclonal IgG1 pagibaximab described in Baker (Nat Biotechnol. 2006 Dec; 24 (12): 1491-3) and Weisman et al. (Int Immunopharmacol. 2009 May; 9 (5): 639-44) BSYX-A110; Biosynexus) and the like. Example 14 discloses a specific embodiment using S. aureus antibody mutants containing the E345R mutation. However, other mutations in Table 1, including but not limited to E430G and S440W, can also be applied in a similar manner to enhance the CDC-mediated ability of antibodies to bacterial antigens.

  Suitable antigens for viral or fungal infections may be any of those described herein.

  In one embodiment, the antigen to which the variant binds is not human EphA2. In another embodiment, the variant is not derived from human EphA2 mAb 12G3H11 (described in Dall'Acqua et al., Supra, which is hereby incorporated by reference in its entirety). In another embodiment, the antigen to which the variant binds is not IL-9. In another embodiment, the variant is not derived from the Fa-hG1 antibody or Fa-hG4 antibody or any variant thereof described in WO2007005612, which is incorporated herein by reference in its entirety. In one embodiment, the antigen to which the variant binds is not HIV-1 gp120. In another embodiment, the variant is not derived from a b12 human IgG1κ antibody directed against gp120.

  In one particular embodiment, the variant is derived from a bispecific parent antibody. The bispecific antibody may be of any isotype, for example IgG1, IgG2, IgG3 or IgG4, and may be a full-length antibody or an Fc-containing fragment thereof. An exemplary method for preparing bispecific antibodies is described in WO 2008/119353 (Genmab).

All embodiments described herein with reference to a dose parent antibody, first parent antibody or second parent antibody include other parent polypeptides comprising an immunoglobulin Fc domain and a binding region, It should be understood that it may also be applicable to one parent polypeptide or a second parent polypeptide. The effective dose and dosage regimen of an antibody depends on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary, non-limiting range for a therapeutically effective amount of an antibody of the invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as about 0.1-10 mg / kg. For example about 0.5 mg / kg, for example about 0.3, about 1, about 3, about 5 or about 8 mg / kg.

  The antibody variants of the invention can also be administered in combination with one or more complement factors or related components to enhance the therapeutic activity of the variant and / or to supplement complement consumption. . Such complement factors and related components include, but are not limited to, C1q, C4, C2, C3, C5, C6, C7, C8, C9, MBL and factor B. Co-administration may be simultaneous, separate or sequential. In one particular embodiment, the invention comprises a pharmaceutical composition comprising a variant of the invention and at least one complement factor or related component in the same or different pharmaceutical composition together with instructions for use. I will provide a.

  The antibody variants of the invention can also be administered in combination therapy, ie in combination with other therapeutic agents appropriate for the disease or condition being treated. Accordingly, in one embodiment, the antibody-containing medicament is for use in combination with one or more additional therapeutic agents such as cytotoxic agents, chemotherapeutic agents or angiogenesis inhibitors. Such combined administration may be simultaneous, separate or sequential.

  In one further embodiment, the present invention is a method for treating or preventing a disease, such as cancer, for a subject in need thereof in combination with radiation therapy and / or surgery. There is provided a method comprising administering a therapeutically effective amount of a variant or pharmaceutical composition.

Methods of PreparationAll embodiments described herein with reference to a parent antibody, a first parent antibody or a second parent antibody include other parent polypeptides comprising an immunoglobulin Fc domain and a binding region, It should be understood that it may also be applicable to one parent polypeptide or a second parent polypeptide.

  The present invention also provides isolated nucleic acids and vectors encoding variants according to any one of the above aspects, and vectors and expression systems encoding variants. Suitable nucleic acid constructs, vectors and expression systems for antibodies and variants thereof are known in the art and are described in the examples. In embodiments where the variant includes not only the heavy chain (or Fc-containing fragment thereof) but also the light chain, the nucleotide sequence encoding the heavy chain portion and the light chain portion can be on the same or different nucleic acids or vectors.

The present invention also provides a method for producing an antibody variant according to any one of the above aspects in a host cell, wherein the variant comprises at least the Fc region of a heavy chain, wherein the method comprises the following: Provide a method that includes steps:
a) providing a nucleotide construct encoding the Fc region of the variant;
b) expressing the nucleotide construct in a host cell; and
c) recovering the antibody variant from the cell culture of the host cell.

  In some embodiments, the antibody is a heavy chain antibody. However, in most embodiments, the antibody will also contain a light chain, and thus the host cell will also express a construct encoding the light chain, either on the same vector or on a different vector.

  Suitable host cells for recombinant expression of the antibody are well known in the art and include CHO cells, HEK-293 cells, PER-C6 cells, NS / 0 cells and Sp2 / 0 cells. In one embodiment, the host cell is a cell capable of performing Asn-linked glycosylation of a protein, eg, a mammalian cell such as a eukaryotic cell, eg, a human cell. In one further embodiment, the host cell is a non-human cell that has been genetically engineered to produce a glycoprotein with human-like or human glycosylation. Examples of such cells include genetically modified Pichia pastoris (Hamilton et al., Science 301 (2003) 1244-1246; Potgieter et al., J. Biotechnology 139 (2009) 318 -325) and genetically modified Lemna minor (Cox et al., Nature Biotechnology 12 (2006) 1591-1597).

  In one embodiment, the host cell is a host cell that cannot efficiently remove the C-terminal lysine K447 residue from the antibody heavy chain. For example, Table 2 in Liu et al. (2008) J Pharm Sci 97: 2426 (incorporated herein by reference) shows the production of several such antibodies that only result in partial removal of the C-terminal lysine. Lines such as Sp2 / 0, NS / 0 or transgenic mammary glands (goats) are listed. In one embodiment, the host cell is a host cell with altered glycosylation machinery. Such cells have been described in the art and can be used as host cells to express the variants of the invention and thereby produce antibodies with altered glycosylation. For example, Shields, RL et al. (2002) J. Biol. Chem. 277: 26733-26740; Umana et al. (1999) Nat. Biotech. 17: 176-1, and EP 1176195; WO03 / 035835; See WO99 / 54342. Other methods for making engineered glycoforms are also known in the art and are described in Davies et al., 2001, Biotechnol Bioeng 74: 288-294; Shields et al, 2002, J Biol Chem 277: 26733- Shinkawa et al., 2003, J Biol Chem 278: 3466-3473), US6602684, WO00 / 61739A1; WO01 / 292246A1; WO02 / 311140A1; WO02 / 30954A1; Potelligent ™ technology (Biowa , Inc. Princeton, NJ); GlycoMAb ™ glycosylation engineering (GLYCART biotechnology AG, Zurich, Switzerland); US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49 Included without limitation.

  The present invention also relates to an antibody obtained or obtainable by the method of the present invention described above.

  In one further aspect, the present invention relates to a host cell capable of producing the antibody variant of the present invention. In one embodiment, the host cell has been transformed or transfected with a nucleotide construct of the invention.

  The invention is further illustrated by the following examples, which should not be construed as further limiting.

Example 1
Design and production of 7D8 mutant Human monoclonal antibody HuMab-7D8 (described in WO 2004/035607) was used as a model antibody. It belongs to a group of human anti-CD20 IgG1 antibodies including ofatumumab (HuMax-CD20, 2F2). These antibodies target a unique membrane proximal epitope on the CD20 molecule and exhibit strong CDC.

  To investigate the functional relevance of complement activation and oligomeric Fc-Fc interactions in CDC, we aim to impair the Fc-Fc side-on interaction and CDC activity of 7D8. Fc: Amino acids in the hydrophobic patch at the Fc interface were mutated. In the first set of variants (Table 3), to change the charge at the position described in the CH2-CH3 domain exposed in the hydrophobic patch, selected based on the 1HZH crystal structure A mutation was introduced (Burton Mol Immunol 1985 Mar; 22 (3): 161-206)).

  From the first set of mutations, I253D and H433A were found to induce the strongest effect on CDC loss by 7D8 (eg, Example 5). The 1HZH crystal structure indicates that I253 and H433 bind to two different pockets at opposite Fc positions of the partner antibody. Based on these data, a second set of mutations was synthesized around the I253 and H433 positions in the crystal structure to further investigate the importance of residues at the Fc: Fc side-on interface to CDC. The second set of mutations around the I253 and H433 positions that are likely to destabilize the Fc: Fc interface and consequently destabilize the CDC are listed in Table 4.

  Double mutants based on two single mutants that showed loss of CDC to negate the possibility that destruction of the direct binding site for C1q is responsible for the observed effects on CDC Were examined for their ability to restore loss of CDC by a single mutant. This principle is schematically shown in FIG. 1D. Double mutants are listed in Table 5 and structural representations are shown in FIGS. 4 and 5.

  Mutants were prepared using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, a full-length plasmid DNA template encoding a 7D8 heavy chain with IgG1m (f) allotype was replicated using forward and reverse primers encoding the desired mutation. The resulting DNA mixture was digested with DpnI to remove the source plasmid DNA and used for transformation of E. coli. Mutant plasmid DNA isolated from the resulting colonies was examined by DNA sequencing (Agowa, Germany). A plasmid DNA mixture encoding both the heavy and light chains of the antibody is transiently transferred to Freestyle HEK293F cells (Invitrogen, US) using 293fectin (Invitrogen, US) essentially as described by the manufacturer. Transfected.

(=)荷電なし
(-)負荷電
(+)正荷電
(δ+)部分的正荷電 (Table 3) Set 1 mutations introduced into the CH2-CH3 domain of 7D8

(=) No charge
(-) Negative load
(+) Positive charge
(δ +) partial positive charge

(=)荷電なし
(-)負荷電
(+)正荷電
(δ+)部分的正荷電 (Table 4) Set 2 mutations introduced into the CH2-CH3 domain of 7D8

(=) No charge
(-) Negative load
(+) Positive charge
(δ +) partial positive charge

(=)荷電なし
(-)負荷電
(+)正荷電 (Table 5) Double mutations introduced into the CH2-CH3 domain of 7D8 to combine two single mutations, each indicating loss of CDC

(=) No charge
(-) Negative load
(+) Positive charge

Example 2
CD20 binding on cells by 7D8 mutants Binding of purified antibody samples to CD20 positive cells was analyzed by FACS analysis. The first set of mutations (Table 3) was tested in Daudi cells, and the second set of mutations (Table 4) was tested in Raji cells. Serial dilutions of antibody preparations in RPMI 1640 / 0.1% BSA in 50 μL of 10 ⁵ cells in polystyrene 96-well round bottom plates (Greiner bio-one 650101) (first set against Daudi Incubate for 30 minutes at 4 ° C. with a 3-fold dilution in the range of 0.04-10 μg / mL, and for the second set for Raji, a 3-fold dilution in the range of 0.003-10 μg / mL). After washing twice with RPMI 1640 / 0.1% BSA, the cells were incubated with secondary antibody in 100 μL at 4 ° C. for 30 minutes. Secondary antibodies include fluorescein isothiocyanate (FITC) -conjugated rabbit-anti-human IgG (F0056, Dako, Glostrup, Denmark; 1/100) for all experiments on Daudi cells and 7D8 antibody on Raji cells. Using. For experiments with purified 7D8 antibody against Raji cells, R-phycoerythrin (R-PE) -conjugated goat F (ab ') ₂ anti-human kappa light chain (2062-09, SouthernBiotech; 1/500) was used as the secondary antibody . Next, the cells were washed twice with PBS / 0.1% BSA / 0.02% azide, resuspended in 100 μL PBS / 0.1% BSA / 0.02% azide, and then FACS Cantoll (BD Biosciences). analyzed. Binding curves were analyzed using non-linear regression (sigmoidal dose response with variable slope) using GraphPad Prism V5.01 software (GraphPad Software, San Diego, CA, USA).

  The binding of 7D8 antibody to Daudi cells was unaffected by the introduction of point mutations in the CH2-CH3 domain and was identical for all mutants tested and wild type 7D8. Furthermore, the binding of the 7D8 antibody to Raji cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain compared to wild type 7D8, except for E345R. At test concentrations above 0.3 μg / mL, decreased binding of IgG1-7D8-E345R to CD20 positive Raji cells was detected. Similarly for H433D and H433R, a decreased binding was detected at the highest antibody concentration tested (10 μg / mL). Since direct labeling of E345R and H433R resulted in similar or even enhanced binding in Daudi cells, the decreased binding by IgG1-7D8-E345R, H433D and H433R is explained by the shielding of the secondary antibody epitope It is considered possible. This enhancement of binding power can be explained by the enhancement of Fc-Fc side-on binding by E345R and H433R compared to wild type IgG1-7D8.

  Combining the K439E and S440K mutations did not affect the binding of the 7D8 antibody to Raji cells and was identical to that of the single mutant and wild type 7D8.

Example 3
C1q binding ELISA with 7D8 mutant
We tested for C1q binding by the 7D8 mutant in an ELISA that coats the purified antibody onto a plastic surface resulting in random antibody multimerization. Pooled human serum was used as the source of C1q.

A 96-well Microlon ELISA plate (Greiner, Germany) was coated overnight at 4 ° C. with serial dilutions of antibody in PBS (range 1.58 to 10.0 μg / mL at 1.5 fold dilution). Plates were washed and blocked with 200 μL / well 0.5 × PBS with 0.025% Tween 20 and 0.1% gelatin. 1 hour at 37 ° C with 3% pooled human serum (Sanquin, product number M0008), 1 hour at room temperature with 100 μL / well rabbit anti-human C1q (DAKO, product number A0136, 1/4000) with washing between incubations , And 100 μL / well porcine anti-rabbit IgG-HRP (DAKO, P0399, 1: 10,000) as a detection antibody, and sequentially incubated for 1 hour at room temperature. Development was carried out with 1 mg / mL 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfate) (ABTS; Roche, Mannheim, Germany) for about 30 minutes. The reaction was stopped by the addition of 100 μL of 2% oxalic acid. Absorbance was measured at 405 nm with a microplate reader (Biotek, Winooski, VT). Log-transformed data was analyzed by fitting a sigmoidal dose response curve with a variable slope using GraphPad Prism software. Mutant EC ₅₀ values were normalized to wild type IgG1-7D8 per plate and multiplied against the average of all wild type IgG1-7D8 data.

As shown in FIG. 6 and Table 6, the point mutations tested had little effect on C1q binding as measured by ELISA. For the IgG1-7D8-I253D mutant, somewhat less efficient C1q binding was measured by ELISA (higher EC ₅₀ values). The activity of the coating was tested for all antibodies and found to be comparable for all antibodies.

¹ 平均およびSDは、少なくとも3回の実験から算出した。
² 統計：Dunnett多重比較検定（GraphPad Prism 5.01）を用いた対数変換データに対する一元配置ANOVA。有意性は野生型IgG1-7D8との比較で算出した：（na）該当なし （ns）非有意 （^*）p=0.01〜0.05 （^**）p=0.001〜0.01 （^***）p＜0.001。 Table 6 EC ₅₀ for C1q binding in ELISA

¹ Mean and SD were calculated from at least 3 experiments.
² Statistics: One-way ANOVA for log-transformed data using Dunnett's multiple comparison test (GraphPad Prism 5.01). Significance was calculated by comparison with wild type IgG1-7D8: (na) Not applicable (ns) Not significant ( ^* ) p = 0.01-0.05 ( ^** ) p = 0.001-0.01 ( ^*** ) p <0.001 .

Example 4
Coating an antibody on a C1q-binding plastic surface on a cell with the 7D8 variant provides an artificial resting system for antibody binding and Fc tail presentation. Therefore, complement binding was also tested by a cell-based assay. Here, C1q binding in CD20 positive B cells opsonized with antibodies was measured by FACS analysis. In experiments using set 1 mutants, Daudi or Raji cells were suspended on ice in 90 μL RPMI 1640 medium containing 10% FBS (2 × 10 ⁶ cells / mL). A 10 μL C1q concentration series (Complement Technologies, Tyler, TX) was added (the final concentration range varies between 0-60 μg / mL and 0-140 μg / mL, depending on maximum binding). Subsequently, 10 μL of purified antibody (final concentration 10 μg / mL, ie saturating concentration) was added and the reaction mixture was immediately transferred to a 37 ° C. water bath and incubated for 1 hour. In experiments with set 2 mutants, test mAbs were added in large amounts to Daudi cells, followed by various concentrations of C1q in aliquots, and the mixtures were incubated as described above. Cells were washed 3 times with PBS / 1% BSA and incubated with rabbit FITC-labeled anti-C1q antibody (DakoCytomation, 10 ug / mL) for 30 minutes at room temperature. Cells were washed with PBS / 1% BSA and resuspended in PBS or fixed in PBS containing 2% formamide. Flow cytometry was performed on a FACSCalibur flow cytometer (BD Biosciences) and the average fluorescence intensity was converted to soluble fluorophore equivalents (MESF) using calibrated beads (Spherotech). Binding dissociation constant of C1q in designated 7D8 antibody to opsonized CD20-positive cells (K _D value) was calculated using SigmaPlot (R) software (Systat Software Inc., Washington). (4 times in Daudi cells, three times in Raji cells) repeat binding experiment an average K _D values were calculated by comparing the K _D values relating C1q binding on cells opsonized with wild-type 7D8 (Table 7 and Table 8).

The same results were obtained when the set 1 mutants were tested on both Daudi and Raji cells. In contrast to the C1q ELISA results, most of the mutants tested had decreased C1q binding (K _D increased) in both Daudi cells (Table 7A) and Raji cells (Table 8) opsonized with antibodies. showed that. Compared to wild type 7D8, IgG1-7D8-Q311A and H435A show little or no reduction in C1q binding in opsonized Daudi or Raji cells, and I253A, I253Y and N434A show a more pronounced reduction. I253D and H433A showed a very dramatic decline. 7D8-H435R is somewhat higher avidity than wildtype 7D8 in both cell types of the surface with respect to C1q binding showed (lower K _D), it was not significant.

Set 2 mutants were tested in Daudi cells. Compared to wild-type 7D8, IgG1-7D8-E345R, E382R and H433R are exhibited enhanced avidity for opsonized Daudi cells, which is reflected in a lower K _D values (Table 7B). All other variants of Set 2 showed reduced binding strength compared to the wild-type 7D8, G385D, Y436D, Q438D, but K439E and S440K showed dramatically increased K _D values (Table 7B) , it could not be measured K _D values with H433D and Y436C in reliability shown so dramatically reduced binding.

  The double mutant IgG1-7D8-K439E / S440K showed restoration of C1q binding to Daudi cells opsonized with antibodies, whereas both single mutants showed C1q binding compared to wild type 7D8. Showed a decline. The binding power of the K439E / S440K double mutant was even somewhat enhanced compared to wild type 7D8 (Table 7C). A mixture of single mutant IgG1-7D8-K439E and IgG1-7D8-K440E was able to fully restore C1q binding, which was equivalent to that of wild type 7D8 (Table 7C).

  The IgG1-7D8 variant did not alter C1q binding in the ELISA (Example 3), and the C1q binding was affected in the cell-based assay is due to the test CH3 position involved in Fc: Fc interaction between antibody molecules However, it does not directly affect C1q binding, but when bound on a cell, it affects the dynamic placement of the antibody Fc tail and, as a result, is an important determinant that also affects the strength of C1q binding. It shows that there is.

^* 野生型7D8との比較（t-検定）
^**（na）該当なし (Table 7A) K _D values for C1q binding to the Daudi cells opsonized with antibodies (variant Set 1)

^* Comparison with wild type 7D8 (t-test)
^** (na) Not applicable

^* 野生型7D8との比較（t-検定）
^**（na）該当なし
^*** 7D8の平均K_Dは実験1、2、3、4、10および11から算出した。
^**** これらの変異体の結合が弱すぎるため、信頼性のある適合曲線およびK_D値を測定することができなかった。 (Table 7B) K _D values for C1q binding to the Daudi cells opsonized with antibodies (variant Set 2)

^* Comparison with wild type 7D8 (t-test)
^** (na) Not applicable
Mean K _D of ^*** 7D8 was calculated from the experimental 1,2,3,4,10 and 11.
^**** For the binding of these mutants too weak, it was not possible to measure a reliable fit curve and K _D values.

^* 野生型7D8との比較（t-検定）
^**（na）該当なし
^*** 7D8の平均K_Dは実験1、2、3、4、10および11から算出した。 (Table 7C) K _D values for C1q binding to the Daudi cells opsonized with antibodies (double mutant)

^* Comparison with wild type 7D8 (t-test)
^** (na) Not applicable
Mean K _D of ^*** 7D8 was calculated from the experimental 1,2,3,4,10 and 11.

^* 野生型7D8との比較（t-検定）
^**（na）該当なし (Table 8) K _D values for C1q binding to Raji cells opsonized with antibodies (variant Set 1)

^* Comparison with wild type 7D8 (t-test)
^** (na) Not applicable

Example 5
C1q activity by 7D8 mutant in CD20 assay in CD20 positive Raji cells C1q activity using cells opsonized with IgG1-7D8 mutant was examined in order to investigate the effect of observed C1q binding changes on CDC activity. Tested in a CDC assay. That is, a CDC assay was performed using C1q-depleted normal human serum to which a predetermined concentration series of C1q was added. 0.1 × 10 ⁶ Raji cells in round bottom 96-well plates (Nunc, Rochester, NY) in 10 μg / mL purified antibody and human C1q in RPMI 1640 medium with a total volume of 100 μL plus 0.1% BSA And a concentration series (0.005, 0.025, 0.1, 0.3, 1.0, 5.0, 30.0 μg / mL) for 15 minutes at room temperature. Next, 25 μL of C1q-depleted serum (Quidel, San Diego, Calif.) Was added and incubated in a water bath for 30 minutes at 37 ° C. or incubated for 45 minutes in an incubator. After incubation, the reaction was stopped by placing the sample on ice. Cell lysis was determined by FACS by using a propidium iodide (PI, Sigma Aldrich, Zwijndrecht, Netherlands) live cell exclusion assay. The lysis rate (% lysis) was determined as follows: lysis rate = (number of PI positive cells / total number of cells) × 100%.

The lysis with wild type 7D8 in the presence of 30 μg / mL C1q minus the lysis when no C1q was added was set to 100%. CH ₅₀ values (C1q concentration resulting in 50% lysis) were calculated by fitting sigmoidal dose response curves to log-transformed data using GraphPad Prism software. Mutant CH ₅₀ values were normalized to wild-type 7D8 (Table 9).

  The data in Table 9 shows that IgG1-7D8-Q311A, E382R and H435A did not show a decrease in C1q activity, consistent with the measured C1q binding force; I253A, I253Y, G385D, N434A and Y436C has a significant decrease in C1q activity; I253D, H310K, K322A, H433A, H433D, Y436D, Q438D, K439E and S440K almost completely lose the ability to induce CDC at all tested C1q concentrations. It was.

  IgG1-7D8-H435R and H433R utilized C1q somewhat more efficiently than wild type 7D8, resulting in more efficient CDC. IgG1-7D8-E345R showed a dramatic enhancement of C1q activity, which resulted in significantly higher CDC lysis compared to wild type 7D8 (Table 9).

  Figure 7 shows that when both mutations are combined in one molecule (K439E / S440K double mutant) by combining the K439E and S440K mutations, both resulting in CDC loss as a single mutant. Or when both single mutants were combined (K439E + S440K mixture), CDC in the C1q activity assay was restored.

⁽¹⁾(n)実験の数
⁽²⁾平均およびSDは、実施したすべての実験から算出した。
⁽³⁾統計：Dunnett多重比較検定（GraphPad Prism 5.01）を用いた対数変換データに対する一元配置ANOVA。有意性は野生型IgG1-7D8との比較で算出した：（na）該当なし （nd）未決定 （ns）非有意 （^*）p=0.01〜0.05 （^**）p=0.001〜0.01 （^***）p＜0.001。
⁽⁴⁾溶解が50％に達しない場合には、CH₅₀を＞30μg／mLに設定した
⁽⁵⁾溶解度50％に達しなかった変異体についてはP値を決定することができなかった。しかし、これらはIgG1-7D8-WTとは有意に異なるものと推測される。 Table 9 CH ₅₀ for C1q activity in CDC assay in Raji cells

⁽¹⁾ (n) Number of experiments
⁽²⁾ Mean and SD were calculated from all experiments performed.
⁽³⁾ Statistics: One-way ANOVA for log-transformed data using Dunnett's multiple comparison test (GraphPad Prism 5.01). Significance was calculated by comparison with wild type IgG1-7D8: (na) Not applicable (nd) Not determined (ns) Not significant ( ^* ) p = 0.01-0.05 ( ^** ) p = 0.001-0.01 ( ^{** *} ) P <0.001.
^{(4) If} dissolution did not reach 50%, CH ₅₀ was set to> 30 μg / mL
⁽⁵⁾ P values could not be determined for mutants that did not reach 50% solubility. However, these are assumed to be significantly different from IgG1-7D8-WT.

Example 6
CDC with 7D8 mutant in CDC assay on CD20 positive cells
0.1 × 10 ⁶ cells in a round bottom 96-well plate (Nunc, Rochester, NY) in a total volume of 80 μL, antibody concentration series (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / mL) and preincubation for 15 minutes at room temperature on a shaker. Next, 20 μL of normal human serum was added as a source of C1q (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by adding 30 μL ice-cold RPMI medium supplemented with 0.1% BSA. Cell lysis was determined by FACS by using propidium iodide.

For CDC assays on Daudi cells, EC ₅₀ values (antibody concentration that resulted in a 50% lysis rate) were calculated by fitting a sigmoidal dose response curve to log-transformed data using GraphPad Prism software. Mutant EC ₅₀ values were normalized to wild-type 7D8 (Table 10 and Table 11).

Table 10 shows that in Daudi cells IgG1-7D8-I253A, Q311A, E382R, H433R and H435A showed no difference compared to CDC wild type 7D8;
IgG1-7D8-I253D, I253Y, H310K, G385D, H433A, H433D, N434A, Y436C, Y436D, Q438D, K439E, S440K and I253D / H433A have significantly worse CDC (higher EC ₅₀ ) than wild type 7D8 Found, they only induced CDC at high antibody concentrations; the C1q binding-deficient mutant IgG1-7D8-K322A, included as a control, almost lost the ability to induce CDC and was tested The EC ₅₀ was not reached at the concentrations tested; IgG1-7D8-H435R showed more efficient CDC in Daudi cells than wild type 7D8. Importantly, consistent with the C1q activity CDC assay, E345R showed dramatically better CDC in Daudi cells than wild type 7D8, with an EC ₅₀ value that was 10 times lower (Table 10). ). Figure 8 shows that when both mutations are combined in one molecule (K439E / S440K double mutant) by combining the K439E and S440K mutations, both resulting in loss of CDC as single mutants. Or when both single mutants were combined (K439E + S440K mixture), CDC was restored.

  Table 11 shows that similar data were found for the IgG1-7D8 mutant in Raji cells.

⁽¹⁾（n）実験の数
⁽²⁾平均およびSDは、実施したすべての実験から算出した。
⁽³⁾統計：Dunnett多重比較検定（GraphPad Prism 5.01）を用いた対数変換データに対する一元配置ANOVA。有意性は野生型7D8との比較で算出した：（na）該当なし （nd）未決定 （ns）非有意 （^*）p=0.01〜0.05 （^**）p=0.001〜0.01 （^***）p＜0.001。
⁽⁴⁾溶解が50％に達しない場合には、EC₅₀を＞30μg／mLに設定した
⁽⁵⁾EC₅₀に達しなかった変異体についてはP値を決定することができなかった。しかし、これらは野生型7D8-WTとは有意に異なるものと推測される。 Table 10 EC ₅₀ calculated from CDC assay in Daudi cells

⁽¹⁾ (n) Number of experiments
⁽²⁾ Mean and SD were calculated from all experiments performed.
⁽³⁾ Statistics: One-way ANOVA for log-transformed data using Dunnett's multiple comparison test (GraphPad Prism 5.01). Significance was calculated by comparison with wild type 7D8: (na) Not applicable (nd) Not determined (ns) Not significant ( ^* ) p = 0.01-0.05 ( ^** ) p = 0.001-0.01 ( ^*** ) p <0.001.
^{(4) If} dissolution did not reach 50%, EC ₅₀ was set to> 30 μg / mL
⁽⁵⁾ P values could not be determined for mutants that did not reach EC ₅₀ . However, it is speculated that these are significantly different from wild type 7D8-WT.

⁽¹⁾（n）実験の数
⁽²⁾平均およびSDは、実施したすべての実験から算出した。
⁽³⁾統計：Dunnett多重比較検定（GraphPad Prism 5.01）を用いた対数変換データに対する一元配置ANOVA。有意性は野生型7D8との比較で算出した：（na）該当なし （nd）未決定 （ns）非有意 （^*）p=0.01〜0.05 （^**）p=0.001〜0.01 （^***）p＜0.001。
⁽⁴⁾CH₅₀に達しない場合には、CH₅₀を＞30μg／mLに設定した
⁽⁵⁾EC₅₀に達しなかった変異体についてはP値を決定することができなかった。しかし、これらは野生型7D8-WTとは有意に異なるものと推測される。 Table 11 EC ₅₀ calculated from CDC assay in Raji cells

⁽¹⁾ (n) Number of experiments
⁽²⁾ Mean and SD were calculated from all experiments performed.
⁽³⁾ Statistics: One-way ANOVA for log-transformed data using Dunnett's multiple comparison test (GraphPad Prism 5.01). Significance was calculated by comparison with wild type 7D8: (na) Not applicable (nd) Not determined (ns) Not significant ( ^* ) p = 0.01-0.05 ( ^** ) p = 0.001-0.01 ( ^*** ) p <0.001.
^{(4) If} CH ₅₀ was not reached, CH ₅₀ was set to> 30 μg / mL
⁽⁵⁾ P values could not be determined for mutants that did not reach EC ₅₀ . However, it is speculated that these are significantly different from wild type 7D8-WT.

Example 7
Ranking of 7D8 mutants by CDC inducibility For 7D8 mutants tested, between the C1q binding assay in Daudi cells (described in Example 4) and the C1q activity assay in Raji cells (described in Example 5), as well as Daudi A correlation was found between the C1q binding assay in cells and the CDC assay in Daudi and Raji cells (described in Example 6) (correlation data table 13). Therefore, by using the K _D values of C1q binding assay in Daudi cells, all 7D8 variants tested, as shown in Table 12, were ranked according to their ability to induce CDC.

^* 信頼性のある適合曲線がない。イタリック体のK_D値は、これらの変異体の結合が弱すぎるため、測定することができなかった。 (Table 12) were all tested for 7D8 variants, they serve as representing the ability to induce CDC, about C1q binding in Daudi cell surface, the order of ranking from those _the K _D value is high

^* There is no reliable fitting curve. K _D values in italics, because the binding of these mutants too weak, could not be measured.

Table 13: Correlation between C1q binding assay in Daudi cells (Example 4) and C1q activity assay in Raji cells (Example 5), and C1q binding assay in Daudi cells and CDC assay in Daudi and Raji cells Correlation between and (Example 06). The data was log transformed before analyzing for correlation.

Example 8
Design and production of CD38 antibody 005 variants Human monoclonal antibody HuMab 005 is a fully human IgG1, κ antibody described in WO / 2006/099875. It was used herein as a model antibody to verify that the identified Fc mutation enhances CDC activity. The mutations tested are listed in Table 14.

  DNA constructs were prepared for the various mutants and transiently transfected as described in Example 1, using the heavy chain of HuMab 005 with the IgG1m (f) allotype as a template for the mutagenesis reaction. .

(=)荷電なし
(-)負荷電
(+)正荷電
(δ+)部分的正荷電 (Table 14) Set of mutations introduced into CH2-CH3 domain of 005 (HuMax-CD38)

(=) No charge
(-) Negative load
(+) Positive charge
(δ +) partial positive charge

Example 9
CD38 binding on cells by HuMab-005 mutant
Binding of unpurified antibody samples to CD38 positive Daudi cells and Raji cells was analyzed by FACS analysis. 10 ⁵ cells were serially diluted (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 0.01%, 0.03, 0.1, 0.3, 1.0, 3.0) in 100 μL in a 96-well round bottom plate made of polystyrene in RPMI 1640 / 0.1% BSA. 10.0, 30.0 μg / mL) and incubation at 4 ° C. for 30 minutes. After washing twice with RPMI 1640 / 0.1% BSA, the cells were combined with FITC-conjugated rabbit F (ab ′) ₂ anti-human IgG (Catalog No. F0056; DAKO; 1: 150) in 50 μL at 4 ° C. for 30 minutes. Incubated. The cells were then washed twice with PBS / 0.1% BSA / 0.02% azide, resuspended in 100 μL PBS / 0.1% BSA / 0.02% azide and analyzed with FACS Cantoll (BD Biosciences). . The binding curve was analyzed using GraphPad Prism V5.01 software. As a negative control, mock-transfected cell supernatant was used.

  HuMab 005 binding to Daudi cells was not significantly affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound Daudi cells in a dose-dependent manner. Except for 005-E345R, which showed somewhat reduced binding, all of the mutants tested had similar binding to wild-type HuMab-005. However, although not bound by any theory, the lower binding may be the result of reduced binding by the secondary antibody, similar to IgG1-7D8-E345 in Example 2. Although the actual binding force with 005-E345R may be similar or even enhanced compared to 005-WT, we do not have this because there is no directly labeled antibody. I could not confirm.

  HuMab-005 binding to Raji cells was also not significantly affected by the introduction of point mutations in the CH2-CH3 domain. All tested antibodies bound Raji cells in a dose-dependent manner. Maximum binding was comparable for the 005-I253D and H433A mutants to that of wild type 005, and was lower for the 005-E435R, K439E, S440K mutant and 005-K439E + 005-S440K combinations. However, although not bound by any theory, the lower binding may be the result of decreased binding by the secondary antibody, similar to IgG1-7D8-E345R (epitope masking) in Example 2. There is.

Example 10
CDC assay on CD38 positive cells with a variant of CD38 antibody 005
0.1 × 10 ⁶ Daudi cells or Raji cells in a round bottom 96-well plate with a total volume of 100 μL and a concentration series of unpurified antibodies (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / ML) and preincubation for 15 minutes at room temperature on a shaker. Next, 25 μL of normal human serum was added as a source of C1q (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

Further analysis of the CDC-enhancing ability of the E435R mutation shown to enhance the CDC activity of both 7D8 and 005 antibodies in Daudi and Raji cells using various concentrations of normal human serum (NHS) in Wien133 cells did. 0.1 × 10 ⁶ Wien133 cells in a round bottom 96-well plate with a total volume of 50 μL and a concentration series of non-purified antibodies (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / mL) and preincubation for 15 minutes at room temperature on a shaker. NHS was then added as a source of C1q in a total volume of 100 μL until a final concentration of either 20% or 50% NHS was reached. The reaction mixture was incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  Mutations identified in the CH2-CH3 region that resulted in loss or enhancement of CDC activity with respect to the CD20 antibody 7D8 were found to have the same effect on the 005 antibody that recognizes CD38. FIG. 9 shows that 005-I253D, H443A, K439E and S440K show a complete loss of CDC activity in both Daudi cells (FIG. 9A) and Raji cells (FIG. 9B), while the 005-E345R mutant is in any cell This shows that the strain also showed strongly enhanced CDC activity. Equivalent to the 7D8 data, the combination of 005-K439E + 005-S440K, both resulting in loss of CDC as a single mutant, resulted in recovery of CDC. Surprisingly, 005-E435R also strongly induced CDC in Wien133 cells, where wild type 005 cannot induce CDC lethality (FIG. 9C). CDC lethality by 005-E345R in Wien133 cells was observed at both 20% and 50% serum concentrations (FIG. 9C). Also in Raji cells, both 7D8-E345R and 005-E345R showed in vitro CDC enhancement in 50% serum, with similar activity in 20% serum (FIG. 9D).

  The E345R mutation in the CH2-CH3 region resulted in enhanced CDC activity in both the CD20 antibody 7D8 and CD38 antibody 005 tested, so the E345R mutation can be applied to induce or enhance CDC. It is thought that.

Example 11
IgG1 antibody containing CDC enhancing mutation E345R is less sensitive to inhibition of CDC by Fc-binding peptide DCAWHLGELVWCT (SEQ ID NO: 19) than wild-type antibody
It has been found that by mutating amino acid positions in hydrophobic patches at the Fc: Fc interface of IgG, the activity of CDC is impaired or enhanced. We further investigated the interaction at the Fc-Fc interface, as observed in the b12 crystal structure, and possibly the formation of oligomer (eg, hexamer ring) structure on the activity of CDC. That is, a 13-residue peptide (DCAWHLGELVWCT (SEQ ID NO: 19)) that targets a consensus binding site in the hydrophobic patch region on the surface of wild-type IgG Fc was used (Delano et al., Science 2000 Feb). 18; 287 (5456): 1279-83). In fact, the entity of a consensus binding site on the surface of IgG Fc as an adaptation region provided for interaction with a variety of different molecules (Delano et al., Science 2000 Feb 18; 287 (5456): 1279- 83) is consistent with the identity of the core amino acid in the hydrophobic patch involved in the Fc-Fc interaction in the IgG1 b12 crystal structure (Saphire et al., Science 2001 Aug 10; 293 (5532): 1155-9). The interactions present at all of the binding interfaces are a common set of six amino acids (Met-252, Ile-253, Ser-254, Asn-434, His-435 and Tyr-436), as well as common skeletal contacts (Delano et al., Science 2000 Feb 18; 287 (5456): 1279-83). Therefore, Fc-binding peptides are expected to affect the Fc-Fc interaction and consequently to CDC activity.

0.1 × 10 ⁶ Daudi cells were preincubated in a round bottom 96-well plate in 75 μL with 1.0 μg / mL unpurified antibody for 10 minutes on a shaker at room temperature. 25 μL of a concentration series of Fc-binding peptide DCAWHLGELVWCT (SEQ ID NO: 19) (final concentration range 0.06-60 μg / mL) was added to the opsonized cells and incubated at room temperature on a shaker for 10 minutes. Next, 25 μL NHS was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by adding 25 μL ice-cold RPMI medium supplemented with 0.1% BSA. 15 μL of propidium iodide was added and cell lysis was determined by FACS analysis.

  CDC mediated by wild type 005 (FIG. 10A) or 7D8 (FIG. 10B) was found to be inhibited in a dose-dependent manner by the Fc-binding peptide DCAWHLGELVWCT (SEQ ID NO: 19). These competition data also suggest that the Fc-Fc interaction in the hydrophobic patch of IgG is involved in CDC activity. Both CD1-enhanced IgG1-005-E345R and IgG1-7D8-E345R mutants are less sensitive to competition by Fc-binding peptides compared to their corresponding wild-type antibodies, indicating that the E345R mutation It suggests that it enhances the stability of the Fc-Fc interaction, resulting in enhanced CDC.

Example 12
Antibody-dependent cell-mediated cytotoxicity (ADCC) of CD38-expressing cells by a variant of CD38 antibody HuMAb 005
Daudi cells were collected (5 × 10 ⁶ cells / ml), washed (twice with PBS, 1200 rpm, 5 minutes), and then 10% cosmic calf serum (CCS) (HyClone, Logan , UT, USA) in 1 mL RPMI1640 medium plus 200 μCi ⁵¹ Cr (Chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, Netherlands). The mixture was incubated for 1 hour at 37 ° C. in a shaking water bath. After cell washing (twice in PBS, 1200 rpm, 5 min), cells were resuspended in RPMI 1640 medium supplemented with 10% CCS and counted by trypan blue exclusion, 1 × 10 ⁵ cells / Dilute to a concentration of mL.

Meanwhile, from a fresh buffy coat (Sanquin, Amsterdam, Netherlands) using standard Ficoll density gradient centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France), peripheral blood mononuclear cells ( PBMC) was isolated. After resuspension of the cells in RPMI 1640 medium supplemented with 10% CCS, the cells were counted by trypan blue exclusion and concentrated to 1 × 10 ⁷ cells / mL.

For ADCC experiments, 50 μL of ⁵¹ Cr-labeled Daudi cells (5,000 cells) in a 96-well microtiter plate with a total volume of 100 μL, 15 μg / mL CD38 antibody IgG1 in RPMI medium supplemented with 10% CCS Pre-incubated with -005 or mutant IgG1-005-E345R. After 10 minutes at room temperature, 50 μL of PBMC (500,000) was added to achieve an effector-target ratio of 100: 1. The maximum amount of cell lysis was determined by incubating 50 μL of ⁵¹ Cr labeled Daudi cells (5,000) with 100 μL of 5% Triton-X100. The amount of spontaneous lysis was determined by incubating 5,000 ⁵¹ Cr-labeled Daudi cells in 150 μL medium without antibodies or effector cells. The level of cell lysis unrelated to antibody was determined by incubating 5,000 Daudi cells with 500,000 PBMC without antibody. The cells were then incubated for 4 hours at 37 ° C., 5% CO ₂ . To determine the amount of cell lysis, the cells were centrifuged (1200 rpm, 3 minutes), 75 μL of the supernatant was transferred to a micronic tube, and then the released ⁵¹ Cr was counted using a gamma counter . Using the measured counts per minute (cpm), the percentage of antibody-mediated lysis was calculated as follows:
(Sample cpm-cpm of lysis unrelated to Ab) / (maximum lysis cpm-spontaneous lysis cpm) x 100%

Table 15 shows the calculated EC ₅₀ values for IgG1-005-wt and IgG1-005-E345R in the ADCC assay performed. Four samples were tested. IgG1-005-E345R shows a significantly lower EC ₅₀ value than IgG1-005-wt in all four samples tested.

(Table 15) Calculated EC50 values of the four experiments performed

  FIG. 11 clearly shows that the mutant IgG1-005-E345R showed enhanced ADCC ability activity compared to the wild-type antibody HuMab-005, and was able to induce ADCC at lower concentrations.

Example 13
FcRn binding and pharmacokinetic analysis of 7D8 mutants compared to wild-type 7D8 The neonatal Fc receptor (FcRn) contributes to the long plasma half-life of IgG by protecting it from degradation. After antibody intracellularization, FcRn binds to the antibody Fc region within the endosome, but in a mild acidic environment (pH 6.0) where the interaction is stable. When the environment is recycled to a neutral (pH 7.4) plasma membrane, the interaction is lost and the antibody is released back into the bloodstream. This affects the plasma half-life of IgG.

  The ability of the 7D8 mutant IgG1-7D8-E354R to interact with FcRn from mice, cynomolgus monkeys and humans was tested in ELISA. All incubations were at room temperature. By biotinylated extracellular domain (FcRnECDHis-B2M-BIO) of recombinantly prepared FcRn (mouse, human or cynomolgus) diluted in PBST with 0.2% BSA, 5 μg / mL (100 μL / well) A 96-well plate was coated for 1 hour. Plates were washed 3 times with PBST and wild type IgG1-7D8 or IgG1-7D8-E354R diluted in 3-fold series (in PBST / 0.2% BSA, pH 6.0) was added and the plates were incubated for 1 hour. Plates were washed with PBST / 0.2% BSA, pH 6.0. Goat-anti-human IgG (Fab'2) -HRP (Jackson Immuno Research, catalog number: 109-035-097) diluted in PBST / 0.2% BSA, pH 6.0 was added and the plates were incubated for 1 hour. After washing, ABTS was added as a substrate and the plate was incubated for 30 minutes in the dark. Absorbance was read at 405 using an EL808 ELISA reader.

  The mice in this study were housed in a barrier unit of the Central Laboratory Animal Facility (Utrecht, The Netherlands), placed in a filter top cage, and freely fed water and feed. All experiments were approved by the Animal Ethics Committee at Utrecht University.

  To analyze the in vivo pharmacokinetics of 7D8 mutants, 100 μg (5 mg / kg) of wild-type 7D8, IgG1-7D8-E354R, -S440K or K322A were used in SCID mice (CB-17 / IcrCrl-scid-BR, Charles-River) was injected intravenously; 3 mice per group.

  Blood samples of 50 μL were collected from the saphenous vein at 10 minutes, 4 hours, 24 hours, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected in heparin containing vials and centrifuged at 10,000g for 5 minutes. Plasma was stored at -20 ° C until determination of mAb concentration.

  Human IgG concentration was determined using a sandwich ELISA. Mouse mAb anti-human IgG-κ clone MH16 (# M1268, CLB Sanquin, Netherlands) coated at a concentration of 2 μg / mL on a 96-well Microlon ELISA plate (Greiner, Germany) was used as a capture antibody. After blocking the plate with PBS containing 2% chicken serum, add serially diluted samples in ELISA buffer (PBS containing 0.05% Tween 20 and 2% chicken serum) and shake at room temperature (RT) Incubated on the machine for 1 hour. The plate was then incubated with goat anti-human IgG immunoglobulin (# 109-035-098, Jackson, West Grace, PA) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfate) (ABTS Developed in Roche, Mannheim, Germany). Absorbance was measured at 405 nm with a microplate reader (Biotek, Winooski, VT).

  The reason for choosing SCID mice is that their plasma IgG concentration is low and thus the clearance of IgG is relatively slow. This provides a very sensitive PK model for detecting clearance changes due to reduced binding between the Fcγ moiety and the neonatal Fc receptor (FcRn).

  Statistical tests were performed using GraphPad PRISM version 4 (Graphpad Software).

  FIG. 12 shows that both wild-type HuMab-7D8 and IgG1-7D8-E345R bound well to mouse, human and cynomolgus FcRn. The binding of IgG1-7D8-E345R was somewhat better than that of wild type 7D8.

  FIG. 13 shows plasma concentrations over time. There was no difference between the wild-type HuMab-7D8 and IgG1-7D8-E345R, -S440K, or K322A with respect to changes in plasma concentration (clearance) over time.

Example 14
Use of the Fc-Fc stabilizing mutation E345R to enhance the bactericidal activity of IgG antibodies against bacteria expressing Fc-binding surface proteins The complement cascade system is an important host defense mechanism against pathogens, It can be divided into different activation pathways: i) the antibody-mediated classical pathway activated by C1q binding to pathogen-bound antibodies, ii) lectins, and iii) the complement system directly recognizes pathogens Alternative pathways, induced by pathogens without antibodies. The three pathways converge at the stage of C3 cleavage and C3b deposition. Microorganisms have generated multiple mechanisms of complement evasion, one of which is mediated by protein A (Joiner Ann. Rev. Microbiol. (1988) 42: 201-30; Foster Nat Rev Microbiol (2005) Dec; 3 (12): 948-58). Protein A was first identified in the cell wall of S. aureus and is well known to bind to the Fc region of IgG (Deisenhofer et al., Biochem (1981) 20, 2361-70; Uhlen et al., J Biol. Chem (1984) 259,1695-1702). So far, its anti-phagocytic effect and its role in the pathogenic action of S. aureus has been explained by the interaction between protein A and IgG, which is neutrophil Fc receptor The antibody directed by the body is misdirected (Foster Nat Rev Microbiol (2005) Dec; 3 (12): 948-58).

  Example 11 shows that CDC mediated by B cell specific IgG1 antibody was inhibited by the competitive Fc binding peptide DCAWHLGELVWCT (SEQ ID NO: 19). This peptide targets the consensus binding site on IgG Fc, which is consistent with the binding sites for protein A, protein G and rheumatoid factor (Delano et al., Science 2000 Feb 18; 287 (5456): 1279-83) . Based on these data, the protein A-mediated bacterial complement escape mechanism works by competing for binding to Fc, resulting in destabilization of the Fc-Fc interaction of microorganism-specific antibodies, It is thought to result in inhibition of antibody-mediated complement activation. Furthermore, Example 11 shows that the B cell specific IgG1 antibody containing the E345R mutation that enhances CDC is more resistant to inhibition of CDC by the competitive Fc binding peptide DCAWHLGELVWCT (SEQ ID NO: 19) than the wild-type parent antibody. It also shows low sensitivity. Based on these results, the Fc binding protein expressed on the surface of microorganisms is estimated, and the stabilization of IgG1 Fc-Fc interaction by the E345R mutation is mediated by competition for binding to Fc by microbial surface proteins such as protein A. It is thought to lead to microorganism-specific antibodies that are less susceptible to complement inhibition by pathogen avoidance strategies. As a result, introduction of the E345R mutation into IgG antibodies directed against bacteria is thought to result in increased C3b deposition and enhanced bactericidal activity on bacteria compared to the wild-type parent antibody.

  As an in vitro measure of complement-mediated bacterial killing, both phagocytosis by neutrophils and production of C3a in plasma consistent with C3b deposition on the bacterial surface are determined as described below can do. Indeed, it has been described that C3b deposition on the surface of S. aureus results in enhanced phagocytosis and correlates with bacterial killing (Rooijakkers et. Al., Nature Immunology 2005: 6,920-927).

S. aureus is labeled with FITC by incubating exponentially growing bacterial cultures in 0.1 M carbonate buffer (pH 9.6) with 100 μg / mL FITC for 1 hour at 37 ° C. Isolate human polymorphonuclear cells (PMN) using Ficoll gradient. FITC-labeled bacteria are opsonized by a specific antibody concentration series with or without the mutation E345R. Incubate 1x10 ⁸ opsonized FITC-labeled bacteria with human PMN for 25 minutes at 37 ° C in vigorous shaking in the presence of 25% IgG-depleted serum as a complement source in a total volume of 200 μL To cause phagocytosis in vitro. Cells are fixed and red blood cells are lysed by incubation with BD FACS lysis solution for 15 minutes at room temperature. After washing, phagocytosis is measured by FACS. Neutrophil populations are sorted by forward and side scatter gating and phagocytosis is expressed as mean fluorescence in the neutrophil population. Alternatively, C3a production in the sample is measured by ELISA as a measure of complement activation and C3b deposition.

  S. aureus-specific antibodies containing the E345R mutation are thought to induce complement activation and phagocytosis by neutrophils to a greater extent than the wild-type parent antibody. An example of an antibody that could be used in such experiments is the chimeric monoclonal IgG1 pagibaximab (BSYX-A110; Biosynexus) that targets lipoteichoic acid (LTA) incorporated into the cell wall of staphylococci (Baker, Nat Biotechnol. 2006 Dec; 24 (12): 1491-3; Weisman et al., Int Immunopharmacol. 2009 May; 9 (5): 639-44).

Example 15
Use of CDC inhibitory mutations to limit CDC activation to target cells that were simultaneously bound by a mixture of two different therapeutic monoclonal antibodies As described in Example 6, mutations K439E and S440K of CD20 antibody 7D8 are monoclonal antibodies. As the CDC activity decreased. CDC was restored by mixing 7D8 antibody containing these mutations. That is, efficient CDC was limited to cells that received binding by both mutant antibodies simultaneously. As described in Example 10, mutations K439E and S440K of CD38 antibody 005 reduced the activity of CDC as a monoclonal antibody. CDC was restored by mixing 005 antibodies containing these mutations. That is, efficient CDC was limited to cells that received binding by both mutant antibodies simultaneously.

It may be advantageous to limit efficient CDC induction to target cells that simultaneously express two specific antigens and use their combined expression to improve the selectivity of CDC induction. is there. In order to limit CDC induction to cells that were simultaneously bound by CD20 and CD38 antibodies, a 7D8-K439E and 005-S440K pair, or a 7D8-S440K and 005-K439E pair, It can be added separately or mixed 1: 1. 0.1 × 10 ⁶ Daudi cells or Raji cells in a round bottom 96 well plate in a total volume of 100 μL, a concentration series of unpurified antibody or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / mL) for 15 minutes at room temperature on a shaker. Next, 25 μL of normal human serum is added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. Stop the reaction by placing the plate on ice. 10 μL of propidium iodide is added and cell lysis is determined by FACS. 7D8-K439E, 005-S440K, 7D8-S440K and 005-K439E are expected to exhibit limited CDC activity. Simultaneous addition of 7D8-K439E and 005-S440K is expected to specifically restore efficient CDC to cells expressing both CD20 and CD38. Similarly, a mixture of 7D8-S440K and 005-K439E is expected to specifically restore efficient CDC against cells expressing both CD20 and CD38.

Example 16
Enhanced specificity of CDC by combining E345R with complementary inhibitory mutations K439E and S440K in a mixture of two different monoclonal antibodies As described in Example 6, mutations K439E of CD20 antibody 7D8 and S440K reduced the activity of CDC as a monoclonal antibody. CDC was restored by mixing 7D8 antibody containing these mutations. That is, efficient CDC was limited to cells that received binding by both mutant antibodies simultaneously. As described in Example 10, mutations K439E and S440K of CD38 antibody 005 reduced the activity of CDC as a monoclonal antibody. CDC was restored by mixing 005 antibodies containing these mutations. That is, efficient CDC was limited to cells that received binding by both mutant antibodies simultaneously.

  It may be advantageous to limit the enhancement of CDC induction to target cells that simultaneously express two specific antigens and to use their combined expression to selectively enhance the enhancement of CDC induction. is there. Also, the enhancement of CDC induction is due to a mixture of at least two different antibodies, which are antibodies that bind to the same cell surface antigen simultaneously at two different epitopes, or bind at two cross-competing similar or identical epitopes. It may also be advantageous to limit to target cells that have received binding simultaneously.

Thus, to limit the enhancement of CDC induction to cells that simultaneously received binding by CD20 and CD38 antibodies, antibodies 7D8-E345R / K439E, 7D8-E345R / S440K, 005-E345R / S440K and 005-E345R / In K439E, the CDC enhancing mutation E345R was combined with the CDC inhibitory mutation. These antibodies were added separately or mixed 1: 1 in CDC experiments as follows. 0.1 × 10 ⁶ Wien133 cells (other cell types such as Daudi cells or Raji cells may be used) in a round bottom 96-well plate in a total volume of 100 μL (non-purified antibody concentration series ( For 7D8-E345R / K439E, 7D8-E345R / S440K, 005-E345R / S440K, or 005-E345R / K439E, a 3-fold dilution to a final concentration of 0.056-10,000 ng / mL) or antibody mixture (final concentration of 0.01 μg / mL) CD20 antibody mixed with a 3-fold dilution of 0-333 ng / mL CD38 antibody; or 3.3 μg / mL CD38 antibody mixed with a 3-fold dilution of 0.0056-1,000 ng / mL CD20 antibody) on a shaker For 15 minutes at room temperature. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  Concentration series of 005-E345R / K439E antibody or 005-E345R / S440K antibody was used as a single agent against Wien133 cells as a single drug, as determined from a fixed concentration of 0.01 μg / mL 7D8 double mutant antibody (Figure 14A). Mixed with the highest concentration at which CDC is minimal) to produce complementary combinations of 005-E345R / K439E + 7D8-E345R / S440K or 005-E345R / S440K + 7D8-E345R / K439E. FIG. 14C shows that 005 double mutant CD38 antibody induced CDC in a dose-dependent manner in the presence of each of a fixed concentration of complementary 7D8-E345R / K439E or 7D8-E345R / S440K CD20 antibody. Yes. The activity of CDC with these complementary combinations (FIG. 14C) was comparable to the 005-E345R single mutant (enhancer) antibody as a single agent (FIG. 14B). In contrast, in the presence of irrelevant antibody b12, both 005-E345R / K439E and 005-E345R / S440K showed almost no CDC in the concentration series tested (single single as shown in FIG. 14B). Equivalent to 005-E345R / K439E or 005-E345R / S440K as a drug).

  A concentration series of 7D8-E345R / K439E or 7D8-E345R / S440K antibody was obtained in Wien133 cells as a single agent in a fixed concentration of 005 double mutant antibody (as determined from FIG. 14B) (But with limited CDC shown) to create complementary combinations of 7D8-E345R / K439E + 005-E345R / S440K or 7D8-E345R / S440K + 005-E345R / K439E. FIG. 14D shows the lowest test corresponding to several 7D8 double mutant antibody molecules per cell in the presence of each of the complementary 005-E345R / K439E or 005-E345R / S440K CD38 antibodies. Even at concentrations, it shows that the 7D8 double mutant CD20 antibody induced CDC very efficiently. To negate the contribution of increased Fc tail density on the cell membrane to the CDC enhancement observed by a mixture of the 7D8 and 005 antibodies and complementary K439E and S440K mutations, the antibody with non-complementary mutations Combinations were also tested. FIG. 14D shows that the non-complementary combination showed much weaker CDC activity than the complementary combination as a result of less efficient Fc-Fc interactions than the complementary combination.

  These data can limit the induction of (enhanced) CDC by therapeutic antibodies to cells that bind simultaneously with a mixture of two complementary antibodies, in this case having different antigen specificities. This suggests that target cell specificity can be increased by requiring co-expression of both antigens.

  As seen in Figures 14A and 14B, 7D8-E345R / K439E, 005-E345R / S440K, 7D8-E345R / S440K and 005-E345R / K439E have limited CDC efficiency compared to 7D8-E345R alone. Was presented. It can also be seen that the mixture of 7D8-E345R / K439E and 7D8-E345R / S440K allowed enhanced efficiency CDC compared to wild type 7D8 antibody as a single agent. Similarly, it was also observed that a mixture of 005-E345R / K439E and 005-E345R / S440K allowed enhanced efficiency of CDC compared to wild-type 005 antibody as a single agent (data Not shown).

Example 17
Use of CDC inhibitory mutations to limit efficient CDC activation to antibody conjugates consisting only of therapeutically administered antibodies As described in Example 6, double mutant K439E of CD20 antibody 7D8 / S440K restored CDC efficiency reduced by K439E or S440K single point mutants. As described in Example 10, double mutant K439E / S440K of CD38 antibody 005 restored CDC efficiency inhibited by K439E or S440K single point mutants. As observed, these single point mutations impaired Fc: Fc interactions with unmutated amino acids on the opposite side of the Fc: Fc interface. Fc: CDC efficiency was restored by the introduction of compensatory mutations on the opposite face of the Fc interface. Thus, efficient CDC was apparently limited to antibody complexes consisting of antibodies containing both mutations.

In another example, the induction of CDC is limited to antibody conjugates consisting only of therapeutically administered antibodies. To limit CDC induction only to cells that are bound by CD20 or CD38 antibody for therapy, combine CDC inhibitory mutations K439E and S440K in the form of antibodies 7D8-K439E / S440K or 005-K439E / S440K Can be considered. These antibodies could be administered separately in a CDC experiment in the presence or absence of non-target specific IgG as follows. 0.1 × 10 ⁶ Daudi cells or Raji cells in a round bottom 96 well plate in a total volume of 100 μL, a concentration series of unpurified antibody or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / mL) for 15 minutes at room temperature on a shaker. Next, 25 μL of normal human serum is added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. Stop the reaction by placing the plate on ice. 10 μl of propidium iodide is added and cell lysis is determined by FACS.

  7D8-K439E / S440K is expected to induce CDC with the same efficiency as wild type 7D8 antibody. The addition of non-specific IgG to 7D8-K439E / S440K is not expected to affect the efficiency of CDC induction for this antibody. Similarly, 005-K439E / S440K is expected to enable CDC with similar efficiency as wild-type HuMAb 005. The addition of non-specific IgG to 005-K439E / S440K is not expected to affect the efficiency of CDC induction for this antibody.

Example 18
Use of CDC inhibitory mutations to limit the enhancement of CDC activation to antibody conjugates consisting only of therapeutically administered antibodies As described in Example 6, CD20 antibody 7D8 double mutant K439E / S440K Restored CDC efficiency reduced by K439E or S440K single point mutants. As described in Example 10, double mutant K439E / S440K of CD38 antibody HuMAb 005 restored CDC efficiency inhibited by K439E or S440K single point mutants. As observed, these single point mutations impaired Fc: Fc interactions with unmutated amino acids on the opposite side of the Fc: Fc interface. Fc: CDC efficiency was restored by the introduction of compensatory mutations on the opposite face of the Fc interface. Thus, efficient CDC was apparently limited to antibody complexes consisting only of antibodies containing both mutations.

In another example, the enhancement of CDC induction is limited to antibody conjugates consisting only of therapeutically administered antibodies. Screening and selecting mutations that stimulate Fc: Fc interactions used for CDC stimulation identify mutations that can form CDC-inducible antibody complexes with serum antibodies that are not specific for the antigenic target of interest. It is thought to be possible. In order to limit the enhancement of CDC induction to only cells that are bound by CD20 antibody or CD38 antibody for therapeutic use, the CDC enhancing mutation E345R can be treated with antibodies 7D8-E345R / K439E / S440K or 005-E345R / K439E / S440K. In combination with CDC inhibitory mutations. These antibodies could be administered separately in a CDC experiment in the presence or absence of non-target specific IgG as follows. 0.1 × 10 ⁶ Daudi cells or Raji cells in a round bottom 96 well plate in a total volume of 100 μL, a concentration series of unpurified antibody or antibody mixture (0.01, 0.03, 0.1, 0.3, 1.0, 3.0, 10.0, 30.0 μg / mL) for 15 minutes at room temperature on a shaker. Next, 25 μL of normal human serum is added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. Stop the reaction by placing the plate on ice. 10 μL of propidium iodide is added and cell lysis is determined by FACS.

  7D8-E345R / K439E / S440K is expected to induce enhanced efficiency CDC compared to wild-type HuMAb 7D8. Addition of non-specific IgG to 7D8-E345R / K439E / S440K is not expected to affect the efficiency of CDC induction compared to wild type 7D8 antibody. Similarly, 005-E345R / K439E / S440K is expected to allow for enhanced efficiency CDC as compared to wild type 005 antibody. The addition of non-specific IgG to 005-E345R / K439E / S440K is not expected to affect the efficiency of CDC compared to wild type 005 antibody.

Example 19
Use of mutant screening approaches to identify mutations that stimulate antibody oligomerization via Fc: Fc interactions detected by CDC assays. As described in Examples 6 and 10, two different CD20 and CD38 Amino acid mutations have been identified that enhance CDC for antibodies that recognize the target antigen against multiple cell lines that express the antigen at various levels. Surprisingly, the single point mutation E345R is sufficient to result in CDC-dependent cytolysis of Wien133 cells against anti-CD38 antibody 005, but these cells cannot be lysed by CDC in the wild-type IgG1 format It was proved.

  It is possible that other mutations on or around the Fc: Fc interface could stimulate oligomerization and CDC in a similar manner. Alternatively, mutations may be able to indirectly stimulate oligomerization, for example by inducing Fster: Fc interactions allosterically.

  In order to determine whether other amino acid mutations can stimulate Fc-mediated antibody oligomerization, anti-CD38 IgG1-005 mutant libraries can be interacted individually and even across, for example, the Fc: Fc interface The CDC assay was used to screen both as a pairwise format for selecting amino acid pairs to be mixed. However, the same strategy can be applied to other antibodies, such as another IgG1 or IgG3 antibody.

  A focused library of mutations at the positions indicated in Table 16 was generated. Mutations were introduced into the IgG1-005 Fc region using the Quikchange site-directed mutagenesis kit (Stratagene, US). Briefly, for each desired mutation position, a 005 heavy chain full-length plasmid DNA template with IgG1m (f) allotype is generated using forward and reverse primers encoding degenerate codons at the desired location. Duplicated. The resulting DNA mixture was digested with DpnI to remove the source plasmid DNA and used for transformation of E. coli. The resulting colonies were pooled and cultured, and plasmid DNA was isolated from these pools and transformed into E. coli to obtain clonal colonies. Mutant plasmid DNA isolated from the resulting colonies was verified by DNA sequencing (LGC genomics, Berlin, Germany). Amplification of the expression cassette from plasmid DNA by PCR, and a DNA mixture containing both the mutated heavy and wild type light chains of the IgG1-005 antibody, essentially as described by the manufacturer, 293fectin (Invitrogen, US) Were used to transiently transfect Freestyle HEK293F cells (Invitrogen, US). The supernatant of the transfected cells containing the antibody variant was collected. Mutant antibody supernatants were screened in the CDC assay both individually and as a pairwise mixture as follows.

0.1 × 10 ⁶ Daudi cells or Wien-133 cells (other cell types such as Raji cells may be used) in a round bottom 96 well plate in a total volume of 100 μL, 1.0 ug / ml Preincubated with unpurified antibody for 15 minutes on a shaker at room temperature. Next, 30 μL of normal human serum was added as a complement source (final concentration 30%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μl of propidium iodide was added and cell lysis was determined by FACS.

  Mutations described in Table 16, Table 17 and Table 18 are detected by CDC efficiency as a single mutant or mixed with other mutants facing mutations across the Fc: Fc interface, for example Oligomer formation was screened for their ability to enhance. Mutations can optionally be further screened for their ability to not impair FcRn, protein A or protein G binding, ADCC, ADCP, or other effector functions mediated by the Fc domain. Combining such point mutations in one Fc domain can further stimulate oligomerization and CDC efficiency.

  Mutations in the CH2-CH3 region incorporated into CD38 antibody 005 were tested for their ability to inhibit oligomerization as determined by CDC on Daudi cells. Mutant antibody lysis was compared to wild type 005 and the latter lysis was set at 100%. The cutoff for inhibition was set at a dissolution rate of 66% or less. When measured in this way, most of the tested mutations inhibited CDC (see Table 16).

  Mutations in the CH2-CH3 region incorporated into CD38 antibody 005 were tested for their ability to enhance oligomerization as determined by CDC on Wien133 cells (Table 17). Wild type CD38 antibody 005 is unable to induce CDC on Wien133 cells. Mutants exhibiting more than 39% cell lysis were evaluated as potent. Quite unexpectedly, virtually all of the substitutions obtained from amino acids E345 and E430 stimulated cell lysis by CDC. To verify this result, amino acids E345, E430, and S440 were replaced with their respective possible mutations by site-directed mutagenesis, and using new human serum batches, they were determined by CDC of Wien133 cells. When tested for their ability to enhance oligomer formation, somewhat more efficient dissolution occurred (Table 18). Again, all substitutions of E345 and E430 induced efficient CDC in Wien133 cells.

  The following preferred mutations caused more than 39% cell lysis of Wien133 cells: P247G, I253V, S254L, Q311L, Q311W, E345A, E345C, E345D, E345F, E345G, E345H, E345I, E345K, E345L, E345M, E345N , E345P, E345Q, E345R, E345S, E345T, E345V, E345W, E345Y, D / E356G, D / E356R, T359R, E382L, E382V, Q386K, E430A, E430C, E430D, E430F, E430G, E430H, E430I, E430L, E430M , E430N, E430P, E430Q, E430R, E430S, E430T, E430V, E430W, E430Y, Y436I, S440Y and S440W.

^*「del」は、表記の位置にアミノ酸残基の欠失があったことを意味する。 (Table 16) Lysis rate of daudi cells in the presence of 1.0 μg / ml IgG1-005 antibody point mutant. IgG1-005 wild type lysed 66% of the cells under these conditions. Each individual position substituted with another amino acid is noted in the left outer column. A row of individual positions indicates the amino acid substituted at each specific position, followed by the measured dissolution rate in parentheses.

^* “Del” means that there was a deletion of an amino acid residue at the indicated position.

Table 17: Lysis rate of Wien-133 cells in the presence of 1.0 μg / ml IgG1-005 antibody point mutant. IgG1-005 wild type lysed 3% of the cells under these conditions. Each individual position substituted with another amino acid is noted in the left outer column. A row of individual positions indicates the amino acid substituted at each specific position, followed by the measured dissolution rate in parentheses.

Table 18: Lysis rate of Wien-133 cells in the presence of 1.0 μg / ml IgG1-005 antibody point mutant. IgG1-005 wild type lysed 12% of the cells under these conditions. Each individual position substituted with another amino acid is noted in the left outer column. A row of individual positions indicates the amino acid substituted at each specific position, followed by the measured dissolution rate in parentheses.

Example 20
In vivo activity of IgG1-7D8-E345R in a B cell lymphoma subcutaneous xenograft model
The in vivo antitumor activity of IgG1-7D8-E345R antibody was evaluated in a subcutaneous model with Raji-luc # 2D1 cells. These cells show approximately 300,000 CD20 molecules per cell (determined by QIFIKIT analysis, data not shown) and a high degree of complement protective receptor expression. Cells in RPMI with 10% cosmic calf serum (HyClone, Logan, UT), penicillin and streptomycin, 1% (v / v) sodium pyruvate and 1 μg / mL puromycin (P-8833, Sigma, Zwijndrecht) Was cultured. Cells were harvested at logarithmic growth phase (confluence approximately 70%). Female SCID mice (CB-17 / IcrPrkdc-scid / CRL) 6-11 weeks old were used (Charles-River). On day 0, 5 × 10 ⁶ Raji-luc # 2D1 cells in 200 μL PBS were injected subcutaneously into the right flank of each mouse. Tumor development was monitored by caliper measurements. When the mean tumor volume reached 100 mm ³ (around day 7), the mice were divided into multiple groups (n = 9) and intraperitoneal in a single dose of 50 μg antibody (2.5 mg / kg) per mouse (Ip) An injection was performed. Unrelated antibody b12 was added to all antibody samples to give a total antibody concentration of 0.5 mg / mL. The treatment groups are shown in Table 18. Seven days after treatment, blood samples were obtained to determine human IgG serum levels in order to verify the appropriateness of antibody administration. Tumor measurements were performed at least twice weekly with calipers (PLEXX) until an endpoint tumor volume of 1500 mm ³ was reached, the tumors showed ulceration, or severe clinical signs were observed.

Table 18: Treatment groups and administration

  FIG. 15A shows the mean tumor growth at day 22 when all groups were still in place. Wild type antibody IgG1-7D8 somewhat inhibited tumor growth compared to negative control antibody IgG1-b12, but this was not statistically significant. Only IgG1-7D8-E345R significantly inhibited tumor growth compared to the negative control antibody IgG1-b12 (p <0.01 by one-way ANOVA analysis).

FIG. 15B shows a Kaplan-Meier plot for the percentage of mice with tumor size smaller than 700 mm ³ . Tumor formation was significantly delayed in mice administered IgG1-7D8-E345R antibody (p <0.01 by Mantel-Cox analysis) compared to mice administered negative control antibody IgG1-b12, but wild type IgG1-7D8 This was not the case in mice receiving.

  These data indicate that the E345R mutation enhanced the in vivo antitumor activity of the CD20 antibody 7D8.

Example 21
In vivo activity of IgG1-005-E345R in B cell lymphoma subcutaneous xenograft model
The in vivo anti-tumor activity of the IgG1-005-E345R antibody was evaluated in a subcutaneous model with Raji-luc # 2D1 cells. These cells show approximately 150,000 CD38 molecules per cell (determined by QIFIKIT analysis, data not shown) and high complement defense receptor expression. The protocol for tumor inoculation and measurement is basically the same as that described in Example 20. On day 0, 5 × 10 ⁶ Raji-luc # 2D1 cells in 200 μL PBS were injected subcutaneously into the right flank of SCID mice. At the time when the mean tumor volume reached 100 mm ³ (around day 7), the mice were divided into multiple groups (n = 7) and intraperitoneally administered at a single dose of 500 μg antibody (25 mg / kg) per mouse ( ip) Injection was performed. The treatment groups are shown in Table 19. To avoid great distress, tumor measurements were taken until an endpoint tumor volume of 1500 mm ³ was reached, until the tumor showed ulceration, or until severe clinical signs were observed.

  FIG. 16A shows the mean tumor growth on day 21 when all groups were still in place. Wild type antibody IgG1-005 somewhat inhibited tumor growth, which was not statistically significant. Only IgG1-005-E345R significantly inhibited tumor growth compared to an irrelevant antibody control on day 21 (p <0.05 by one-way ANOVA analysis).

FIG. 16B shows a Kaplan-Meier plot for the percentage of mice with tumor size smaller than 500 mm ³ . Tumor formation in mice treated with IgG1-005-E345R antibody compared to mice administered negative control antibody IgG1-b12 (p <0.001 in Mantel-Cox analysis) or wild-type IgG1-005 (p <0.05) Was significantly delayed.

  These data indicate that the E345R mutation in CD38 antibody 005 resulted in enhanced in vivo anti-tumor activity.

Table 19: Treatment groups and administration

Example 22
Monovalent target binding further enhances CDC activity of E345R antibody
The molecular surface of the IgG1 hexamer ring observed in the b12 crystal structure is that for each IgG in the hexamer ring, one of the two C1q binding sites is upward with respect to the ring structure and the other is downward. Furthermore, one Fab arm of each antibody is oriented upwards, one is oriented downwards, so that it clearly shows that only one Fab arm per antibody is involved in antigen binding. This suggests that the binding per antibody molecule in the hexamer antibody ring is monovalent. By being monovalent, the antibody may have an orientation suitable for hexamerization upon antigen binding. To test this hypothesis, the CDC activity of the bispecific CD38 / EGFR antibody with the E345R mutation was shown to be CD38 positive and EGFR negative that the bispecific antibody can only bind monovalently through CD38. Were tested in Wien133 cells and compared to the CDC activity of a bivalent binding CD38 antibody, also carrying the E345R mutation. The human monoclonal antibody HuMax-EGFr (2F8, described in WO 2004/056847) was used as the basis for the EGFR antibody described in this example.

  Bispecific antibodies were generated in vitro by the DuoBody ™ platform, ie by 2-MEA-induced Fab arm exchange as described in WO 2011/147986. Underlying this method is the use of complementary CH3 domains that promote heterodimer formation under specific assay conditions. In order to be able to generate bispecific antibodies by this method, an IgG1 molecule carrying certain mutations in the CH3 domain was generated: one of the parent IgG1 antibodies has the F405L mutation and the other parent IgG1 The antibody has a K409R mutation. To make bispecific antibodies, these two parent antibodies were combined with 25 mM 2-mercaptoethylamine-HCl (in a total volume of 100 μL of TE so that each antibody had a final concentration of 0.5 mg / mL). 2-MEA) and incubated at 37 ° C. for 90 minutes. The reduction reaction is stopped when the reducing agent 2-MEA is removed by using a spin column (Microcon centrifugal filter, 30k, Millipore) using the manufacturer's protocol.

For the CDC assay, 0.1 × 10 ⁶ Wien133 cells were placed in a round-bottom 96-well plate in a total volume of 100 μL at room temperature on a shaker with antibody concentration series (0.01-10.0 μg / mL). For 15 minutes. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  FIG. 17 shows that, as expected, CD38 antibodies without the E345R mutation (wild type IgG1-005 and IgG-b12-K409R × IgG1-005-F405L) did not induce lethality of Wien133 cells. Yes. Similarly, EGFR antibody IgG1-2F8-E345R / F405L (data not shown) that did not bind to EGFR negative Wien133 cells did not induce CDC, as expected. The introduction of the K409R mutation did not affect the ability of the IgG1-005-E345R antibody to induce nearly 60% lethality in Wien133 cells (described in Example 10). Interestingly, IgG1-005-E345R / K409R x IgG1-2F8-E345R / F405L, a bispecific CD38 / EGFR antibody that can only bind monovalently in CD38 positive and EGFR negative Wien133 cells, is the largest It showed enhanced CDC lethality (from nearly 60% lethality to almost 100%).

  These data indicate that monovalent targeting can further enhance the maximum lethality of antibodies containing the CDC-enhancing E345R mutation. In addition, these data indicate that E345R oligomerization enhancing mutations as measured by enhanced CDC activity can be applied to other antibody formats such as DuoBody.

Example 23
The oligomerization enhancing E345R mutation may be applied to other antibody formats such as DuoBody ™
The effect of the E345R mutation was tested on a bispecific antibody in DuoBody format. CDC assays were performed with CD20 / CD38 bispecific antibodies in CD20 positive and CD38 positive Wien133 and Raji cells.

Bispecific antibodies were made as described in Example 22. For CDC assay, 0.1 × 10 ⁶ Wien133 cells or Raji cells were placed on a shaker with antibody concentration series (0.01-30.0 μg / mL) in a round bottom 96-well plate in a total volume of 100 μL. For 15 minutes at room temperature. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  Figure 18 shows that the introduction of the E345R mutation enhanced CDC of the bispecific IgG1-005-F405L x IgG1-7D8-K409R antibody on Wien 133 cells (Figure 18A) and Raji cells (Figure 18B). ing. These data indicate that E345R oligomerization enhancing mutations can be applied to other antibody formats to enhance CDC activity.

Example 24
E345R can restore CDC by EGFR antibody 2F8 and further enhance it by monovalent target binding As described in Examples 6, 10 and 26, E345R is an antibody that recognizes various blood tumor targets (CD20 And CDC for CD38) were enhanced or restored. To extend this analysis to solid tumor antigens, the effect of E345R on the CDC ability of EGFR antibody 2F8 was tested in A431 epidermoid carcinoma cells. Furthermore, the effect of monovalent EGFR targeting on E345R-mediated CDC induction was also demonstrated in bispecific EGFR x CD20 antibodies (IgG1-2F8-E345R / F405L x IgG1-7D8-E345R /) in EGFR positive and CD20 negative A431 cells. K409R).

Bispecific antibodies were made as described in Example 22. For the CDC assay, 5 × 10 ⁶ cells / mL A431 cells were labeled with 100 μCi ⁵¹ Cr for 1 hour at 37 ° C. Cells were washed 3 times with PBS and resuspended in media at a concentration of 1 × 10 ⁵ cells / mL. 25,000 labeled cells were incubated for 15 minutes at room temperature in a round bottom 96-well plate with a concentration series of unpurified antibody (0-30 μg / mL at 3-fold dilution) in a total volume of 100 μL. Next, 50 μL of normal human serum dilution was added as a complement source (final concentration 25%) and incubated for 1 hour in a 37 ° C. incubator. Cells are spun down (300 xg for 3 min) and 25 μL of supernatant is added to 100 μL microscint in white 96 well optiplate (PerkinElmer) for 15 min on a shaker (750 rpm) Incubation of ⁵¹ Cr release was determined as counts per minute (cpm) in a scintillation counter. Maximum lysis (100%) was determined by ⁵¹ Cr levels measured in the supernatant of cells treated with Triton X-100. Spontaneous lysis was determined by ⁵¹ Cr levels measured in the supernatant of cells incubated without antibody. Specific cell lysis is the formula: specific lysis = 100 x (sample cpm-spontaneous cpm) / (maximum cpm-spontaneous cpm).

  FIG. 19 shows that IgG1-2F8-E345R / F405L can lyse A431 cells by CDC, whereas wild type 2F8 cannot kill A431 cells. These data indicate that CDC activity can be restored in EGFR antibody 2F8 by introducing E345R mutation. This indicates the possibility that the applicable range of the CDC-enhanced E345R mutation can be extended to antibodies targeting solid tumor antigens.

  The bispecific EGFR × CD20 antibody IgG-2F8-E345R / F405L × IgG1-7D8-E345R / K409R showed further enhancement of CDC in EGFR positive and CD20 negative A431 cells.

  These data further support the hypothesis that monovalence facilitates the formation of Fc-Fc interactions and subsequent CDC induction as hypothesized for the CD38 binding antibody described in Example 22. It is.

Example 25
E345R enhances or restores CDC by CD38 antibody 003 and CD20 antibody 11B8 and rituximab As described in Examples 6, 10 and 24, E345R is a plurality of cells that express the antigen at various levels. As tested against strains, the CDC activity of some antibodies is enhanced or induced with different target specificities (CD20, CD38 and EGFR). From this, the introduction of the E345R mutation was judged to be a general mechanism for enhancing or restoring CDC for existing antibodies. To further support this, the effect of the E345R mutation on CDC was further investigated with additional antibodies with varying intrinsic CDC activity on Daudi and Wien133 cells: CD38 antibody 003 described in WO 2006/099875, and WO 2005 / The CD20 antibodies rituximab (type I) and 11B8 (type II) described in 103081 were also tested. CD20 antibodies can be divided into two subgroups (Beers et al. Seminars in Hematology 47, (2) 2010, 107-114). Type I CD20 antibodies activate complement and induce CDC by redistributing CD20 molecules in the plasma membrane into lipid rafts, thereby clustering the antibody Fc region and improving C1q binding Exhibits remarkable ability. Type II CD20 antibodies do not appreciably change the CD20 distribution and are not associated with clustering, which are relatively ineffective on CDC.

0.1 × 10 ⁶ Daudi cells or Raji cells in a round bottom 96-well plate in a total volume of 70 μL, a concentration series of unpurified antibodies (0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1.0, 3.0 , 10.0 μg / mL) for 15 minutes at room temperature on a shaker. Next, 30 μL of normal human serum was added as a source of C1q (final concentration 30%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  FIG. 20 shows that the E345R mutation enhanced CDC on both (A) Daudi and (B) Wien133 cells for all tested antibodies. Interestingly, all antibodies did not induce CDC in the wild-type format at the concentrations used, but efficiently induced CDC after the introduction of the E345R mutation: CD38 mAb 003 and CD20 type II mAb 11B8 on Daudi cells And CD38 mAb 005 and 003 and CD20 type II mAb 11B8 against Wien133 cells. These data suggest that enhanced antibody oligomerization, more specifically by introducing the E345R mutation, is a common mechanism to enhance or restore CDC by existing antibodies .

Example 26
E345R enhances intracellular translocation of tissue factor antibodies. To investigate whether enhanced oligomerization induces enhanced intracellular translocation of antibodies, wild-type and E345R mutant tissue factor (TF) antibodies Was co-localized by confocal microscopy using the lysosomal marker LAMP1.

SK-OV-3 cells were grown in standard tissue culture media for 1 day at 37 ° C. on coverslips (1.5 microns thick, Thermo Fisher Scientific, Braunschweig, Germany). To block lysosomal activity, cells were preincubated with 50 μg / mL leupeptin (Sigma) for 1 hour, after which 10 μg / mL tissue factor (TF) antibody (WO 2010/066803) was added. Cells were incubated at 37 ° C. for an additional hour, 3 hours or 16 hours. Subsequently, the cells were washed with PBS and incubated with 4% formamide (Klinipath) for 30 minutes at room temperature (RT). Slides were washed with blocking buffer (PBS with 0.1% saponin [Roche] and 2% BSA [Roche]) and incubated for 20 minutes with blocking buffer containing 20 mM NH ₄ Cl to inactivate formamide . Slides are washed again with blocking buffer and mixed with mouse-anti-human CD107a-APC (BD Pharmingen) to identify lysosomal LAMP1 and goat-anti-human IgG-FITC (Jackson) to identify TF antibodies The solution was incubated for 45 minutes at room temperature. The slides were washed again with blocking buffer, and 20 μL of mounting medium (6 g glycerol [Sigma] and 2.4 g Mowiol 4-88 [Omnilabo] was added to 6 mL distilled water, to which 12 mL 0.2 M was added. Tris [Sigma] pH 8.5, followed by incubation at 50-60 ° C for 10 minutes; mount medium was quantified and stored at -20 ° C). I mounted it overnight. Slides were imaged using a Leica SPE-II confocal microscope (Leica Microsystems) equipped with a 63x 1.32-0.6 oil immersion objective and LAS-AF software.

  12-bit grayscale TIFF images were analyzed for co-localization using MetaMorph® software (Meta Series version 6.1, Molecular Devices Inc, Sunnyvale California, USA). Images were captured as a stack and background was subtracted. The same threshold setting was used for all FITC images and all APC images (manually set). Co-localization is shown as FITC pixel intensity in the region of interest (ROI), where ROI is composed of all of the APC positive regions. In order to compare different slides stained with different TF antibodies, images were normalized using APC pixel intensity. Mouse-anti-human CD107a-APC was used to stain the lysosomal marker LAMP1 (CD107a). The pixel intensity of LAMP1 should not differ between the various imaged TF antibodies.

The standardized values for the co-localization of FITC and APC are expressed as arbitrary units according to the formula [(FITC TPI × co-localization rate) / 100] × [1 / APC TPI].
Colocalization rate = FITC TPI co-localized with APC pixels / APC TPI
TPI, total pixel intensity

  FIG. 21 shows the amount of FITC pixel intensity of wild-type and E345R mutant TF antibodies overlapping with APC-labeled lysosomal markers. For each antibody or condition tested, three different images from one slide containing approximately 1, 3, or more than 5 cells were analyzed. Differences were observed between the different images in each slide. However, it was still clear that the E345R mutation for antibodies 011 and 098 resulted in enhanced lysosomal colocalization after 1 hour incubation when compared to wild type 011 and 098. These results indicate that mutant E345R can induce faster intracellular translocation and lysosomal colocalization, thereby enhancing antibody drug conjugates.

Example 27
Enhancement of CDC by E345R mutation in rituximab in various B cell lines with similar levels of CD20 but different levels of membrane-bound complement regulatory proteins Examples 25 and 28 are wild-type for Daudi and Wien133 cells It shows that the CDC activity of rituximab was enhanced by introducing the E345R mutation. This enhanced CDC activity is due to E345R-mediated stabilization of the Fc-Fc interaction. Subsequently, the accompanying hexamer antibody ring structure on the target cell membrane facilitates the capture and enrichment of activated complement components in the vicinity of the cell membrane, thereby efficiently producing a membrane-damaging complex. Can be promoted. As a result of this efficient complement activation, it is believed that the inhibitory effect by membrane-bound complement regulatory protein (mCRP) can be partially overcome. Overexpression of mCRP such as CD55, CD46 and CD59 is thought to be an obstacle for the response of immunotherapy with monoclonal anti-tumor antibodies (Jurianz et al., Mol Immunol 1999 36: 929-39; Fishelson et al. Mol Immunol 2003 40: 109-23, Gorter et al., Immunol Today 1999 20: 576-82, Zell et al., Clin Exp Immunol. 2007 Dec 150 (3): 576-84). This compared the activity of rituximab-E345R against that of wild-type rituximab against a series of B cell lines with different levels of the mCRPs CD46, CD55 and CD59 but equivalent levels of CD20 target expression.

  The B cell lines Daudi, WIL2-S, WSU-NHL, MEC-2 and ARH-77 express equivalent amounts of CD20 molecules as determined by QIFIKIT analysis (specific antibody binding capacity-sABC is almost 250,000) (Data not shown). To compare the expression levels of complement regulatory proteins between these cell lines, CD46 (mouse anti-human CD46, CBL488, clone J4.48 Chemicon), CD55 (mouse anti-human CD55, CBL511, clone BRIC216, Chemicon) ) And CD59 (mouse anti-human CD59, MCA1054x, clone MEM-43, Serotec) were subjected to QIFIKIT analysis.

For the CDC assay, 0.1 × 10 ⁶ cells were placed in a round bottom 96-well plate in a total volume of 100 μL with a saturating antibody concentration series (0.002-40.0 μg / mL at 4-fold dilution) Pre-incubated for 15 minutes at room temperature on a shaker. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS. Maximum CDC-mediated lethality was calculated from two independent experiments using the top of the best fit values for non-linear fit in GraphPad PRISM 5.

Figures 22A-D show that introduction of E345R into wild-type rituximab resulted in enhanced CDC activity, as observed by enhanced maximal lysis and reduced EC ₅₀ for all B cell lines tested. It is shown that.

  Figure 22E shows that maximal CDC-mediated lethality induced by the rituximab-E345R mutant was always higher than wild-type rituximab, regardless of the expression level of membrane-bound complement regulatory protein . These data indicate that the introduction of E345R enhances the therapeutic potential of monoclonal antibodies because tumor cells are less effective in avoiding antibody-mediated complement attack by antibodies containing E345R.

Example 28
Comparison of CDC kinetics for wild type antibody and E345R antibody Example 6 (CD20 antibody 7D8 against Daudi and Raji), Example 10 (CD38 antibody 005 against Daudi, Raji and Wien133) and Example 25 (CD38 antibody 003 against Daudi and Wien133) And the introduction of the E345R mutation that stabilizes the Fc: Fc interaction, as observed by reduced EC ₅₀ values and enhanced maximal lysis for various antibodies against the various cell lines described in CD20 antibodies rituximab and 11B8) Has been shown to enhance or restore CDC. Next, to further elucidate the difference in CDC activity between the wild type antibody and the E345R antibody, the kinetics of the CDC reaction was analyzed.

0.1 × 10 ⁶ Raji cells were preincubated for 15 minutes at room temperature on a shaker with a saturating concentration (10.0 μg / mL) of antibody in a round bottom 96-well plate in a total volume of 100 μL. Next, 25 μL of normal human serum was added as a source of complement (final concentration 20%) and incubated for various periods ranging from 0-60 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  FIG. 23A shows that wild type CD20 antibody IgG1-7D8 showed a maximum CDC-mediated lethality of 80% of Raji cells, which was already reached after 5 minutes under the test conditions. However, for IgG-7D8-E345R, 80% lethality of Raji cells was observed more rapidly after 3 minutes. In addition, the maximum dissolution (95%) by IgG-7D8-E345R was reached after 5 minutes.

  FIG. 23B also shows that the introduction of the E345R mutation resulted in faster killing of target cells for the wild-type CD20 antibody rituximab, which is less potent than 7D8 in inducing CDC in the Raji cells used . Wild-type rituximab showed maximum CDC-mediated lethality of 32%, which was reached after 20 minutes. Rituximab-E345R has already reached 32% lethality after approximately 3 minutes, and notably, maximum lysis (85%) with rituximab-E345R was also reached after 20 minutes.

  FIG. 23C + D shows that Raji cells used, resistant to CDC-mediated lethality by wild-type CD38 antibodies IgG1-003 and IgG1-005, could be rapidly killed by introducing the E345R mutation. Is shown. IgG1-003-E345R and IgG1-005-E345R showed maximum CDC (50% and 60%, respectively) already after 5 minutes.

In summary, E345R antibodies are more potent than their wild-type counterparts, a combination of higher activity (lower EC ₅₀ ), enhanced maximum lysis, and faster kinetics of the CDC response caused by.

Example 29
Comparison of CDC kinetics for bispecific antibodies with or without E345R mutation Example 23 includes a CD38 × CD20 bispecific antibody IgG1-005-F405L × IgG1-7D8 produced by the DuoBody platform It has been described that applying the E345R mutation to -K409R can result in enhanced lethality as observed by a decrease in EC ₅₀ in CDC assays in Raji and Wien133 cells. Next, to further elucidate the difference in CDC activity between CD38 × CD20 bispecific antibody with E345R and without it, the kinetics of the CDC reaction was analyzed.

0.1 × 10 ⁶ Raji cells were preincubated for 15 minutes at room temperature on a shaker with a saturating concentration (10.0 μg / mL) of antibody in a round bottom 96-well plate in a total volume of 100 μL. Next, 25 μL of normal human serum was added as a source of complement (final concentration 20%) and incubated for various periods ranging from 0-60 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  FIG. 24 shows that the bispecific antibody IgG1-005-F405L × IgG1-7D8-K409R induced maximal CDC-mediated lethality of 83% and reached it after 10 minutes. The introduction of E345R resulted in a maximal lethal enhancement with IgG1-005-E345R-F405L x IgG1-7D8-E345R-K409R (98%), which was already reached after 2 minutes. These data indicate that introduction of an E345R mutation that stabilizes Fc-Fc into a bispecific antibody results in faster CDC-mediated lethality of target cells.

Example 30
Comparison of CDC kinetics for monovalent binding antibodies with and without E345R Example 22 observed by enhanced maximal lysis by CD38 x EGFR bispecific antibody against CD38 positive and EGFR negative Wien133 cells by monovalent target binding As shown, it was shown that the CDC activity of the E345R antibody was further enhanced. Next, to further elucidate the difference in CDC-mediated lethal capacity between monovalent binding antibodies with E345R and those without it, the kinetics of the CDC reaction was analyzed.

Bispecific CD38 × EGFR and CD20 × EGFR antibodies with or without the E345R mutation were generated in vitro by the DuoBody platform as described in Example 22. The CDC activity of the CD38 × EGFR bispecific antibody was tested in CD38 positive and EGFR negative Raji cells, where the bispecific antibody can only bind monovalently through CD38. 0.1 × 10 ⁶ Raji cells were preincubated for 15 minutes at room temperature on a shaker with a saturating concentration (10.0 μg / mL) of antibody in a round bottom 96-well plate in a total volume of 100 μL. Next, 25 μL of normal human serum was added as a source of complement (final concentration 20%) and incubated for various periods ranging from 0-60 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  Figure 25 shows that the bispecific antibody CD38 x EGFR (IgG1-005-K409R x IgG1-2F8-F405L) induced a maximum CDC-mediated lethality of 55% and reached it after approximately 10 minutes. ing. The introduction of E345R resulted in an increase in maximum lethality (96%), which was already reached within 5 minutes.

  FIG. 25 shows that the bispecific antibody CD20 × EGFR (IgG1-7D8-K409R × IgG1-2F8-F405L) induced a maximum CDC-mediated lethality of 85% and reached it after approximately 5 minutes. ing. However, 85% lysis was observed more rapidly after 2 minutes when using CD20xEGFR antibody with E345R introduced. In addition, the maximum dissolution (97%) by the E345R CD20 × EGFR antibody was reached after 5 minutes.

  In summary, the introduction of the E345R mutation into these monovalent binding antibodies results in a more potent antibody, which combines the enhancement of maximum lysis with the faster kinetics of the CDC reaction. to cause.

Example 31
CDC with combination of therapeutic antibody and E345R / Q386K antibody
As described in Example 19, a mutant CD38 antibody derived from IgG1-005 is an efficient CDC in Wien133 cells by replacing the E345 position of the wild-type antibody with any amino acid other than glutamic acid (E). Could be induced. This suggests that oligomerization as a prerequisite for CDC is hindered by the presence of glutamate side chains at position 345 of the antibody. Since E345 on one Fc is in close proximity to Q386 on the second Fc moiety that opposes in the hexamer antibody ring structure, E345-mediated hindrance of oligomerization in the first antibody is the Q386 of the second antibody. It is likely that it could be removed by substitution at the position. This would subsequently enable E345 in the first antibody to interact better when both antibodies are combined with the mutated 386 position in the second antibody. To test this hypothesis, for Wien133, wild type antibodies (IgG1-003, IgG1-005 or IgG1-11B8) were used as IgG1-005-E345R / Q386K or IgG1-005-E345R / Q386K / E430G as an example. A mixed CDC assay was performed.

Concentration series of non-purified IgG1-005-E345R / Q386K, IgG1-005-E345R / Q386K / E430G or control antibody in 0.1 × 10 ⁶ Wien133 cells in a round bottom 96-well plate in a total volume of 100 μL At room temperature on a shaker in the presence or absence of 1.0 or 10.0 μg / mL of wild type IgG1-003, IgG1-005 or IgG1-11B8 antibody (0.003 to 20.0 μg / mL at 3.33 dilution) For 15 minutes. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  FIG. 26A / B / C shows that CD38 antibody IgG1-005-E345R / Q386K induced CDC-mediated lysis of Wien133 cells in a dose-dependent manner (dashed line). By combining IgG1-005-E345R / Q386K with 1 μg / mL or 10 μg / mL wild-type CD38 antibody IgG1-003 (FIG. 26A) or wild-type CD20 antibody IgG1-11B8 (FIG. 26B), maximum cell lysis is achieved. An enhancement was brought about. Combining IgG1-005-E345R / Q386K with wild-type IgG1-005 inhibited CDC in a dose-dependent manner, probably due to competition for binding sites (FIG. 26C).

  FIG. 26D / E / F shows similar data for the CD38 antibody IgG1-005-E345R / Q386K / E430G.

  These data are related to antibody oligomerization and CDC activation when wild-type antibodies IgG1-003 and IgG1-11B8 are combined with IgG1-005-E345R / Q386K or IgG1-005-E345R / Q386K / E430G. It points to that. In such a combination, it is believed that the blockage of oligomer formation due to the E345 position present in the wild type antibody can be at least partially removed by Q386K substitution in the mutant antibody. The application is particularly interesting in improving therapies using antibodies that are wild-type at the E345 position, such as rituximab, ofatumumab, daratumumab or trastuzumab. Such oligomerization-inducing antibodies may also promote the formation of cell-bound complexes with the patient's own antibodies directed against target cells such as tumor cells or bacteria.

  Example 19 shows that these specific mutations induced efficient CDC against Wien133 cells when incorporated into the CD38 antibody IgG1-005, and other amino acids other than E345 that enhance CDC when subjected to the mutation. For example, E430 and S440 are described. With the exception of the I253 and Y436 mutants, the identified oligomerization enhancing mutation is in contact with the unmutated amino acid on the opposing second Fc moiety in the hexamer ring structure. Thus, the identified oligomerization-enhancing mutations can be expected to promote oligomer formation with unmutated antibodies, either alone or in combination, and a selection strategy similar to that applied in Example 19. It is believed that further optimization of such variants can be achieved.

Example 32
In order to investigate whether E345R can stimulate CDC activity in non-IgG1 antibody isotypes by introducing CDC-induced oligomerization-induced mutations in IgG2, IgG3 and IgG4 antibody isotypes, it is performed by methods known in the art. , Isotype variants of the CD38 antibody IgG1-005 were generated using human IgG2, IgG3 or IgG4 constant domains to yield IgG2-005, IgG3-005 and IgG4-005. In addition, the oligomerization enhancing E345R mutation was introduced into all of these antibodies to yield IgG2-005-E345R, IgG3-005-E345R and IgG4-005-E345R. In a similar manner, CD38 antibodies IgG1-003 to IgG2-003 and IgG2-003-E345R were also produced. The CDC activity of various isotypes was compared in an in vitro CDC assay.

0.1 × 10 ⁶ Wien133 cells were preincubated for 15 minutes at room temperature on a shaker with 10 μg / mL unpurified antibody in a round bottom 96-well plate in a total volume of 100 μL. IgG1-005-E345R was added at 3.0 μg / mL. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS.

  FIG. 27 shows that IgG2-005, IgG2-003, IgG3-005 and IgG4-005 could not efficiently lyse (A) Daudi cells or (B) Wien133 cells under the conditions tested. (Almost 20% lysis observed was considered background). The introduction of the E345R mutation allowed for powerful CDC on Daudi cells for all tested IgG isotypes. Although these results were confirmed using CDC on Wien133 cells, IgG3-005-E345R exhibited limited CDC activity compared to other isotype mutants. These data indicate that oligomerization enhancing mutations such as E345R can be adapted to promote CDC activity of IgG2, IgG3 and IgG4 antibodies in addition to IgG1.

Example 33
CDC with IgG1-005 and IgG1-005-E345R in an ex vivo CDC assay on patient-derived CD38-positive B-cell chronic lymphocyte leukemia (CLL) cells
Cryopreserved primary cells from CLL patient samples were obtained from CDB-IDIBAPS-Hospital Clinic (Dr. Elias Campo, Hematopathology Unit, Department of Pathology, Hospital Clinic, Institut d'Investigacions Biomediques August Pi i Sunyer (IDIBAPS), University of From the hematopathology biobank from Barcelona, Barcelona, Spain or from the National Heart, Lung and Blood Institute (NHLBI) (Dr. Adrian Wiestner, NHLBI, Hematology Branch of the National Institutes of Health (NIH), Bethesda). Informed consent from all patients in accordance with the Hospital Clinic (Barcelona, Spain) Institutional Ethics Committee or NIH Institutional Review Board and Declaration of Helsinki Got. All samples were characterized genetically and immunophenotypically.

  CLL samples were categorized into two groups according to their CD38 expression determined by FACS: 5 samples were included in the high CD38 group (CD38 expression on Daudi cells 50% -98%), 4 samples Were included in the low CD38 group (CD38 expression on Daudi cells 0.5% to 3%).

  Fluorescently labeled CLL cells (labeled with 5 μM calcein AM) were incubated with an antibody concentration series (0.01-10 μg / mL at 10-fold dilution). Next, normal human serum was added as a complement source (10% final concentration) to cells opsonized with antibodies (100,000 cells / well) and incubated at 37 ° C. for 45 minutes. The supernatant was collected and fluorescence was read on a Synergy ™ HT fluorometer as a measure of cell lysis. Cell lethality was calculated as follows: specific lysis = 100 x (sample-spontaneous lysis) / (maximum lysis-spontaneous lysis), where maximum lysis is a sample of cells treated with 1% Triton. Spontaneous lysis is determined from samples incubated with cells in the presence of 10% NHS without antibody.

  FIG. 28 shows that IgG1-005-E345R has stronger CDC activity compared to wild-type IgG1-005 in both CLL primary cells with high CD38 expression and CLL primary cells with mild CD38 expression. It shows that it has increased.

Example 34
Compared to wild-type IgG1-005, the IgG1-005 mutant FcRn-binding neonatal Fc receptor (FcRn) is responsible for the long plasma half-life of IgG by protecting IgG from degradation. After antibody intracellularization, FcRn binds to the antibody Fc region within the endosome, but in a mild acidic environment (pH 6.0) where the interaction is stable. When reused at the plasma membrane where the environment is neutral (pH 7.4), the interaction is lost and the antibody is released again into the bloodstream. This affects the plasma half-life of IgG.

  The ability of IgG1-005 mutants E345K, E345Q, E345R, E345Y, E430F, E430G, E430S, E430T, S440Y, K439E and S440K to interact with FcRn from mice, cynomolgus monkeys and humans was tested in ELISA. In the mouse FcRn ELISA, mutants P247G and I253D were also tested. I253D was used as a negative control for binding to FcRn. All incubations were at room temperature. 96-well plates diluted in PBST supplemented with 0.2% BSA, biotinylated extracellular domain (FcRnECDHis-B2M-BIO) of recombinantly produced FcRn (mouse, human or cynomolgus) 5 μg / mL (100 μL / mL) Well) and coated for 1 hour. Plates were washed 3 times with PBST and wild type IgG1-005 or 005 variants diluted in 3-fold series (in PBST / 0.2% BSA, pH 6.0) were added and the plates were incubated for 1 hour. Plates were washed with PBST / 0.2% BSA, pH 6.0. Goat-anti-human IgG (Fab'2) -HRP (Jackson Immuno Research, catalog number: 109-035-097) diluted in PBST / 0.2% BSA, pH 6.0 was added and the plates were incubated for 1 hour. After washing, ABTS was added as a substrate and the plate was incubated for 30 minutes in the dark. Absorbance was read at 405 nm using an EL808 ELISA reader. Data obtained with the mouse FcRn ELISA was analyzed using best fit values for non-linear agonist dose response fits using log-transformed concentrations in GraphPad PRISM 5 to calculate apparent affinity (EC50) (Table 20). This experiment showed that FcRn binding was not altered by any of the IgG1-005 mutants compared to wild type IgG1-005.

Table 20 Apparent affinity of IgG1-005 and mutants for mouse FcRn (EC50) (μg / ml units)

  FIG. 29 shows that wild type IgG1-005 and all tested variants of IgG1-005 bound well to mouse, human and cynomolgus FcRn at pH 6.0. At pH 7.4, no significant binding to FcRn was detected (data not shown).

Example 35
Enhancement of CDC by various mutations in rituximab in B cell lines Ramos and SU-DHL-4 As described in Example 19, the oligomerization and CDC activity of anti-CD38 antibody IgG1-005 is on the Fc: Fc interface Or it may be stimulated by a single mutation at a particular residue in the vicinity. Oligomerization may also be indirectly stimulated by other types of mutations at residues remote from the Fc: Fc interface that enhance the Fc: Fc interaction allosterically. This was also tested for the IgG1 anti-CD20 antibody rituximab against two B cell lines (Ramos and SU-DHL-4). The following mutations were tested (essentially as described in Example 19): E345K, E345Q, E345R, E345Y, E430G, E430S, E430T and S440Y.

For the CDC assay, 0.1 × 10 ⁶ cells (Ramos or SU-DHL-4) were placed in a round bottom 96-well plate in a total volume of 100 μL with a saturating antibody concentration series (0.0001 at 3-fold dilution). ˜10.0 μg / mL) for 15 minutes at room temperature on a shaker. Next, 25 μL of normal human serum was added as a complement source (final concentration 20%) and incubated for 45 minutes in a 37 ° C. incubator. The reaction was stopped by placing the plate on ice. 10 μL of propidium iodide was added and cell lysis was determined by FACS. Data were analyzed using best fit values for non-linear agonist dose responses using log-transformed concentrations in GraphPad PRISM 5. FIG. 30 shows that all rituximab mutants tested were able to enhance CDC activity in both B cell lines.

Example 36
Complement activation in the target-independent fluid phase: Comparison of IgG1-005 mutants with wild-type IgG1-005 Target-independent complement activation involves antibodies such as bloodstream or organ tissue Can be a safety issue when activating complement. This can lead to unwanted complement activation products or unwanted complement deposition. 100 μg / ml IgG1-005 mutants E345K, E345Q, E345R, E345Y, E430F, E430G, E430S, E430T, S440Y, wild-type IgG1 to investigate complement activation in target-independent liquid phase -005 or heat-aggregated IgG (HAG, positive control) was incubated for 1 hour at 37 ° C in 90% normal human serum. Samples were then transferred to an ELISA kit for measuring C4d production (Micro Vue C4d-fragment, Quidel, San Diego, CA, USA). C4d is an activated fragment of C4, a marker of classical complement pathway activation.

  FIG. 31 shows that wild type IgG1-005, IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345Y, IgG1-005-E430G, IgG1-005-E430S and IgG1-005-S440Y have little C4 activity While IgG1-005-E345R, IgG1-005-E430F and IgG1-005-E430T showed increased C4d production (C4 activation) compared to wild-type IgG1-005 ing.

Example 37
Plasma clearance rate of IgG1-005 mutant compared to wild type IgG1-005 The mice in this study were housed in a barrier unit of the Central Laboratory Animal Facility (Utrecht, The Netherlands) and placed in a filter top cage. Ad libitum access to water and feed. All experiments were approved by the Utrecht University Animal Ethics Committee. Three mice per group were used to inject 500 μg of antibody to SCID mice (CB-17 / Icr-Prkdc <Scid> / IcrIcoCrl, Charles-River).

  50 μL blood samples were collected from the saphenous vein at 10 minutes, 4 hours, 1 day, 2 days, 7 days, 14 days and 21 days after antibody administration. Blood was collected in heparin containing vials and centrifuged at 10,000g for 5 minutes. Plasma was stored at −20 ° C. until determination of antibody concentration.

  The concentration of specific human IgG was determined using a total hIgG ELISA and a CD38 specific sandwich ELISA.

  For total hIgG ELISA, mouse mAb anti-human IgG-κ clone MH16 (# M1268, CLB Sanquin, Netherlands) coated on a 96-well Microlon ELISA plate (Greiner, Germany) at a concentration of 2 μg / mL as capture antibody Using. After blocking the plate with PBS supplemented with 0.2% bovine serum albumin, samples were added and serially diluted in ELISA buffer (PBS supplemented with 0.05% Tween 20 and 0.2% bovine serum albumin) at room temperature. Incubate for 1 hour on a plate shaker at (RT). The plate was then incubated with goat anti-human IgG immunoglobulin (# 109-035-098, Jackson, West Grace, PA) and 2,2'-azino-bis (3-ethylbenzthiazoline-6-sulfate) (ABTS Developed in Roche, Mannheim, Germany). Absorbance was measured at 405 nm with a microplate reader (Biotek, Winooski, VT).

  For specific CD38 ELISA, 96-well Microlon ELISA plates (Greiner, Germany) were coated with CD38 extracellular domain (concentration 2 μg / mL) labeled with a His tag. After blocking the plate with ELISA buffer, samples diluted serially with ELISA buffer were added and incubated for 1 hour on a plate shaker at room temperature (RT). The plate was then incubated with 30 ng / ml mouse anti-human IgG1-HRP (Sanquin M 1328, clone MH161-1) to give 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfate) (ABTS Developed in Roche, Mannheim, Germany). Absorbance was measured at 405 nm with a microplate reader (Biotek, Winooski, VT).

  FIG.32A shows wild type reference antibody IgG1-005 and antibody variants IgG1-005-E345K, IgG1-005-E345Q, IgG1-005-E345R, IgG1-005-E345Y, IgG1-005-E430F, IgG1-005-E430G IgG clearance rates of IgG1-005-E430S, IgG1-005-E430T, IgG1-005-S440Y are shown. Mutants IgG1-005-E430S, IgG1-005-E430G and IgG1-005-S440Y, IgG1-005-E430T, IgG1-005-E345K, IgG1-005-E345Q and IgG1-005-E345Y are wild-type IgG1-005 The clearance rate was almost the same as in the case of. Mutants IgG1-005-E430F and IgG1-005-E345R exhibited faster clearance rates. Plasma clearance rate was calculated as dose / AUC (mL / day / kg). The AUC value (area under the curve) was determined from the concentration-time curve.

  FIG. 32B shows wild type reference antibody IgG1-005 and antibody variants IgG1-005-E345K, IgG1-005- when injected intravenously one day after intraperitoneal administration of 8.0 mg of an irrelevant IgG1-B12 control antibody. The IgG clearance rates determined by CD38-specific ELISA of E345R, IgG1-005-E430G, IgG1-005-E430S and IgG1-005-S440Y are shown. A wild type reference antibody IgG1 in the absence of an irrelevant b12 control was included as a control. Mutants IgG1-005-E430S, IgG1-005-E430G, IgG1-005-S440Y and IgG1-005-E345K showed almost the same clearance rate as the wild type. Mutant IgG1-005-E345R exhibited faster clearance.

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims. Any and all combinations of the embodiments disclosed in the dependent claims are intended to be within the scope of the present invention.

Claims (76)

A method for enhancing complement dependent cytotoxicity (CDC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the following steps:
Introducing into the parent polypeptide a mutation in one or more amino acid residues selected from the group corresponding to E430X, E345X and S440W in the Fc region of the human IgG1 heavy chain.   The mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain. The method described in 1.   The mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain. Method. A method for enhancing CDC and antibody-dependent cell-mediated cytotoxicity (ADCC) of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising the following steps:
Introducing a mutation in one or more amino acid residues corresponding to E430X, E345X and S440W in the Fc region of human IgG1 heavy chain into the parent polypeptide.   5. The method of claim 4, wherein the mutation in one or more amino acid residues corresponds to E345R, E430T and E430F in the Fc region of a human IgG1 heavy chain.   6. The method according to any one of claims 1 to 5, wherein the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.   7. The method of claim 6, wherein the parent antibody is a monospecific antibody, bispecific antibody or multispecific antibody. A first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and a second polypeptide comprising an immunoglobulin second CH2-CH3 region and a second antigen-binding region; A method for enhancing the complement dependent cytotoxicity (CDC) of a parent antibody, which is a bispecific antibody comprising a peptide, comprising:
The first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens, and the method is directed against the first and / or second CH2-CH3 regions, Introducing a mutation in one or more amino acid residues selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the IgG1 heavy chain; and the first CH2-CH3 region comprises: A further amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; and the second CH2-CH3 region is human An additional amino acid mutation at a position selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the IgG1 heavy chain; and the additional amino acid in the first CH2-CH3 region Mutations in the second CH2-CH3 region The method, which is different from an amino acid mutation.   The mutation in one or more amino acid residues is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain. Item 9. The method according to Item 8.   10. The method according to any one of claims 8 or 9, comprising introducing mutations into both the first and second polypeptides of the bispecific antibody.   The further amino acid mutation of the first CH2-CH3 region is in a position corresponding to K409 in the Fc region of a human IgG1 heavy chain, for example K409R; and the further amino acid of the second CH2-CH3 region The method according to any one of claims 8 to 10, wherein the mutation is at a position corresponding to F405 in the Fc region of the human IgG1 heavy chain, for example, F405L. Introducing a mutation other than S440Y and S440W at one or more positions, and (i) a mutation in each of the amino acid residues corresponding to K439 and S440 in the Fc region of the human IgG1 heavy chain, provided that S440 A mutation, provided that the mutation in is not S440Y or S440W,
(Ii) mutations in each of the amino acid residues corresponding to K447 and 448 in the Fc region of the human IgG1 heavy chain, for example K447K / R / H and 448E / D, preferably human IgG1 heavy chain in the Fc region of the human IgG1 heavy chain (Iii) mutations in each of the amino acid residues corresponding to K447, 448 and 449 in the Fc region of the human IgG1 heavy chain, eg, K447D / E, 448K in the Fc region of the human IgG1 heavy chain / R / H and 449P, preferably K447E, 448K and 449P in the Fc region of the human IgG1 heavy chain
12. The method according to any one of claims 1 to 11, further comprising the step of introducing.   Introducing a mutation other than S440Y and S440W at one or more positions, and further introducing a mutation into each of the amino acid residues corresponding to K439 and / or S440 in the Fc region of the human IgG1 heavy chain, However, the method according to claim 12, provided that the mutation in S440 is neither S440Y nor S440W.   The mutation at a position corresponding to K439 in the Fc region of human IgG1 heavy chain is K439D / E, and / or the mutation at a position corresponding to S440 in the Fc region of human IgG1 heavy chain is S440K / R, The method according to claim 13.   A method of enhancing the complement dependent cytotoxicity (CDC) of a combination of at least a first and a second parent polypeptide, wherein the at least first and second parent polypeptides are each an immunoglobulin Fc domain and One or more selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of the human IgG1 heavy chain with respect to the at least first and / or second parent polypeptide. The method comprising introducing mutations in the amino acid residues of   For said at least first and / or second parent polypeptide, selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain 16. The method of claim 15, comprising introducing mutations in said one or more amino acid residues.   17. The method of claim 16, comprising introducing mutations that may be the same or different for both the first and second parent polypeptides. (I) 1 selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide Introducing mutations in one or more amino acid residues;
(Ii) including mutations in one or more amino acid residues selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain Providing the second parent polypeptide,
The method of claim 16 comprising: The mutation at one or more positions is different from S440Y and S440W, and the method comprises:
(I) introducing a second mutation into the amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide; and (ii) the second parent poly A step of introducing a second mutation into the amino acid residue corresponding to S440 in the Fc region of the human IgG1 heavy chain with respect to the peptide, provided that the mutation is neither S440Y nor S440W Further comprising steps (i) and (ii)
(Iii) a step of introducing a second mutation into an amino acid residue corresponding to position S440 in the Fc region of the human IgG1 heavy chain with respect to the first parent polypeptide, provided that the mutation is S440Y Stage, provided that it is not S440W;
(Iv) The second parent polypeptide may be a step of introducing a second mutation at an amino acid residue corresponding to position K439 in the Fc region of the human IgG1 heavy chain. The method as described in any one of.   The mutation at a position corresponding to K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at a position corresponding to S440 in the Fc region of a human IgG1 heavy chain is S440K / R Item 20. The method according to Item 19.   21. The first and second parent antibodies according to any one of claims 15 to 20, wherein the first and second parent polypeptides are first and second parent antibodies, each comprising an immunoglobulin Fc domain and an antigen binding region. Method.   24. The method of claim 21, wherein the first and second parent antibodies are monospecific antibodies, bispecific antibodies or multispecific antibodies.   The first and / or second parent antibody comprises a first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and a second CH2-CH3 region and a second A bispecific antibody comprising a second polypeptide comprising an antigen binding region, wherein the first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens, and The first CH2-CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; and Two CH2-CH3 regions contain additional amino acid mutations at positions selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain; and The additional amino acid mutation in the CH2-CH3 region of the second CH2-CH3 region 23. The method of claim 22, wherein the method differs from the amino acid mutation.   The first CH2-CH3 region comprises a further amino acid mutation, for example K409R, at a position corresponding to K409 in the Fc region of a human IgG1 heavy chain, and the second CH2-CH3 region is a human IgG1 heavy chain 24. The method of claim 23, comprising an additional amino acid mutation, such as F405L, at a position corresponding to F405 in the Fc region.   25. The method of any one of claims 1-3 and 6-24, wherein the method does not alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the parent polypeptide or the parent antibody.   26. The method of any one of claims 1-25, wherein the binding of the parent polypeptide or parent antibody to the neonatal Fc receptor (FcRn) determined by the method disclosed in Example 34 is not altered.   More than 30% of the binding of the parent polypeptide or the parent antibody to the neonatal Fc receptor (FcRn) as determined by the change in absorbance at OD405nm, as determined by the method disclosed in Example 34, for example 26. The method of any one of claims 1-25, wherein the method does not increase or decrease by more than 20%, 10% or 5%.   Does not enhance the apparent affinity of the parent polypeptide or the parent antibody to the mouse neonatal Fc receptor (FcRn) by more than 0.5-fold, as determined by the method disclosed in Example 34, or the parent polypeptide or 26. The method of any one of claims 1-25, wherein the apparent affinity of the parent antibody for mouse FcRn is not reduced more than 2-fold.   29. The method of any one of claims 1-28, wherein the method does not alter the plasma clearance rate of the parent polypeptide or the parent antibody as determined by the method disclosed in Example 37.   Enhancing the plasma clearance rate of the parent polypeptide or antibody determined by the method disclosed in Example 37 by more than 3.0 times, for example, by 2.5 times, 2.0 times, 1.5 times or 1.2 times 29. The method of any one of claims 1-28, wherein the method is not reduced.   31. The method of any one of claims 1-30, wherein the method does not alter complement activation in the target-independent liquid phase of the variant as determined by the method disclosed in Example 36.   32. The method of any one of claims 1-31, wherein the plasma half-life of the parent polypeptide or the parent antibody is not altered.   A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain Said variant comprising one or more mutations selected from: provided that the variant does not contain further mutations in the Fc domain that alter binding of the variant to the neonatal Fc receptor (FcRn).   A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y and S440W in the Fc region of a human IgG1 heavy chain To the neonatal Fc receptor (FcRn) of the mutant as measured by the change in absorbance at OD405nm, as determined by the method disclosed in Example 34, comprising one or more mutations selected from Said mutant, provided that it does not contain further mutations in the Fc domain that increase or decrease binding by more than 30%, for example more than 20%, 10% or 5%.   35. The variant according to any one of claims 33 or 34, wherein the one or more mutations are selected from the group corresponding to E430G, E430S, E345K and E345Q in the Fc region of the human IgG1 heavy chain.   36. The variant according to any one of claims 33 to 35, which does not comprise further mutations in the Fc domain that alter the antibody-dependent cell-mediated cytotoxicity (ADCC) of the variant.   37. A variant according to any one of claims 33 to 36, which does not contain further mutations in the Fc domain that alter the plasma clearance rate of the variant as determined by the method disclosed in Example 37.   Further increase in the plasma clearance rate of the mutant as determined by the method disclosed in Example 37 by more than 3.0 fold, for example 2.5 fold, 2.0 fold, 1.5 fold or 1.2 fold or more in the Fc domain 37. The variant according to any one of claims 33 to 36, which does not contain a mutation.   37. A variant according to any one of claims 33 to 36, which does not contain further mutations in the Fc domain that alter the serum half-life of the variant.   40. Any of the claims 33-39, comprising no further mutations in the Fc domain that alter complement activation in the target-independent fluid phase of the mutant as determined by the method disclosed in Example 36. The variant according to one item.   41. A variant according to any one of claims 33 to 40, which does not comprise further mutations in the Fc domain.   42. A variant according to any one of claims 33 to 41 comprising only one mutation.   34. A combination of two mutations in an amino acid residue selected from the group corresponding to E345X / E430X, E345X / S440Y, E345X / S440W, E430X / S440Y and E430X / S440W in the Fc region of a human IgG1 heavy chain 43. A variant according to any one of -42. A variant of a parent polypeptide comprising an immunoglobulin Fc domain and a binding region, comprising:
A first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain; and selected from the group corresponding to Said variant comprising a second mutation:
(I) amino acid residues corresponding to K439 and S440 in the Fc region of human IgG1 heavy chain, provided that the mutation in S440 is neither S440Y nor S440W, and the first mutation is S440Y or S440W 2 mutations are in the amino acid residue corresponding to K439 in the Fc region of the human IgG1 heavy chain;
(Ii) amino acid residues corresponding to K447D / E or corresponding to K447K / R / H and 448P in the Fc region of human IgG1 heavy chain; or (iii) K447D / in the Fc region of human IgG1 heavy chain Amino acid residues corresponding to E or corresponding to K447K / R / H and 448K / R / H and 449P.   Corresponds to the first mutation selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R and E345Y in the Fc region of human IgG1 heavy chain, and K439 and S440 in the Fc region of human IgG1 heavy chain 45. A variant according to claim 44, comprising a second mutation in the amino acid residue to be obtained provided that the mutation in S440 is not S440Y or S440W.   46. The variant according to claim 45, wherein the mutation in the amino acid residue corresponding to K439 is K439D / E, and the amino acid residue corresponding to S440 is S440K / R.   47. The variant according to any one of claims 33 to 46, wherein the parent polypeptide is a parent antibody comprising an immunoglobulin Fc domain and an antigen binding region.   48. The variant of claim 47, selected from monospecific antibodies, bispecific antibodies or multispecific antibodies. A first polypeptide comprising an immunoglobulin first CH2-CH3 region and a first antigen-binding region and a second polypeptide comprising an immunoglobulin second CH2-CH3 region and a second antigen-binding region; A variant of a parent antibody that is a bispecific antibody comprising a peptide, wherein the first and second antigen binding regions bind to different epitopes on the same antigen or on different antigens; and And / or the second CH2-CH3 region is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain or A further mutation in an amino acid residue comprising a plurality of mutations and wherein the first polypeptide is selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain And wherein the second polypeptide is a Fc region of a human IgG1 heavy chain Further mutations in amino acid residues selected from those corresponding to F405, T366, L368, K370, D399, Y407 and K409, and wherein the further mutation in the first polypeptide comprises the second poly Said variant different from said further mutation in a peptide. (I) the first polypeptide comprises a further mutation in the amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain, for example K409R; and (ii) the second polypeptide comprises human IgG1 heavy A further mutation in an amino acid residue corresponding to F405 in the Fc region of the chain, eg F405L; or alternatively (iii) the first polypeptide is an amino acid corresponding to F405 in the Fc region of a human IgG1 heavy chain A further mutation at a residue, for example F405L; and (iv) the second polypeptide comprises a further mutation at an amino acid residue corresponding to K409 in the Fc region of the human IgG1 heavy chain, for example K409R;
50. A variant according to claim 49.   51. A variant according to any one of claims 33 to 50 which is conjugated to a drug, toxin or radiolabel, for example conjugated to a toxin via a linker.   52. A variant according to any one of claims 33 to 51 which is part of a fusion protein.   53. A human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally a human full length antibody, such as a human full length IgG1 antibody. Mutants.   A composition comprising first and second variants of a parent polypeptide each comprising an immunoglobulin Fc domain and a binding region, wherein the first and / or second variant comprises a human IgG1 heavy chain The composition comprising one or more mutations selected from the group corresponding to E430X, E345X, S440Y and S440W in the Fc region of:   The first and / or second variant is selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain 55. The composition of claim 54, comprising a plurality of mutations.   56. The composition of claim 55, wherein both the first and second variants comprise one or more mutations that may be the same or different. Said first variant comprises one or more mutations selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of human IgG1 heavy chain And the second variant is in an amino acid residue selected from the group corresponding to E430G, E430S, E430F, E430T, E345K, E345Q, E345R, E345Y, S440Y and S440W in the Fc region of the human IgG1 heavy chain 56. The composition of claim 55, wherein the composition does not comprise one or more mutations. (I) the first mutant further comprises a mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain; and (ii) the second mutant is in the Fc region of the human IgG1 heavy chain. Further comprising a mutation at a position corresponding to S440, provided that the mutation is neither S440Y nor S440W; or (i) and (ii) alternatively
(Iii) the first variant further comprises a mutation at a position corresponding to S440 in the Fc region of the human IgG1 heavy chain, provided that the mutation is not S440Y or S440W; and (iv) the The second variant further comprises a mutation at a position corresponding to K439 in the Fc region of the human IgG1 heavy chain;
58. The composition according to any one of claims 54 to 57, which may be   59. The composition of claim 58, wherein the mutation at position K439 in the Fc region of a human IgG1 heavy chain is K439D / E and / or the mutation at position S440 in the Fc region of a human IgG1 heavy chain is S440K / R. object. (I) the first variant further comprises a prodrug, and (ii) the second variant comprises an activator for the prodrug on the first variant; or (i) And (ii) is alternatively
(Iii) the second variant comprises a prodrug, and (iv) the first variant may comprise an activator for the prodrug on the second variant, 60. The composition according to any one of claims 54 to 59.   61. The first and second parent antibodies according to any one of claims 54 to 60, wherein the first and second parent polypeptides are first and second parent antibodies, each comprising an immunoglobulin Fc domain and an antigen binding region. Composition.   Said first and second antibodies are each human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgM or IgE antibody, optionally each human full length antibody, for example each human full length IgG1 antibody 62. The composition of claim 61.   64. The composition of claim 62, wherein the first and second antibodies are each selected from monospecific antibodies, bispecific antibodies, or multispecific antibodies.   The first and / or second parent antibody comprises a first polypeptide comprising a first CH2-CH3 region and a first antigen-binding region of an immunoglobulin and a second CH2-CH3 region and a second, respectively. A bispecific antibody comprising a second polypeptide comprising a plurality of antigen-binding regions, wherein the first and second antigen-binding regions bind to different epitopes on the same or different antigens, and The first CH2-CH3 region comprises an additional amino acid mutation at a position selected from those corresponding to K409, T366, L368, K370, D399, F405 and Y407 in the Fc region of the human IgG1 heavy chain; and Two CH2-CH3 regions contain additional amino acid mutations at positions corresponding to F405, T366, L368, K370, D399, Y407 and K409 in the Fc region of the human IgG1 heavy chain, and the first CH2-CH3 region Wherein said further amino acid mutation in said second CH2-CH3 region 64. The composition of claim 63, wherein the composition is different from the mutation.   The further amino acid mutation of the first CH2-CH3 region is in a position corresponding to K409 in the Fc region of a human IgG1 heavy chain, for example K409R; and the further amino acid of the second CH2-CH3 region 65. The composition of claim 64, wherein the mutation is at a position corresponding to F405 in the Fc region of a human IgG1 heavy chain, such as F405L.   64. The composition of any one of claims 54 to 63, wherein the first and second variants of the composition bind to different epitopes on the same antigen or on different antigens.   67. Any of the first and second variants are conjugated to a drug, toxin or radiolabel, eg, conjugated to a toxin via a linker. The composition according to one item.   68. The composition according to any one of claims 54 to 67, wherein one or both of the first and second variants are part of a fusion protein.   69. The composition of any one of claims 54-63 and 66-68, wherein the first and / or second variant of the composition comprises only one mutation.   70. A composition comprising the variant according to any one of claims 33 to 53 or the composition according to any one of claims 54 to 69 and a pharmaceutically acceptable carrier.   54. A kit of parts comprising a first variant and a second variant as defined in any one of claims 33 to 53 for simultaneous, separate or sequential use in therapy.   72. A variant, composition or kit of parts according to any one of claims 33 to 71 for the treatment of diseases such as cancer.   72. A method for the treatment of a disease in a human comprising administering a variant, composition or kit of parts according to any one of claims 33-71.   72. A method for the treatment of cancer in a human comprising administering a variant, composition or kit of parts according to any one of claims 33-71.   72. A variant, composition or kit of parts according to any one of claims 33 to 71 for use in imaging at least part of a human or other mammalian body.   72. A method for imaging at least part of a human or other mammalian body comprising administering a variant, composition or kit of parts according to any one of claims 33-71. .

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