WO2013118858A1 - 抗体のFc領域改変体 - Google Patents
抗体のFc領域改変体 Download PDFInfo
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Definitions
- the present invention provides an antibody Fc region whose amino acid sequence is modified from a naturally occurring antibody Fc region, an antibody containing the Fc region, a pharmaceutical composition containing the antibody, and a method for producing them.
- Antibodies are attracting attention as pharmaceuticals because of their high stability in plasma and few side effects. Among them, many IgG-type antibody drugs are on the market, and many antibody drugs have been developed (Non-patent Documents 1 and 2).
- Non-patent Document 3 Fc region with improved pharmacokinetics
- Fc region with enhanced ADCC activity which is an effector function
- Fc region with reduced ADCC activity in neutralizing antibodies have been studied (Non-patent Document 3).
- modification of the Fc region is known to adversely affect the physical properties of the antibody.
- the midpoint of heat denaturation is reduced by about 20 ° C. (Non-patent Document 6).
- Non-patent Documents 7-9 Furthermore, in the modified Fc region with improved blood retention, a decrease in thermal stability and storage stability has been reported (Patent Document 1). That is, many of the modified Fc regions found so far have lost good stability, which is one of the advantages of antibodies, as functions are enhanced.
- Non-patent Document 10 a method of introducing a cysteine by modifying the amino acid of the CH2 domain has been reported. It has been reported that the formation of a new disulfide bond by introduction of cysteine increases the thermal stability by about 10 ° C to 20 ° C. However, since this report evaluates the thermal stability only with the CH2 domain, the thermal stability in the form of IgG is unknown. In addition, an increase in heterogeneity is expected with the formation of new disulfide bonds. As described above, no Fc region excellent in both activity and stability has been reported so far.
- the present invention has been made in view of such circumstances, and an object thereof is to provide a polypeptide having improved stability by altering the amino acid of the antibody Fc region.
- the inventors of the present invention may have enhanced function while maintaining antibody stability, or may have recovered stability that has decreased with increased function. I thought it was desirable.
- the present inventors have found that a polypeptide having an Fc region of an antibody is stable compared to the parent polypeptide by modifying at least one amino acid in the loop site of the Fc region.
- a polypeptide whose binding activity to Fc ⁇ R is maintained or enhanced while improving thermal stability and improving thermal stability compared to the parent polypeptide.
- a polypeptide with reduced Fc ⁇ R binding activity was successfully obtained.
- the present inventors succeeded not only in adjusting the binding activity to Fc ⁇ R while improving the thermal stability, but also obtaining a polypeptide having a reduced aggregate content.
- the present invention relates to the following.
- EU numbering 234, EU numbering 235th, EU numbering 239th, EU numbering 266th, EU numbering 267th, EU numbering 268th, EU numbering 269th, EU numbering 270th in the loop region of the Fc region , EU numbering 295, EU numbering 296, EU numbering 298, EU numbering 300, EU numbering 324, EU numbering 325, EU numbering 326, and EU numbering 330 The polypeptide according to any one of [1] to [5], wherein at least one or more amino acid mutations are introduced.
- the polypeptide according to Item Any one of [1] to [4] and [6], wherein at least one amino acid mutation is introduced at an amino acid site selected from the group consisting of EU numbering 326 and EU numbering 330 The polypeptide according to Item.
- the amino acid modification in the loop region of the Fc region includes substitution of the EU numbering 234th amino acid with Ile, substitution of the EU numbering 266th amino acid with Ile, substitution of the EU numbering 268th amino acid with Gln, EU numbering 269th amino acid substitution to Asp, EU numbering 270th amino acid substitution to Glu, EU numbering 295th amino acid substitution to Met or Leu, EU numbering 300th amino acid substitution to Glu, EU numbering at least one selected from the group consisting of substitution of amino acid at position 324 with His, substitution of amino acid at position 326 with EU or Ser or Ala, and substitution of amino acid at position 330 with His or Tyr.
- EU numbering 234, EU numbering 235th, EU numbering 239th, EU numbering 267th, EU numbering 268th, EU numbering 270th, EU numbering 295th, EU numbering 296th in the loop region of the Fc region [1] to [3], [5], [6], wherein at least one amino acid mutation is introduced at an amino acid site selected from the group consisting of EU numbering 298th and EU numbering 325th The polypeptide according to any one of the above.
- the amino acid modification in the loop region of the Fc region includes the substitution of the 234th amino acid of EU numbering to Lys or Arg, the substitution of the 235th amino acid of EU numbering to Lys or Arg, the Lys of the 239th amino acid of EU numbering , EU numbering 267th amino acid to Pro, EU numbering 268th amino acid to Met or Lys, EU numbering 270th amino acid to Phe, EU numbering 295th amino acid Met
- substitution of the amino acid number 296 of EU numbering to Gly substitution of amino acid number 298 of EU numbering to Gly, and substitution of amino acid number 325 of EU numbering to any of Gly, His or Met
- the polypeptide according to any one of [1] to [3], [5], [6], and [9], wherein the polypeptide is at least one amino acid modification selected from the above.
- At least one or more amino acid mutations are introduced in an amino acid site selected from the group consisting of 295th EU numbering, 326th EU numbering, and 330th EU numbering in the loop region of the Fc region, [1] The polypeptide according to any one of [4].
- the amino acid modification in the loop region of the Fc region includes substitution of the 295th amino acid of EU numbering with Met or Leu, substitution of the 326th amino acid of EU numbering with Ser or Ala, and the amino acid of the EU numbering 330th amino acid.
- at least one amino acid mutation has been introduced at an amino acid site selected from the group consisting of EU numbering 325th, [1] to [3], [5] peptide.
- the amino acid modification in the loop region of the Fc region includes substitution of EU numbering 234th amino acid with Lys, EU numbering 235th amino acid substitution with Lys or Arg, EU numbering 239th amino acid Lys or Ser Substitution to EU numbering 268th amino acid to Lys or His, EU numbering 270th amino acid to Phe or Asp, EU numbering 295th amino acid to Met, EU numbering 296th amino acid At least one amino acid modification selected from the group consisting of substitution of Gly with EU, substitution of amino acid at position 298 with Gly, and substitution of amino acid at position 325 with EU or His or Gly.
- the polypeptide according to any one of [1] to [3], [5] and [13].
- the amino acid modification in the loop region of the Fc region includes substitution of the EU numbering 234th amino acid with Lys, substitution of the EU numbering 235th amino acid with Lys or Arg, and EU numbering 239th amino acid with Lys.
- the amino acid modification at the loop site of the Fc region is the substitution of the EU numbering 234th amino acid with Lys, the EU numbering 235th amino acid with Lys or Arg, the EU numbering 239th amino acid with Lys Substitution, EU numbering 295th amino acid to Met, EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, and EU numbering 325th amino acid to His or Gly
- the polypeptide according to any one of [1] to [3], [5], and [17] which is at least one amino acid modification selected from the group consisting of: [19] EU numbering 234, EU numbering 235th, EU numbering 268th, EU numbering 270th, EU numbering 295th, EU numbering 296th, EU numbering 298th, and EU numbering 325 in the loop region of the Fc region
- the amino acid modification in the loop region of the Fc region includes substitution of the 234th amino acid of EU numbering to Lys, substitution of the 235th amino acid of EU numbering to Lys or Arg, and substitution of the 268th amino acid of EU numbering 268 to Lys. Substitution, substitution of EU numbering 270 amino acid to Phe, EU numbering 295th amino acid to Met, EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, And at least one amino acid modification selected from the group consisting of substitution of the amino acid number 325 of EU numbering with His or Gly, any one of [1] to [3], [5], [19] The polypeptide according to Item.
- the amino acid modification site in the loop region of the Fc region is selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325
- the polypeptide according to any one of [1] to [3], [5], wherein at least one or more amino acid mutations are introduced at the amino acid site.
- the amino acid modification in the loop region of the Fc region is the substitution of the EU numbering 234th amino acid with Lys, the EU numbering 235th amino acid with Lys or Arg, the EU numbering 295th amino acid with Met.
- At least one selected from the group consisting of substitution, substitution of EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, and EU numbering 325th amino acid to His or Gly The polypeptide according to any one of [1] to [3], [5], and [21], which is the above amino acid modification.
- EU numbering 247, EU numbering 250th, EU numbering 307th, EU numbering 309th, EU numbering 315th, EU numbering 360th, EU numbering 385th, EU numbering 386th in the loop region of the Fc region At least one amino acid mutation has been introduced in an amino acid site selected from the group consisting of EU numbering 387th, EU numbering 389th, EU numbering 428th, and EU numbering 433th, [1] to [ 4].
- the polypeptide according to any one of 4).
- the amino acid modification in the loop site of the Fc region is substitution of EU numbering 247th amino acid to Val, EU numbering 250th amino acid to Phe, Ile, Met, Val, Trp, or Tyr.
- At least one amino acid mutation has been introduced at the amino acid site at EU numbering 298 or EU numbering 309 in the loop region of the Fc region, [1] to [3], [5]
- At least one or more amino acid modifications in the loop region of the Fc region are selected from the group consisting of substitution of EU numbering 298th amino acid with Gly and substitution of EU numbering 309th amino acid with Asp
- the polypeptide according to any one of [1] to [3], [5], and [25], which is an amino acid modification of [27] A method for improving stability of a polypeptide having an Fc region of an antibody by adding at least one amino acid modification to a loop site of the Fc region as compared with a parent polypeptide.
- a method for producing a polypeptide comprising the following steps, comprising an antibody Fc region, wherein at least one amino acid in a loop site of the Fc region is modified, and stability is improved compared to the parent polypeptide; (A) in a polypeptide having an Fc region of an antibody, a step of modifying at least one amino acid at a loop site of the Fc region; (B) measuring the stability of the polypeptide modified in step (a), and (C) selecting a polypeptide with improved stability compared to the parent polypeptide.
- a method for producing a polypeptide having the Fc region of an antibody comprising the following steps, wherein at least one amino acid in a loop site of the Fc region is modified, and stability is improved compared to the parent polypeptide; (A) modifying the nucleic acid encoding the polypeptide so that the stability is improved compared to the parent polypeptide; (B) introducing the modified nucleic acid into a host cell and culturing so as to express; (C) recovering the polypeptide from the host cell culture.
- EU numbering 234, EU numbering 235th, EU numbering 239th, EU numbering 266th, EU numbering 267th, EU numbering 268th, EU numbering 269th, EU numbering 270th in the loop part of the Fc region , EU numbering 295, EU numbering 296, EU numbering 298, EU numbering 300, EU numbering 324, EU numbering 325, EU numbering 326, and EU numbering 330 The method according to any one of [27] to [33], wherein at least one amino acid mutation is introduced.
- the amino acid modification in the loop region of the Fc region is substitution of EU numbering 234th amino acid with Ile, substitution of EU numbering 266th amino acid with Ile, substitution of EU numbering 268th amino acid with Gln, EU numbering 269th amino acid substitution to Asp, EU numbering 270th amino acid substitution to Glu, EU numbering 295th amino acid substitution to Met or Leu, EU numbering 300th amino acid substitution to Glu, EU numbering at least one selected from the group consisting of substitution of amino acid at position 324 with His, substitution of amino acid at position 326 with EU or Ser or Ala, and substitution of amino acid at position 330 with His or Tyr.
- the amino acid modification in the loop region of the Fc region includes the substitution of the 234th amino acid of EU numbering to Lys or Arg, the substitution of the 235th amino acid of EU numbering to Lys or Arg, the Lys of the 239th amino acid of EU numbering , EU numbering 267th amino acid to Pro, EU numbering 268th amino acid to Met or Lys, EU numbering 270th amino acid to Phe, EU numbering 295th amino acid Met Substitution of EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, and EU numbering 325th amino acid to Gly, His or Met
- the method according to any one of [27] to [31], [33], [34], and [37], wherein the method is at least one amino acid modification selected from [39] At least one or more amino acid mutations are introduced at an amino acid site selected from the group consisting of EU numbering 295th, EU numbering 326th, and EU numbering 330th in the loop region of the Fc region
- the amino acid modification at the loop site of the Fc region is substitution of Met or Leu at EU numbering 295, substitution of Ser or Ala at EU numbering 326, and substitution of His or Tyr at EU numbering 330
- the method according to any one of [27] to [31], [33] wherein at least one amino acid mutation is introduced at an amino acid site selected from the group consisting of EU numbering 325.
- the amino acid modification in the loop region of the Fc region includes substitution of the EU numbering 234th amino acid with Lys, substitution of the EU numbering 235th amino acid with Lys or Arg, EU numbering 239th amino acid Lys or Ser Substitution to EU numbering 268th amino acid to Lys or His, EU numbering 270th amino acid to Phe or Asp, EU numbering 295th amino acid to Met, EU numbering 296th amino acid At least one amino acid modification selected from the group consisting of substitution of Gly with EU, substitution of amino acid at position 298 with Gly, and substitution of amino acid at position 325 with EU or His or Gly.
- the amino acid modification in the loop region of the Fc region is substitution of EU numbering 234th amino acid with Lys, EU numbering 235th amino acid substitution with Lys or Arg, EU numbering 239th amino acid with Lys Substitution, substitution of EU numbering 268th amino acid to Lys, EU numbering 270th amino acid substitution to Phe, EU numbering 295th amino acid substitution to Met, EU numbering 296th amino acid substitution to Gly, [27] to [31], which is at least one amino acid modification selected from the group consisting of substitution of the 298th amino acid of EU numbering with Gly and substitution of the 325th amino acid of EU numbering with His or Gly. [33] The method according to any one of [43].
- the amino acid modification in the loop region of the Fc region is the substitution of the EU numbering 234th amino acid with Lys, the EU numbering 235th amino acid with Lys or Arg, the EU numbering 268th amino acid with Lys. Substitution, substitution of EU numbering 270 amino acid to Phe, EU numbering 295th amino acid to Met, EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, And any one of [27] to [31], [33], [47], which is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 325 of EU numbering with His or Gly The method according to item.
- amino acid site selected from the group consisting of EU numbering 234, EU numbering 235, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325, in the loop region of the Fc region
- the method according to any one of [27] to [31], [33], wherein at least one amino acid mutation is introduced.
- the amino acid modification in the loop region of the Fc region includes substitution of the 234th amino acid of EU numbering to Lys, substitution of the 235th amino acid of EU numbering to Lys or Arg, and substitution of the 295th amino acid of EU numbering 295 to Met.
- At least one selected from the group consisting of substitution, substitution of EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, and EU numbering 325th amino acid to His or Gly The method according to any one of [27] to [31], [33], [49], which is the above amino acid modification.
- EU numbering 247th, EU numbering 250th, EU numbering 307th, EU numbering 309th, EU numbering 315th, EU numbering 360th, EU numbering 385th, EU numbering 386th in the loop part of the Fc region At least one amino acid mutation is introduced at an amino acid site selected from the group consisting of EU numbering 387, EU numbering 389, EU numbering 428, and EU numbering 433, [27] to [32] The method as described in any one of. [52]
- the amino acid modification in the loop region of the Fc region is substitution of EU numbering 247th amino acid to Val, EU numbering 250th amino acid to Phe, Ile, Met, Val, Trp, or Tyr.
- Substitution substitution of EU numbering 307 amino acid to any of Ala, Gln, or Pro, EU numbering substitution of 309th amino acid to any of Ala, Arg, or Pro, EU numbering 315th amino acid of Ala EU numbering 360th amino acid substitution to His, EU numbering 385th amino acid substitution to Asp, EU numbering 386th amino acid substitution to Pro, EU numbering 387th amino acid substitution to Glu Substitution, substitution of EU numbering 389th amino acid to Ser, EU numbering 428th amino acid substitution of any of His, Trp, Tyr, or Phe, and EU numbering 433th amino acid At least one or more amino acid modifications selected from the group consisting of substitution of Lys acid, [27] - [32] The method according to any one of [51].
- any one of [27] to [31], [33], wherein at least one amino acid mutation is introduced at the amino acid site at EU numbering 298 or EU numbering 309 in the loop region of the Fc region The method according to one item.
- At least one or more amino acid modifications in the loop region of the Fc region are selected from the group consisting of substitution of EU numbering 298th amino acid with Gly and substitution of EU numbering 309th amino acid with Asp
- the method according to any one of [27] to [54], wherein the modification is modification in a polypeptide having an Fc region of human IgG.
- a vector comprising the nucleic acid according to [56].
- a host cell transformed with the vector according to [57].
- a pharmaceutical composition comprising the polypeptide according to any one of [1] to [26] or the polypeptide produced by the method according to any one of [27] to [55].
- a therapeutic or prophylactic agent for an immunoinflammatory disease or cancer comprising the pharmaceutical composition according to [59].
- ischemic reperfusion trauma atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, and staining due to malgias, aplastic anemia, hemolysis Anemia, idiopathic thrombocytopenia, Goodpasture syndrome, Guillain-Barre syndrome, Hashimoto disease, pemphigus, IgA nephropathy, hay fever, antiphospholipid antibody syndrome, polymyositis, Wegener's sarcoma, nodular arteritis, mixed
- the therapeutic or prophylactic agent according to [60], which is sexual connective tissue disease or fibromyalgia.
- Cancer is pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatocellular carcinoma, cervical cancer, endometrial cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer, bile duct cancer , Bladder cancer, ureter cancer, thyroid cancer, adrenal cancer, kidney cancer, cancer of other glandular tissues, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, hemangiosarcoma, fibrosarcoma, malignant peripheral nerve
- the therapeutic or prophylactic agent according to [60] which is a tumor, gastrointestinal stromal tumor, tendonoma, Ewing sarcoma, osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma, or other parenchymal tumor .
- An immunization comprising the step of administering to the subject the polypeptide according to any one of [1] to [26] or the polypeptide produced by the method according to any one of [27] to [55] A method of treating or preventing an inflammatory disease or cancer.
- polypeptide according to any one of [1] to [26] or the method according to any one of [27] to [55] for use in the treatment or prevention of an immunoinflammatory disease or cancer A polypeptide produced by [65] The polypeptide according to any one of [1] to [26] or the method according to any one of [27] to [55] in the manufacture of an agent for treating or preventing an immunoinflammatory disease or cancer Use of a polypeptide produced by [66] An immunoinflammatory property comprising the step of using the polypeptide according to any one of [1] to [26] or the polypeptide produced by the method according to any one of [27] to [55] A method for producing a therapeutic or prophylactic agent for a disease or cancer.
- FIG. 6 is a chromatogram for measuring aggregate content of TS20 to TS27 in which multiple modifications are combined.
- FIG. 10 is a chromatogram of aggregate content measurement of TS28 to TS39 in which multiple modifications are combined.
- FIG. 6 is a chromatogram of measurement of aggregate content of TS40 to TS43 in which multiple modifications are combined. It represents the relationship between the amino acid residues constituting the Fc regions of IgG1, IgG2, IgG3 and IgG4 and the EU numbering of kabat (also referred to herein as EU INDEX).
- the present invention provides a polypeptide having the Fc region with improved stability compared to the parent polypeptide by introducing an amino acid substitution at the loop site of the antibody Fc region. Furthermore, the present invention provides a method for improving the stability of a polypeptide having an Fc region, as compared with a parent polypeptide, by introducing an amino acid substitution at the loop site of the antibody Fc region. Furthermore, the present invention provides a method for producing a polypeptide having an Fc region with improved stability compared to the parent polypeptide by introducing an amino acid substitution at the loop site of the antibody Fc region.
- the polypeptide in the present invention usually refers to peptides and proteins having a length of about 10 amino acids or more. Moreover, although it is normally a polypeptide derived from a living organism
- the polypeptide may be an antibody.
- a preferred example of the polypeptide of the present invention is human IgG. When human IgG is used as an antibody, the type of the constant region is not limited, and it is possible to use isotype (subclass) human IgG such as IgG1, IgG2, IgG3, IgG4.
- the “parent polypeptide” means a polypeptide serving as a basis or reference for producing a polypeptide having the Fc region of the antibody of the present invention. That is, it can be a polypeptide having an Fc region of an antibody, and the polypeptide before modification of at least one amino acid in the Fc region.
- the parent polypeptide in the present invention may be, for example, a polypeptide having an Fc region of natural IgG, or a polypeptide having an Fc region of IgG obtained by adding a modification other than the amino acid modification of the present invention to natural IgG. It may be.
- Native IgG refers to a polypeptide that includes the same amino acid sequence as an IgG found in nature and belongs to the class of antibodies substantially encoded by immunoglobulin gamma genes.
- natural human IgG means natural human IgG1, natural human IgG2, natural human IgG3, natural human IgG4, and the like.
- Naturally-occurring IgG includes naturally occurring mutants.
- the Fc region of natural IgG means an Fc region including the same amino acid sequence as an Fc region originating from IgG found in nature.
- the Fc region of natural IgG is shown in FIG. 3 (SEQ ID NOs: 11 to 14).
- Fc region originating from natural human IgG1 Fc region originating from natural human IgG2, natural human IgG3
- the Fc region of natural IgG includes mutants that naturally occur therefrom.
- amino acid positions of the present invention are defined according to Kabat (Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., 1987 and 1991). The site is expressed according to EU numbering according to the amino acid position of Kabat.
- the portion connecting the ⁇ helix and ⁇ sheet is called a loop, and its length and structure are not determined.
- the part connecting the ⁇ sheet and the ⁇ sheet of the CH2 domain is called a loop, a loop site, a loop part, or a loop structure.
- the amino acid positions of the specific loop sites subjected to the modification in the present invention are EU numbering 234th, EU numbering 235th, EU numbering 236th, EU numbering 237th, EU numbering 238th, EU numbering 239th, EU numbering 247, EU numbering 250, EU numbering 265th, EU numbering 266th, EU numbering 267th, EU numbering 268th, EU numbering 269th, EU numbering 270th, EU numbering 271th, EU numbering 295th, EU numbering 296th, EU numbering 298th, EU numbering 300th, EU numbering 307th, EU numbering 309th, EU numbering 315th, EU numbering 324th, EU numbering 325th, EU numbering 326th, EU numbering 327th, EU numbering 329th, EU numbering 330th, EU number From numbering 333, EU numbering 335, EU numbering 337, EU numbering 360, EU numbering 385th, EU numbering 386th, EU numbering
- Fc region refers to a region containing a fragment consisting of a hinge region or a part thereof, CH2 and CH3 domains in an antibody molecule.
- the IgG class Fc region is Kabat's EU numbering (also referred to herein as EU INDEX) (see Fig. 3), for example, from 226th cysteine to C-terminus, or 230th proline to C-terminus.
- EU INDEX also referred to herein as EU INDEX
- the Fc region can be suitably obtained by partially eluting IgG1, IgG2, IgG3, IgG4 monoclonal antibodies and the like with a protease such as pepsin and then re-eluting the fraction adsorbed on the protein A column.
- proteolytic enzymes are not particularly limited as long as full-length antibodies can be digested so that Fab and F (ab ') 2 can be produced in a limited manner by appropriately setting the reaction conditions of the enzyme such as pH.
- pepsin, papain, etc. can be illustrated.
- polypeptide of the present invention examples include polypeptides having an Fc region with improved stability compared to the parent polypeptide.
- a preferred embodiment of a polypeptide having an Fc region with improved stability compared to the parent polypeptide is, for example, a polypeptide in which at least one amino acid is modified at the loop site of the Fc region.
- the present invention relates to the Fc region loop region of an antibody (eg, human IgG (IgG1, IgG2, IgG3, IgG4)), EU numbering 234th, EU numbering 235th, EU numbering 236th, EU numbering 237 of the IgG.
- an antibody eg, human IgG (IgG1, IgG2, IgG3, IgG4)
- EU numbering 234th EU numbering 235th
- EU numbering 236th EU numbering 237 of the IgG.
- the present invention relates to the Fc region loop site of an antibody (for example, human IgG (IgG1, IgG2, IgG3, IgG4)), the EU numbering 234th, EU numbering 235th, EU numbering 239th, EU, Numbering 266th, EU numbering 267th, EU numbering 268th, EU numbering 269th, EU numbering 270th, EU numbering 295th, EU numbering 296th, EU numbering 298th, EU numbering 300th, EU numbering 324th, EU
- an antibody Fc region comprising an Fc region into which at least one amino acid mutation has been introduced at an amino acid site selected from the group consisting of numbering 325, EU numbering 326, and EU numbering 330.
- thermodynamic stability means, for example, the thermodynamic stability of a polypeptide, but is not limited thereto.
- the thermodynamic stability of a polypeptide can be evaluated or judged using, for example, the thermal denaturation midpoint (Tm) of the CH2 region as an index. That is, the polypeptide of the present invention is preferably evaluated or judged using the heat denaturation midpoint (Tm) as an index.
- Tm can be measured by CD (circular dichroism), DSC (differential scanning calorimetry), and DSF (differential scanning fluorometry).
- IgG maintains a highly controlled three-dimensional structure, and the three-dimensional structure and physical stability of each region affect each other. That is, a modification introduced into a certain region may affect other regions, and may change the three-dimensional structure and physical stability of the whole IgG. For this reason, in evaluating the effect of the modification introduction, it is desirable to evaluate in the form of IgG. For the above reasons, in this specification, the thermal stability of the CH2 region of the modified antibody prepared in the form of IgG was evaluated.
- CD calculates Tm by observing the change in mean residue molar ellipticity ( ⁇ ) with increasing temperature.
- An example of a CD measuring instrument is a circular dichroism dispersometer (JASCO).
- JASCO circular dichroism dispersometer
- Tm is a temperature that takes a midpoint value of ⁇ at low temperature and ⁇ at high temperature.
- a protein solution prepared with a citric acid, tris, phosphoric acid solution or the like can be used, and can be used for the measurement at a concentration of several hundred ⁇ g / mL.
- DSC calculates Tm by observing the amount of heat change with increasing temperature.
- DSC measuring instruments include MicroCal-VP-DSC and Micro Cal Capillary DSC (both are GE Healthcare).
- Tm The temperature at this time is Tm.
- DSF calculates Tm by observing the exposure of hydrophobic residues as the temperature rises using a fluorescent reagent that specifically binds to hydrophobic residues (eg SYPRO®Orange).
- a fluorescent reagent that specifically binds to hydrophobic residues
- Examples of DSF measuring instruments include Rotor-Gene Q (QIAGEN) and CFX96 real-time PCR analysis system (Bio-Rad).
- a protein solution prepared with PBS, histidine buffer or the like can be used, and can be used for the measurement at a concentration of several tens ⁇ g / mL to several hundreds ⁇ g / mL.
- the stability of the polypeptide is improved, for example, compared to the Tm of the CH2 region in the Fc region of the parent polypeptide as a control obtained based on the above measurement method.
- Tm of CH2 region in peptide Fc region is 0.1 degree or more, preferably 0.2 degree or more, 0.3 degree or more, 0.4 degree or more, 0.5 degree or more, 1 degree or more, 2 degree or more, 3 degree or more, 4 degree or more, 5 More than 10 degrees, 10 degrees, 20 degrees or more.
- polypeptide of the present invention not only has improved stability as compared to the parent polypeptide, but is also referred to as Fc ⁇ receptor (referred to herein as Fc ⁇ receptor or Fc ⁇ R) compared to the parent polypeptide. It may be a polypeptide having an Fc region in which the binding activity to (some) is maintained or enhanced.
- polypeptide having an Fc region in which the binding activity to Fc ⁇ R is maintained or enhanced compared to the parent polypeptide for example, the following implementation Examples include polypeptides having the amino acid site described in any of TS1-TS8, TS20-TS27, TS44-TS50, TS52-TS55, or TS57-TS67.
- polypeptide that maintains or enhances the binding activity to Fc ⁇ R as compared with the parent polypeptide include, for example, EU numbering 234, EU numbering 266, EU numbering 268, EU numbering 269 in the loop region of the Fc region. At least one amino acid mutation is introduced at an amino acid site selected from the group consisting of EU numbering 270, EU numbering 295, EU numbering 300, EU numbering 324, EU numbering 326, and EU numbering 330 The polypeptide which is made is mentioned.
- the amino acid modification site in the loop region of the Fc region is EU numbering 234th, EU numbering 266th, EU numbering 268th, EU numbering 269th, EU numbering 270th, EU numbering 295th , EU numbering 300th, EU numbering 324th, EU numbering 326th, and EU numbering 330th polypeptide.
- the amino acid modification in the loop region of the Fc region comprises substitution of the EU numbering 234th amino acid with Ile, EU numbering 266th amino acid substitution with Ile, EU numbering 268th Substitution of amino acid to Gln, EU numbering 269th amino acid to Asp, EU numbering 270th amino acid to Glu, EU numbering 295th amino acid to Met or Leu, EU numbering 300th A group consisting of amino acid substitution with Glu, EU numbering 324th amino acid substitution with His, EU numbering 326th amino acid substitution with Ser or Ala, and EU numbering 330th amino acid substitution with His or Tyr.
- Polypeptides that are at least one or more amino acid modifications selected from the above are exemplified.
- polypeptide in this embodiment the amino acid modification in the loop region of the Fc region, the substitution of the EU numbering 234th amino acid to Ile, the EU numbering 266th amino acid substitution to Ile, EU numbering 268th amino acid Substitution of Gln, EU numbering 269th amino acid to Asp, EU numbering 270th amino acid to Glu, EU numbering 295th amino acid to Met or Leu, EU numbering 300th amino acid
- the polypeptide that maintains or enhances the binding activity to Fc ⁇ R compared to the parent polypeptide is selected from the group consisting of EU numbering 295th, EU numbering 326th and EU numbering 330th, for example, in the loop region of the Fc region And a polypeptide having at least one amino acid mutation introduced at the amino acid site.
- a preferred polypeptide in this embodiment is a polypeptide whose amino acid modification sites in the loop region of the Fc region are EU numbering 295th, EU numbering 326th and EU numbering 330th.
- the amino acid modification at the loop site of the Fc region includes substitution of the 295th amino acid of EU numbering 295 with Met or Leu, substitution of the 326th amino acid of EU numbering 326 with Ser or Ala, and A polypeptide that is at least one amino acid modification selected from the group consisting of substitution of the amino acid at position 330 of EU number with His or Tyr.
- polypeptides in this embodiment the amino acid modification in the loop region of the Fc region, the substitution of the amino acid number 295 of EU numbering to Met or Leu, substitution of the amino acid number 326 of EU numbering to Ser or Ala, and EU It is a polypeptide that is a substitution of the numbering 330th amino acid to His or Tyr.
- polypeptide that maintains or enhances the binding activity to Fc ⁇ R compared to the parent polypeptide include, for example, EU numbering 247, EU numbering 250th, EU numbering 307th, EU numbering 309th in the loop region of the Fc region. , EU numbering 315, EU numbering 360, EU numbering 385, EU numbering 386, EU numbering 387, EU numbering 389, EU numbering 428, and EU numbering 433 And a polypeptide into which at least one amino acid mutation has been introduced.
- a preferred polypeptide in this embodiment the amino acid modification site in the loop region of the Fc region, EU numbering 247th, EU numbering 250th, EU numbering 307th, EU numbering 309th, EU numbering 315th, EU numbering 360th, Polypeptides that are EU numbering 385, EU numbering 386, EU numbering 387, EU numbering 389, EU numbering 428, and EU numbering 433.
- amino acid modification in the loop site of the Fc region substitution of the 247th amino acid of the EU numbering to Val, Phe of the EU numbering 250th amino acid, Phe, Ile, Met, Val, Trp, Or substitution to either Tyr, EU numbering 307th amino acid to any of Ala, Gln, or Pro, EU numbering 309th amino acid to any of Ala, Arg, or Pro, EU Numbering 315th amino acid substitution to Ala, EU numbering 360th amino acid substitution to His, EU numbering 385th amino acid substitution to Asp, EU numbering 386th amino acid substitution to Pro, EU numbering 387 Substitution of amino acid Glu, EU numbering 389 amino acid substitution to Ser, EU numbering 428 amino acid His, Trp, Tyr, or Phe Substitution of Re to or, and at least include the polypeptide is one or more amino acid modifications selected from the group consisting of substitution of Lys in the EU numbering system 433 amino acids.
- the amino acid modification in the loop region of the Fc region substitution of the 247th amino acid of the EU numbering to Val, Phe of the amino acid number of EU numbering 250, Phe, Ile, Met, Val, Trp, or Substitution to any of Tyr, EU numbering 307th amino acid substitution to any of Ala, Gln, or Pro, EU numbering 309th amino acid substitution to any of Ala, Arg, or Pro, EU numbering 315th amino acid substitution to Ala, EU numbering 360th amino acid substitution to His, EU numbering 385th amino acid substitution to Asp, EU numbering 386th amino acid substitution to Pro, EU numbering 387th Substitution of amino acid to Glu, EU numbering 389th amino acid to Ser, EU numbering 428th amino acid to His, Trp, Tyr, or Phe Substitution, and a polypeptide is a substitution of Lys for EU numbering 433 amino acids.
- the polypeptide is an antibody
- the antibody is preferably used as a cancer antibody whose effector function is important.
- polypeptide of the present invention is a polypeptide having an Fc region that not only has improved stability compared to the parent polypeptide but also has reduced Fc ⁇ receptor binding activity compared to the parent polypeptide. There may be. In the present invention, not only the stability is improved compared to the parent polypeptide, but also a polypeptide having an Fc region with reduced Fc ⁇ R binding activity compared to the parent polypeptide. Polypeptides having the amino acid modification sites of TS9-TS19, TS28-TS43, TS51, or TS56 shown.
- Polypeptides that have reduced Fc ⁇ R binding activity compared to the parent polypeptide include, for example, EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 267, EU numbering in the loop region of the Fc region, EU At least one amino acid mutation is introduced at an amino acid site selected from the group consisting of numbering 268th, EU numbering 270th, EU numbering 295th, EU numbering 296th, EU numbering 298th, and EU numbering 325th Polypeptides that are present.
- the amino acid modification site in the loop region of the Fc region is EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 267th, EU numbering 268th, EU numbering 270th , EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325.
- More preferred polypeptides in this aspect include amino acid modification in the loop region of the Fc region, substitution of the EU numbering 234th amino acid with Lys or Arg, EU numbering 235th amino acid substitution with Lys or Arg, EU numbering 239th amino acid substitution to Lys, EU numbering 267th amino acid substitution to Pro, EU numbering 268th amino acid substitution to Met or Lys, EU numbering 270th amino acid substitution to Phe, EU numbering 295th amino acid substitution to Met, EU numbering 296th amino acid substitution to Gly, EU numbering 298th amino acid substitution to Gly, and EU numbering 325th amino acid Gly, His or Met And a polypeptide that is at least one amino acid modification selected from the group consisting of substitutions.
- polypeptide in this embodiment the amino acid modification in the loop site of the Fc region, the substitution of the EU numbering 234th amino acid to Lys or Arg, the EU numbering 235th amino acid substitution to Lys or Arg, EU numbering 239th amino acid substitution to Lys, EU numbering 267th amino acid substitution to Pro, EU numbering 268th amino acid substitution to Met or Lys, EU numbering 270th amino acid substitution to Phe, EU numbering 295th amino acid substitution to Met, EU numbering 296th amino acid substitution to Gly, EU numbering 298th amino acid substitution to Gly, and EU numbering 325th amino acid Gly, His or Met A polypeptide that is a substitution for.
- Polypeptides that have reduced Fc ⁇ R binding activity compared to the parent polypeptide include, for example, EU numbering 234, EU numbering 235, EU numbering 239th, EU numbering 268th, EU numbering in the loop region of the Fc region, EU A polypeptide in which at least one amino acid mutation is introduced at an amino acid site selected from the group consisting of numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 Can be mentioned.
- a preferred polypeptide in this embodiment the amino acid modification site in the loop region of the Fc region, EU numbering 234th, EU numbering 235th, EU numbering 239th, EU numbering 268th, EU numbering 270th, EU numbering 295th, A polypeptide which is EU numbering 296th, EU numbering 298th, and EU numbering 325th.
- amino acid modification in the loop region of the Fc region includes substitution of the 234th amino acid of EU numbering to Lys, substitution of the 235th amino acid of EU numbering to Lys or Arg, and EU numbering 239.
- Substitution of amino acid Lys or Ser, EU numbering 268th amino acid substitution to Lys or His, EU numbering 270th amino acid substitution to Phe or Asp, EU numbering 295th amino acid substitution to Met At least one selected from the group consisting of substitution of the EU numbering 296th amino acid with Gly, EU numbering 298th amino acid substitution with Gly, and EU numbering 325th amino acid substitution with His or Gly.
- polypeptide in this embodiment the amino acid modification in the loop site of the Fc region, the substitution of the 234th amino acid of the EU numbering to Lys, the substitution of the 235th amino acid of the EU numbering to Lys or Arg, the 239th EU numbering Substitution of amino acid to Lys or Ser, EU numbering 268th amino acid to Lys or His, EU numbering 270th amino acid to Phe or Asp, EU numbering 295th amino acid to Met, It is a polypeptide that is substitution of EU numbering 296th amino acid with Gly, EU numbering 298th amino acid with Gly, and EU numbering 325th amino acid with His or Gly.
- Polypeptides that have reduced Fc ⁇ R binding activity compared to the parent polypeptide include, for example, EU numbering 234, EU numbering 235, EU numbering 239th, EU numbering 268th, EU numbering in the loop region of the Fc region, EU A polypeptide in which at least one amino acid mutation is introduced at an amino acid site selected from the group consisting of numbering 270, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325 Can be mentioned.
- the amino acid modification site in the loop region of the Fc region is EU numbering 234th, EU numbering 235th, EU numbering 239th, EU numbering 268th, EU numbering 270th, EU numbering 295th
- EU numbering 296, EU numbering 298, and EU numbering 325 is EU numbering 234th, EU numbering 235th, EU numbering 239th, EU numbering 268th, EU numbering 270th, EU numbering 295th.
- amino acid modification in the loop region of the Fc region includes substitution of the 234th amino acid of EU numbering to Lys, substitution of the 235th amino acid of EU numbering to Lys or Arg, and EU numbering 239.
- substitution of amino acid to Lys, EU numbering 268th amino acid to Lys, EU numbering 270th amino acid to Phe, EU numbering 295th amino acid to Met, EU numbering 296th Poly which is at least one amino acid modification selected from the group consisting of substitution of amino acid with Gly, substitution of EU numbering 298th amino acid with Gly, and substitution of EU numbering 325th amino acid with His or Gly Peptides are mentioned.
- polypeptide in this embodiment the amino acid modification in the loop site of the Fc region, the substitution of the 234th amino acid of the EU numbering to Lys, the substitution of the 235th amino acid of the EU numbering to Lys or Arg, the 239th EU numbering Substitution of amino acid to Lys, substitution of EU numbering 268th amino acid to Lys, substitution of EU numbering 270th amino acid to Phe, substitution of EU numbering 295th amino acid to Met, EU numbering 296th amino acid Is a polypeptide that is a substitution of Gly at the 298th amino acid of EU numbering with Gly, and a substitution of His at the 325th amino acid of EU numbering with His or Gly.
- polypeptide having a reduced binding activity to Fc ⁇ R compared to the parent polypeptide examples include, for example, EU numbering 234, EU numbering 235, EU numbering 239, EU numbering 295, EU numbering in the loop region of the Fc region, EU Examples include a polypeptide having at least one amino acid mutation introduced at an amino acid site selected from the group consisting of 296th numbering, 298th EU numbering, and 325th EU numbering.
- the amino acid modification site in the loop region of the Fc region is EU numbering 234th, EU numbering 235th, EU numbering 239th, EU numbering 295th, EU numbering 296th, EU numbering 298th And a polypeptide which is EU numbering 325.
- amino acid modification in the loop region of the Fc region includes substitution of the 234th amino acid of EU numbering to Lys, substitution of the 235th amino acid of EU numbering to Lys or Arg, and EU numbering 239. Substitution of amino acid Lys, EU numbering 295th amino acid substitution to Met, EU numbering 296th amino acid substitution to Gly, EU numbering 298th amino acid substitution to Gly, and EU numbering 325th A polypeptide that is at least one amino acid modification selected from the group consisting of substitution of amino acids with His or Gly.
- polypeptide in this embodiment the amino acid modification in the loop site of the Fc region, the substitution of the 234th amino acid of the EU numbering to Lys, the substitution of the 235th amino acid of the EU numbering to Lys or Arg, the 239th EU numbering Substitution of amino acid to Lys, EU numbering 295th amino acid to Met, EU numbering 296th amino acid to Gly, EU numbering 298th amino acid to Gly, and EU numbering 325th A polypeptide that is a substitution of an amino acid to His or Gly.
- Polypeptides that have reduced Fc ⁇ R binding activity compared to the parent polypeptide include, for example, EU numbering 234, EU numbering 235, EU numbering 268th, EU numbering 270th, EU numbering in the loop region of the Fc region.
- Examples include polypeptides in which at least one amino acid mutation has been introduced at an amino acid site selected from the group consisting of numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325.
- the amino acid modification site in the loop region of the Fc region is EU numbering 234th, EU numbering 235th, EU numbering 268th, EU numbering 270th, EU numbering 295th, EU numbering 296th A polypeptide which is EU numbering 298th and EU numbering 325th.
- the amino acid modification in the loop region of the Fc region includes substitution of the EU numbering 234th amino acid with Lys, EU numbering 235th amino acid substitution with Lys or Arg, EU numbering 268 Substitution of Lys for the 2nd amino acid, substitution of 270th amino acid for EU numbering to Phe, substitution of 295th amino acid for EU numbering to Met, substitution of 296th amino acid for EU numbering to Gly, 298th EU numbering Examples include polypeptides that are at least one amino acid modification selected from the group consisting of substitution of amino acids with Gly, and substitution of EU numbering 325th amino acid with His or Gly.
- polypeptide in this embodiment the amino acid modification in the loop site of the Fc region, the substitution of the EU numbering 234th amino acid to Lys, the EU numbering 235th amino acid substitution to Lys or Arg, EU numbering 268th Substitution of amino acid with Lys, substitution of EU numbering 270th amino acid with Phe, substitution of EU numbering 295th amino acid with Met, substitution of EU numbering 296th amino acid with Gly, EU numbering 298th amino acid Is a polypeptide that is a substitution of Gly and EU numbering 325th amino acid to His or Gly.
- polypeptide having a reduced binding activity to Fc ⁇ R compared to the parent polypeptide examples include, for example, EU numbering 234, EU numbering 235, EU numbering 295, EU numbering 296, EU numbering 296 in the loop region of the Fc region, EU Examples include a polypeptide having at least one amino acid mutation introduced at an amino acid site selected from the group consisting of 298th numbering and 325th EU numbering.
- the amino acid modification site in the loop region of the Fc region is EU numbering 234, EU numbering 235, EU numbering 295, EU numbering 296, EU numbering 298, and EU numbering 325. Is the second polypeptide.
- the amino acid modification in the loop region of the Fc region includes substitution of the EU numbering 234th amino acid with Lys, EU numbering 235th amino acid substitution with Lys or Arg, EU numbering 295 The group consisting of the substitution of the first amino acid with Met, the substitution of the EU numbering 296th amino acid with Gly, the substitution of the EU numbering 298th amino acid with Gly, and the substitution of the EU numbering 325th amino acid with His or Gly.
- Polypeptides that are at least one or more amino acid modifications selected from the above are exemplified.
- the amino acid modification in the loop region of the Fc region includes substitution of the EU numbering 234th amino acid with Lys, EU numbering 235th amino acid substitution with Lys or Arg, EU numbering 295 Poly of the substitution of the amino acid of Mth to the Met, the substitution of the EU numbering 296th amino acid to Gly, the substitution of the EU numbering 298th amino acid to Gly, and the substitution of the EU numbering 325th amino acid to His or Gly It is a peptide.
- polypeptide having a reduced binding activity to Fc ⁇ R compared to the parent polypeptide include, for example, at least one amino acid at the amino acid site at EU numbering 298 and EU numbering 309 at the loop site of the Fc region. Mention may be made of polypeptides into which mutations have been introduced.
- Preferred polypeptides in this embodiment include polypeptides in which the amino acid modification at the loop site of the Fc region is EU numbering 298th and EU numbering 309th.
- the amino acid modification in the loop region of the Fc region is selected from the group consisting of substitution of the EU numbering 298th amino acid with Gly, and EU numbering 309th amino acid substitution with Asp And a polypeptide that is at least one or more amino acid modifications.
- a more preferred polypeptide in this embodiment is a polypeptide in which the amino acid modification at the loop site of the Fc region is substitution of the EU numbering 298th amino acid with Gly, and EU numbering 309th amino acid with Asp. Can be mentioned.
- the polypeptide is an antibody
- the antibody is preferably used as a neutralizing antibody.
- Fc ⁇ receptor refers to a receptor that can bind to the Fc region of IgG1, IgG2, IgG3, and IgG4 monoclonal antibodies, and substantially means any member of the protein family encoded by the Fc ⁇ receptor gene.
- this family includes Fc ⁇ RI (CD64), including isoforms Fc ⁇ RIa, Fc ⁇ RIb and Fc ⁇ RIc; isoforms Fc ⁇ RIIa (including allotypes H131 (H) and R131 (R)), Fc ⁇ RIIb (Fc ⁇ RIIb-1 and Fc ⁇ RIIb- 2) and Fc ⁇ RII (CD32) including Fc ⁇ RIIc; and Fc ⁇ RIII (CD16) including isoforms Fc ⁇ RIIIa (including allotypes V158 and F158) and Fc ⁇ RIIIb (including allotypes Fc ⁇ RIIIb-NA1 and Fc ⁇ RIIIb-NA2), and any undiscovered Human Fc ⁇ Rs or Fc ⁇ R isoforms or all
- the Fc ⁇ R of the present invention includes not only those derived from the above-mentioned humans, but also those derived from mice, rats, rabbits and monkeys, but are not limited thereto, and may be derived from any organism.
- Mouse Fc ⁇ Rs include Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), Fc ⁇ RIII (CD16) and Fc ⁇ RIII-2 (CD16-2), as well as any undiscovered mouse Fc ⁇ Rs or Fc ⁇ R isoforms or allotypes. It is not limited to.
- Fc ⁇ receptor in the present invention include human Fc ⁇ I (CD64), Fc ⁇ IIA (CD32), Fc ⁇ IIB (CD32), Fc ⁇ IIIA (CD16) and / or Fc ⁇ IIIB (CD16).
- the polynucleotide sequence and amino acid sequence of Fc ⁇ I are shown in SEQ ID NOs: 1 (NM_000566.3) and 2 (NP_000557.1), respectively.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ IIA are shown in SEQ ID NOs: 3 (BC020823.1) and 4 (AAH20823.1), respectively.
- the polynucleotide sequence and amino acid sequence of Fc ⁇ IIB are shown in SEQ ID NOs: 5 (BC146678.1) and 6 (AAI46679.1), respectively.
- the polynucleotide and amino acid sequences of Fc ⁇ IIIA are shown in SEQ ID NOs: 7 (BC033678.1) and 8 (AAH33678.1), respectively.
- Fc ⁇ RIIa has two gene polymorphisms in which the 131st amino acid of Fc ⁇ RIIa is substituted with histidine (H type) or arginine (R type) (J. Exp. Med, 172, 19-25, 1990).
- Fc region binding activity to Fc ⁇ receptors includes ELISA and FACS (fluorescence activated cell sorting), ALPHA screen (Amplified Luminescent Proximity Homogeneous Asssay), BIACORE method using surface plasmon resonance (SPR) phenomenon, etc. (Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010).
- ALPHA screen is implemented based on the following principle by ALPHA technology using two beads of donor and acceptor.
- a luminescent signal is detected only when the molecule bound to the donor bead interacts biologically with the molecule bound to the acceptor bead and the two beads are in close proximity.
- a photosensitizer in the donor bead excited by the laser converts ambient oxygen into excited singlet oxygen. Singlet oxygen diffuses around the donor bead, and when it reaches the adjacent acceptor bead, it causes a chemiluminescence reaction in the bead, and finally light is emitted.
- the chemiluminescence reaction does not occur because the singlet oxygen produced by the donor bead does not reach the acceptor bead.
- biotin-labeled polypeptide aggregates bind to donor beads, and Fc ⁇ receptors tagged with glutathione S-transferase (GST) bind to acceptor beads.
- GST glutathione S-transferase
- the polypeptide aggregate having a wild type Fc region interacts with the Fc ⁇ receptor to produce a signal of 520-620 nm.
- Polypeptide aggregates with untagged mutant Fc regions compete with the interaction between polypeptide aggregates with wild-type Fc regions and Fc ⁇ receptors. Relative binding affinity can be determined by quantifying the decrease in fluorescence that results from competition.
- a polypeptide aggregate such as an antibody is biotinylated using Sulfo-NHS-biotin or the like.
- a method of tagging the Fc ⁇ receptor with GST it is expressed in a cell or the like holding a fusion gene in which a polynucleotide encoding the Fc ⁇ receptor and a polynucleotide encoding GST are fused in-frame.
- a method of purification using a glutathione column can be appropriately employed.
- the obtained signal is suitably analyzed by fitting to a one-site competition model using nonlinear regression analysis using software such as GRAPHPAD PRISM (GraphPad, San Diego).
- the Biacore system takes the shift amount, that is, the mass change at the sensor chip surface on the vertical axis, and displays the time change of mass as measurement data (sensorgram).
- the amount of analyte bound to the ligand captured on the sensor chip surface is determined from the sensorgram. Further, the kinetics: association rate constant (ka) and dissociation rate constant (kd) are obtained from the sensorgram curve, and the dissociation constant (KD) is obtained from the ratio of the constants.
- an inhibition measurement method is also preferably used. Examples of inhibition assays are described in Proc. Natl. Acad. Sci. USA (2006) 103 (11), 4005-4010.
- a polypeptide having a reduced binding activity to Fc ⁇ R is a polypeptide (polypeptide having at least one amino acid modification in the Fc region of the parent polypeptide and the parent polypeptide).
- the binding amount to each Fc ⁇ R of the polypeptide variant when the binding amount to each Fc ⁇ R of the parent polypeptide is 100 is preferably 80 or less, preferably 50 or less, 40 or less, 30 or less, 20 or less, particularly preferably 10 or less, 5 or less, 4 or less, 3 or less, 2 or less, 1 or less, or 0.1 or less.
- the polypeptide with enhanced Fc ⁇ R binding activity was assayed with essentially the same amount of parent polypeptide and polypeptide variant. Sometimes it refers to at least one Fc ⁇ R that binds to Fc ⁇ R with a substantially stronger binding affinity than the parent polypeptide.
- the binding amount to each Fc ⁇ R of the polypeptide variant when the binding amount to each Fc ⁇ R of the parent polypeptide is 100 is preferably This means something that has improved to 120 or more, 150 or more, 200 or more, or 300 or more.
- a polypeptide whose binding activity to Fc ⁇ R is maintained (maintained) is a modification of at least one amino acid in the parent polypeptide and the Fc region of the parent polypeptide. That bind to Fc ⁇ R with the same binding affinity that is essentially unchanged from the parent polypeptide when assayed with essentially the same amount of polypeptide (including polypeptide variants) Say.
- the binding amount to each Fc ⁇ R of the polypeptide variant when the binding amount to each Fc ⁇ R of the parent polypeptide is 100 (hereinafter referred to as the binding amount ratio) is preferably Say what is 80 or more and 120 or less.
- a polypeptide for example, human IgG
- a polypeptide for example, human IgG
- another Fc region for example, as a modification to another Fc region, for human IgG (IgG1, IgG2, IgG3, IgG4), a modification that replaces the EU numbering 238th Pro with Asp and / or a EU numbering 328th Leu with Glu Can be mentioned.
- Fc ⁇ RI, Fc ⁇ RIIIa, and Fc ⁇ RIIa were compared to the parent polypeptide by introducing human IgG with a modification in which EU numbering 238th Pro was replaced with Asp and / or a modification in which EU numbering 328th Leu was replaced with Glu. It is possible to provide a polypeptide in which the binding activity is maintained or reduced with respect to both the R-type and H-type gene polymorphisms and the binding activity to Fc ⁇ RIIb is enhanced.
- Fc region modification to human IgG containing a modification in which EU numbering 238th Pro is replaced with Asp and / or a modification in which EU numbering 328th Leu is replaced with Glu.
- the modification means any one of amino acid substitution, deletion, addition, insertion, or a combination thereof.
- additional modifications can be included. Additional alterations can be selected from, for example, amino acid substitutions, deletions, modifications, or combinations thereof.
- a modification that reduces the binding activity to Fc ⁇ RIIa (R type) without reducing the binding activity to Fc ⁇ RIIb is preferable.
- a preferred amino acid substitution as such a modification is Gly at the 237th EU numbering.
- Preferred amino acid substitutions include, for example, EU numbering 237th Gly to Trp, EU numbering 237th Gly to Phe, EU numbering 238th Pro to Phe, EU numbering 325th Asn Met EU numbering 267th Ser to Ile, EU numbering 328th Leu to Asp, EU numbering 267th Ser to Val, EU numbering 328th Leu to Trp Substitution, substitution of EU numbering 267 Ser to Gln, EU numbering 267th Ser to Met, EU numbering 236 Gly to Asp, EU numbering 327th Ala to Asn, EU number 325th Asn to Ser, EU numbering 235th Leu to Tyr, EU numbering 266th Val to Met, EU numbering 328th Leu to Tyr, EU numbering 235 Substitution of Leu to Trp, EU numbering 235th Leu to Phe, EU numbering 239th Ser to Gly, EU numbering 327th Ala to Glu, EU numbering 327th Ala to Gly, EU numbering
- a modification that reduces the binding activity to Fc ⁇ RIIa (R type) without reducing the binding activity to Fc ⁇ RIIb is preferable.
- a preferred amino acid substitution as such a modification is Gly at the 237th EU numbering.
- amino acid substitution J ⁇ ⁇ ⁇ ⁇ Immunol. 2006 Jan 1; 176 (1): 346-56, J Biol Chem. 2006 Aug 18; 281 (33): 23514-, which improves the binding activity to FcRn, for example, in the Fc region of the antibody.
- WO / 2006/019447, WO / 2006/053301, WO / 2009 / 086320 amino acid substitution
- amino acid substitution (WO / 2009/041613) may be added to improve the heterogeneity and stability of the antibody.
- amino acid modifications of the present invention can be appropriately added to the amino acid modifications of the present invention.
- amino acid modification of the present invention with an amino acid modification for altering the isoelectric point value (pI value) of the antibody.
- modification of the constant region for example, EU numbering position 250 described in known literature (J. Immunol. 2006, 176 (1): 346-356, Nat. Biotechnol. 1997 15 (7): 637-640, etc.)
- amino acid modification such as position 428.
- variable region modifications include amino acid modifications described in WO2007 / 114319 and WO2009 / 041643.
- the amino acid to be modified is preferably an amino acid exposed on the surface of a polypeptide having antigen-binding activity.
- substitution of the amino acid at position 196 in the EU numbering in the amino acid sequence of the heavy chain constant region can be mentioned.
- the heavy chain constant region is IgG4, for example, by substituting 196th lysine with glutamine, the pI value can be lowered and the plasma retention can be increased.
- the plasma retention can also be controlled by modifying the binding force to FcRn.
- the amino acid modification for modifying the binding force to FcRn include, for example, known literature (The Journal of Biological Chemistry vol. 276, No. 9 6591-6604, 2001 and Molecular Cell, Vol. 7, 867-877, 2001). And substitution of amino acids in the antibody heavy chain constant region described in.
- Modifications for improving stability under acidic conditions For example, when the polypeptide of the present invention has an IgG4 heavy chain constant region, a stable four-chain structure (H2L2 structure) is suppressed by suppressing IgG4 half-molecule formation under acidic conditions. It is preferable to maintain. Therefore, arginine (Immunology 2002, 105, 9-19), the amino acid at position 409 EU numbering, which plays an important role in maintaining the 4-chain structure, is an IgG1 type that maintains a stable 4-chain structure even under acidic conditions. It is preferable to substitute with lysine. Such modifications can be used in combination with the amino acid modifications of the present invention.
- Modification for reducing heterogeneity It is possible to combine the modification of the amino acid of the present invention with the method described in WO2009 / 041613. Specifically, for example, when the polypeptide of the present invention has an IgG1 heavy chain constant region, a modification that deletes the C-terminal 2 amino acids of the IgG1 heavy chain constant region, that is, the EU numbering 446th glycine and the 447th lysine. It is possible to reduce heterogeneity by combining with modification of amino acids based on the examples of the present invention.
- Modification for inhibiting deamidation reaction It is possible to combine the modification of the amino acid of the present invention with the modification of amino acid for inhibiting the deamidation reaction. It has been reported that the deamidation reaction is likely to occur particularly at the site where asparagine (N) and glycine (G) are adjacent (... NG %) (Geiger et al. J. Bio. Chem. 1987; 262 : 785-794).
- the polypeptide of the present invention has a site where asparagine and glycine are adjacent to each other, the deamidation reaction can be suppressed by modifying the amino acid sequence. Specifically, for example, one or both amino acid sequences of asparagine and glycine are substituted with other amino acids. More specifically, for example, aspartic acid is substituted with aspartic acid.
- polypeptide of the present invention include IgG antibodies.
- an IgG antibody as an antibody, the type of the Fc region is not limited, and it is possible to use IgG of an isotype (subclass) such as IgG1, IgG2, IgG3, and IgG4.
- the IgG antibody of the present invention is preferably human IgG, more preferably human IgG1 or human IgG4, and the amino acid sequences of the heavy chain Fc regions of human IgG1 and human IgG4 are known.
- an amino acid residue can be substituted with another amino acid residue for the purpose of modifying the following points (a) to (c); (a) the backbone structure of the polypeptide in the region of the sheet structure or the helical structure; (b) charge or hydrophobicity at the target site, or (c) Side chain size.
- Amino acid residues are divided into the following groups based on general side chain properties: (1) Hydrophobicity: norleucine, met, ala, val, leu, ile; (2) Neutral hydrophilicity: cys, ser, thr, asn, gln; (3) Acidity: asp, glu; (4) Basicity: his, lys, arg; (5) Residues that affect chain orientation: gly, pro; and (6) Aromaticity: trp, tyr, phe.
- substitution of amino acid residues within each of these groups is called conservative substitution, while the substitution of amino acid residues between other groups is called non-conservative substitution.
- the substitution in the present invention may be a conservative substitution, a non-conservative substitution, or a combination of a conservative substitution and a non-conservative substitution.
- the modification of the amino acid sequence is prepared by various methods known in the art. These methods include, but are not limited to, site-directed mutagenesis (Hashimoto-Gotoh, T, Mizuno, T, Ogasahara, Y, and Nakagawa, M. (1995) An oligodeoxyribonucleotide- directed dual amber method for site-directed mutagenesis. Gene 152, 271-275, Zoller, MJ, and Smith, M.
- the amino acid modifications of the present invention include post-translational modifications.
- post-translational modifications addition or deletion of sugar chains can be shown.
- the 297th amino acid residue of the EU numbering can be modified with a sugar chain.
- the sugar chain structure to be modified is not limited.
- antibodies expressed in eukaryotic cells contain glycosylation in the Fc region. Therefore, antibodies expressed in the following cells are usually modified with some sugar chain.
- Mammalian antibody-producing cells ⁇ Eukaryotic cells transformed with an expression vector containing DNA encoding the antibody
- the eukaryotic cells shown here include yeast and animal cells.
- CHO cells and HEK293H cells are representative animal cells for transformation with an expression vector containing DNA encoding an antibody.
- the Fc region of the present invention includes those having no sugar chain modification at this position.
- An antibody whose Fc region is not modified with a sugar chain can be obtained by expressing a gene encoding the antibody in prokaryotic cells such as Escherichia coli. More specifically, for example, sialic acid may be added to the sugar chain of the Fc region (MAbs. 2010 Sep-Oct; 2 (5): 519-27.).
- the present invention provides an antibody having an Fc region in which any one of the amino acid sequences described above is modified.
- antibody in the present invention is used in the broadest sense, and as long as it exhibits a desired biological activity, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody variants, antibody fragments, multispecificity Any antibody such as an antibody (for example, a bispecific antibody (sometimes referred to as a bispecific antibody)), a chimeric antibody, or a humanized antibody is included.
- the antibody of the present invention is not limited in the type of antigen, the origin of the antibody, etc., and may be any antibody.
- the origin of the antibody is not particularly limited, and examples thereof include a human antibody, a mouse antibody, a rat antibody, and a rabbit antibody.
- monoclonal antibodies can be produced by the hybridoma method (Kohler and Milstein, Nature 256: 495 (1975)) or recombinant methods (US Pat. No. 4,816,567). May be. It may also be isolated from a phage antibody library (Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1991)).
- monoclonal antibodies include humanized antibodies and chimeric antibodies.
- B cell cloning as described in Bernasconi et al.
- each antibody in particular, use for constructing an expression vector for production of IgG1, IgG2, IgG3 or IgG4 can be used as appropriate.
- sugar chain of the antibody of the present invention may be modified.
- antibodies with altered sugar chains include, for example, antibodies modified with glycosylation (WO99 / 54342, etc.), antibodies lacking fucose added to sugar chains (WO00 / 61739, WO02 / 31140, WO2006 / 067847). , WO2006 / 067913), and antibodies having a sugar chain having bisecting GlcNAc (WO02 / 79255).
- Humanized antibodies are also referred to as reshaped human antibodies.
- non-human animals for example, humanized antibodies obtained by grafting mouse antibody CDRs to human antibodies are known.
- General genetic recombination techniques for obtaining humanized antibodies are also known.
- Overlap-Extension-PCR is known as a method for transplanting mouse antibody CDRs into human FRs.
- amino acid residues of FR can be substituted as necessary so that the CDR of the reshaped human antibody forms an appropriate antigen-binding site.
- amino acid sequence mutations can be introduced into FRs by applying the PCR method used for transplantation of mouse CDRs into human FRs.
- Transgenic animals having all repertoires of human antibody genes are used as immunized animals and desired by DNA immunization. Human antibodies can be obtained.
- the V region of a human antibody is expressed as a single chain antibody (scFv) on the surface of the phage by the phage display method.
- Phages expressing scFv that bind to the antigen can be selected.
- the DNA sequence encoding the V region of the human antibody that binds to the antigen can be determined.
- the V region sequence is fused in-frame with the sequence of the desired human antibody C region, and then inserted into an appropriate expression vector, whereby an expression vector can be prepared.
- the human antibody is obtained by introducing the expression vector into a suitable expression cell as described above and expressing the gene encoding the human antibody.
- These methods are already known (see International Publications WO1992 / 001047, WO1992 / 020791, WO1993 / 006213, WO1993 / 011236, WO1993 / 019172, WO1995 / 001438, WO1995 / 015388).
- the antigen is not particularly limited, and any antigen may be used.
- an antigen for example, a ligand (cytokine, chemokine, etc.), a receptor, a cancer antigen, an MHC antigen, a differentiation antigen, an immunoglobulin and an immune complex partially containing an immunoglobulin are preferably exemplified.
- cytokines examples include interleukins 1-18, colony stimulating factors (G-CSF, M-CSF, GM-CSF, etc.), interferons (IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , etc.), growth factors ( EGF, FGF, IGF, NGF, PDGF, TGF, HGF, etc.), tumor necrosis factor (TNF- ⁇ , TNF- ⁇ ), lymphotoxin, erythropoietin, leptin, SCF, TPO, MCAF, BMP.
- chemokines examples include CC chemokines such as CCL1 to CCL28, CXC chemokines such as CXCL1 to CXCL17, C chemokines such as XCL1 to XCL2, and CX3C chemokines such as CX3CL1.
- receptors include, for example, hematopoietic factor receptor family, cytokine receptor family, tyrosine kinase type receptor family, serine / threonine kinase type receptor family, TNF receptor family, G protein coupled receptor family, GPI Examples include receptors belonging to the receptor family such as anchor type receptor family, tyrosine phosphatase type receptor family, adhesion factor family, hormone receptor family and the like. Regarding the receptors belonging to these receptor families and their characteristics, a number of documents such as Cooke BA., King RJB., Van der Molen HJ. Ed. New Comprehesive Biochemistry Vol.
- Specific receptors belonging to the above receptor family include, for example, human or mouse erythropoietin (EPO) receptors (Blood (1990) 76 (1), 31-35, Cell (1989) 57 (2), 277- 285), human or mouse granulocyte colony stimulating factor (G-CSF) receptor (Proc. Natl. Acad. Sci. USA. (1990) 87 (22), 8702-8706, mG-CSFR, Cell (1990) 61 (2), 341-350), human or mouse thrombopoietin (TPO) receptor (Proc Natl Acad Sci U S A. (1992) 89 (12), 5640-5644, EMBO J.
- EPO erythropoietin
- human or mouse leptin receptor human or mouse growth hormone (GH) receptor, human or mouse Interleukin (IL) -10 receptor, human or mouse insulin-like growth factor (IGF) -I receptor, human or mouse leukemia inhibitory factor (LIF) receptor, human or mouse ciliary neurotrophic factor (CNTF) receptor A body etc. are illustrated suitably.
- GH growth hormone
- IL Interleukin
- IGF insulin-like growth factor
- LIF human or mouse leukemia inhibitory factor
- CNTF ciliary neurotrophic factor
- Cancer antigens are antigens that are expressed as cells become malignant and are also called tumor-specific antigens.
- abnormal sugar chains appearing on the cell surface and protein molecules when cells become cancerous are also cancer antigens and are also called cancer sugar chain antigens.
- cancer antigens include, for example, GPC3 (Int J Cancer. (2003) 103 (4) that belongs to the GPI-anchored receptor family as the above receptor but is expressed in several cancers including liver cancer. , 455-65), EpCAM expressed in multiple cancers including lung cancer (Proc Natl Acad Sci U S A. (1989) 86 (1), ⁇ ⁇ 27-31), CA19-9, CA15-3, serial SSEA -1 (SLX) and the like are preferable.
- GPC3 Int J Cancer. (2003) 103 (4) that belongs to the GPI-anchored receptor family as the above receptor but is expressed in several cancers including liver cancer. , 455-65
- EpCAM expressed in multiple cancers including lung cancer Proc Natl Acad Sci U S A. (19
- MHC antigens are mainly classified into MHC class I antigen and MHC class II antigen, which includes HLA-A, -B, -C, -E, -F, -G, -H.
- MHC class II antigens include HLA-DR, -DQ, and -DP.
- Immunoglobulins include IgA, IgM, IgD, IgG, and IgE.
- the immune complex includes at least any component of immunoglobulin.
- variable region constituting the antibody of the present invention can be a variable region that recognizes an arbitrary antigen. As long as the antigen binding activity of the amino acid sequence constituting the antibody variable region is maintained, modification of one or more amino acid residues is allowed.
- the site to be modified and the number of amino acids to be modified are not particularly limited. For example, amino acids present in CDR and / or FR can be appropriately modified.
- the amino acid of the variable region is modified, although not particularly limited, it is preferable that the binding activity is maintained, for example, 50% or more, preferably 80% or more, more preferably 100% or more compared to before modification. It preferably has binding activity. Further, the binding activity may be increased by amino acid modification. For example, the binding activity may be 2 times, 5 times, 10 times or more compared to before the modification.
- KD dissociation constant
- apparent KD Apparent dissociation constant
- KD (dissociation constant) and apparent KD can be measured by methods known to those skilled in the art. For example, Biacore (GE healthcare), Scatchard plot, flow cytometer, etc. It is possible to use.
- k d dissociation rate constant
- apparent k d Apparent dissociation rate constant
- Biacore GE healthcare
- flow cytometer etc.
- the alteration of the amino acid sequence can be at least one of substitution, addition, deletion, and modification of amino acid residues.
- the site to be modified and the number of amino acids to be modified are not particularly limited, and generally within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (eg, within 5 amino acids, within 3 amino acids). is there. Alternatively, for example, 20% or less, specifically 10% or less (for example, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% or less) of amino acid residues in the entire amino acid sequence Modification of is permissible.
- the modification to pyroglutamic acid by pyroglutamylation of N-terminal glutamine of the variable region is a modification well known to those skilled in the art. Therefore, the antibody of the present invention includes a variable region in which, when the N-terminus of the heavy chain is glutamine, it is modified with pyroglutamic acid.
- variable region of the antibody of the present invention may be of any sequence, mouse antibody, rat antibody, rabbit antibody, goat antibody, camel antibody, humanized antibody obtained by humanizing these non-human antibodies, and human It may be a variable region of an antibody of any origin, such as an antibody.
- “Humanized antibody” refers to an antibody derived from a mammal other than a human, also referred to as a reshaped human antibody, such as a complementarity determination region (CDR) of a mouse antibody to the CDR of a human antibody. It is transplanted.
- CDR complementarity determination region
- variable region of the antibody of the present invention has a pH dependence on the binding to the antigen, and may be able to repeatedly bind to the antigen (WO / 2009/125825).
- the light chain constant region of an antibody has a ⁇ chain and ⁇ chain type constant region, but any light chain constant region may be used. Furthermore, in the present invention, the light chain constant region may be a light chain constant region that has been modified by amino acid substitution, deletion, addition, and / or insertion.
- the heavy chain Fc region of the antibody of the present invention for example, the heavy chain Fc region of a human IgG antibody can be used, and preferably the heavy chain Fc region of a human IgG1 antibody or human IgG4 antibody.
- polypeptide of the present invention can be combined with other proteins, bioactive peptides and the like to form Fc fusion protein molecules.
- proteins and bioactive peptides include, but are not limited to, receptors, adhesion molecules, ligands, and enzymes.
- Fc fusion protein molecule of the present invention include a protein in which an Fc domain is fused to a receptor protein that binds to a target.
- a protein in which an Fc domain is fused to a receptor protein that binds to a target For example, TNFR-Fc fusion protein, IL1R-Fc fusion protein, VEGFR-Fc fusion protein, CTLA4 -Fc fusion proteins and the like (Nat Med. 2003 Jan; 9 (1): 47-52, BioDrugs. 2006; 20 (3): 151-60.).
- the protein to be fused to the polypeptide of the present invention may be any molecule as long as it binds to the target molecule.
- scFv molecule WO2005 / 037989
- single domain antibody molecule WO2004 / 058821, WO2003 / 002609
- antibody -Like molecule a multispecific antibody that binds to multiple types of target molecules or epitopes.
- DARPins WO2002 / 020565
- Affibody WO1995 / 001937
- Avimer WO2004 / 044011, WO2005 / 040229
- Adnectin WO2002 / 032925
- the antibody and Fc fusion protein molecule may also be a multispecific antibody that binds to multiple types of target molecules or epitopes.
- the antibodies of the present invention also include modified antibodies.
- modified antibody include antibodies bound to various molecules such as polyethylene glycol (PEG) and cytotoxic substances.
- PEG polyethylene glycol
- Such a modified antibody can be obtained by chemically modifying the antibody of the present invention. Methods for modifying antibodies have already been established in this field.
- the antibody of the present invention may be a bispecific antibody.
- Bispecific antibody refers to an antibody having variable regions that recognize different epitopes in the same antibody molecule, but the epitope may exist in different molecules or in the same molecule. It may be.
- polypeptide of the present invention can be produced by methods known to those skilled in the art.
- the antibody can be prepared by the following method, but is not limited thereto.
- DNA encoding the heavy chain of an antibody the DNA encoding the heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest, and the DNA encoding the light chain of the antibody
- DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest for example, obtain the Fc region portion of the DNA encoding the natural heavy chain, It can be obtained by appropriately introducing substitutions so that a codon encoding a specific amino acid in the region encodes another amino acid of interest.
- DNA encoding a protein in which one or more amino acid residues in the Fc region of the natural heavy chain are substituted with other amino acids of interest By designing a DNA encoding a protein in which one or more amino acid residues in the Fc region of the natural heavy chain are substituted with other amino acids of interest, and chemically synthesizing the DNA, It is also possible to obtain DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest.
- the amino acid substitution site and the type of substitution are not particularly limited. Moreover, it is not restricted to substitution, Any of deletion, addition, insertion, or those combinations may be sufficient.
- DNA encoding a heavy chain in which one or more amino acid residues in the Fc region are substituted with other amino acids of interest can be produced by dividing into partial DNAs.
- Examples of the combination of partial DNAs include DNA encoding a variable region and DNA encoding an Fc region, or DNA encoding an Fab region and DNA encoding an Fc region, but are not limited to these combinations. is not.
- the DNA encoding the light chain can also be produced by dividing it into partial DNAs.
- a DNA encoding a heavy chain variable region is incorporated into an expression vector together with DNA encoding a heavy chain Fc region to construct a heavy chain expression vector.
- a light chain expression vector is constructed by incorporating a DNA encoding a light chain variable region into an expression vector together with a DNA encoding a light chain Fc region.
- the DNA encoding the target antibody When the DNA encoding the target antibody is incorporated into an expression vector, it is incorporated into the expression vector so that it is expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody. In that case, a combination of an appropriate host and an expression vector can be used.
- vectors examples include M13 vectors, pUC vectors, pBR322, pBluescript, and pCR-Script.
- pGEM-T pDIRECT, pT7 and the like can be used in addition to the above vector.
- an expression vector is particularly useful.
- an expression vector for example, when the host is E. coli such as JM109, DH5 ⁇ , HB101, XL1-Blue, a promoter that can be efficiently expressed in E. coli, such as the lacZ promoter (Ward et al., Nature (1989) 341). , 544-546; FASEB J. (1992) 6, 2422-2427, incorporated herein by reference in its entirety, araB promoter (Better et al., Science (1988) 240, 1041-1043, in its entirety by reference) Are incorporated herein), or have a T7 promoter or the like.
- such vectors include pGEX-5X-1 (Pharmacia), “QIAexpress® system” (QIAGEN), pEGFP, or pET (in this case, the host expresses T7 RNA polymerase).
- pGEX-5X-1 Pulacia
- QIAexpress® system QIAGEN
- pEGFP pEGFP
- pET in this case, the host expresses T7 RNA polymerase.
- BL21 is preferred).
- the vector may also contain a signal sequence for polypeptide secretion.
- the signal sequence for polypeptide secretion is the pelB signal sequence (Lei, S. P. et al J. Bacteriol. (1987) 169, 4397, which is incorporated herein by reference in its entirety when produced in the periplasm of E. coli. Built in).
- Introduction of a vector into a host cell can be performed using, for example, the lipofectin method, the calcium phosphate method, or the DEAE-Dextran method.
- vectors for producing the polypeptide of the present invention include mammalian-derived expression vectors (for example, pcDNA3 (manufactured by Invitrogen), pEGF-BOS® (Nucleic® Acids.® Res.
- pEF Bacillus subtilis-derived expression vectors
- pCDM8 Bacillus subtilis-derived expression vectors
- insect cell-derived expression vectors eg, “Bac-to-BACBAbaculovairus expression system” (GIBCO BRL), pBacPAK8
- plant-derived expression vectors eg, pMH1, pMH2
- animal virus-derived expression vectors eg, pHSV, pMV, pAdexLcw
- retrovirus-derived expression vectors eg, pZIPneo
- yeast-derived expression vectors eg, “Pichia® Expression® Kit” (manufactured by Invitrogen), pNV11, SP-Q01
- Bacillus subtilis-derived expression vectors for example, pPL608, pKTH50.
- promoters required for expression in cells such as the SV40 promoter (Mulligan et al., Nature (1979) 277, 108, see MMTV-LTR promoter, EF1 ⁇ promoter (Mizushima et al., Nucleic Acids Res. (1990) 18, 5322, which is hereby incorporated by reference in its entirety), CAG promoter ( Gene. (1991) 108, 193, which is incorporated herein by reference in its entirety, is essential to have a CMV promoter, etc., and a gene (eg, drug (neomycin) for selecting transformed cells. And drug resistance genes that can be discriminated by G418, etc.).
- Examples of such a vector include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
- a vector having a DHFR gene complementary to the CHO cell lacking the nucleic acid synthesis pathway for example, , PCHOI, etc.
- amplifying with methotrexate (MTX) for example, COS with a gene expressing SV40 T antigen on the chromosome
- COS with a gene expressing SV40 T antigen on the chromosome An example is a method of transforming with a vector (such as pcD) having an SV40 replication origin using cells.
- a vector such as pcD
- the replication origin those derived from polyoma virus, adenovirus, bovine papilloma virus (BPV) and the like can also be used.
- the expression vectors are selectable markers: aminoglycoside transferase (APH) gene, thymidine kinase (TK) gene, E. coli xanthine guanine phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase ( dhfr) gene and the like.
- APH aminoglycoside transferase
- TK thymidine kinase
- Ecogpt E. coli xanthine guanine phosphoribosyltransferase
- dhfr dihydrofolate reductase
- Antibody recovery can be performed, for example, by culturing transformed cells and then separating them from the inside of the cell or the culture solution of molecularly transformed cells.
- methods such as centrifugation, ammonium sulfate fractionation, salting out, ultrafiltration, 1q, FcRn, protein A, protein G column, affinity chromatography, ion exchange chromatography, and gel filtration chromatography are used. It can carry out in combination as appropriate.
- the present invention also provides a method for modifying a polypeptide to produce a polypeptide with improved stability compared to the parent polypeptide. That is, the present invention relates to a method for improving the stability of a polypeptide having an Fc region of an antibody as compared with a parent polypeptide by adding at least one amino acid modification to the loop site of the Fc region. In this method, it is preferable to use the thermal denaturation midpoint (Tm) as an index for evaluating or diagnosing stability.
- Tm thermal denaturation midpoint
- the present invention also provides a method for producing a polypeptide with improved stability compared to the parent polypeptide.
- a polypeptide having an Fc region of an antibody, wherein at least one amino acid in the loop site of the Fc region is modified, and stability is improved compared to the parent polypeptide Provide a method.
- a production method including the following steps can be mentioned; (A) in a polypeptide having an Fc region of an antibody, a step of modifying at least one amino acid at a loop site of the Fc region; (B) a step of measuring the stability of the polypeptide modified in the step (a), and (c) a step of selecting a polypeptide having improved stability compared to the parent polypeptide.
- a preferred embodiment is a method for producing a polypeptide having an Fc region of an antibody, wherein at least one amino acid in the loop site of the Fc region is modified, and has improved stability as compared to the parent polypeptide,
- a production method comprising the steps of: (A) modifying the nucleic acid encoding the polypeptide so that the stability is improved compared to the parent polypeptide; (B) introducing the nucleic acid into a host cell and culturing so as to express it; (C) recovering the polypeptide from the host cell culture.
- polypeptides antibodies
- Fc fusion protein molecules produced by the production method are also included in the present invention.
- EU numbering 234th EU numbering 235th, EU numbering 236th, EU numbering 237th, EU numbering 238th, EU numbering 239th, EU numbering 247th, EU numbering 250th, EU numbering 265th, EU numbering 266th, EU numbering 267th, EU numbering 268th, EU numbering 269th, EU numbering 270th, EU numbering 271th, EU numbering 295th, EU numbering 296th, EU numbering 298th, EU numbering 300th, EU numbering 307th, EU numbering 309th, EU numbering 315th, EU numbering 324th, EU numbering 325th, EU numbering 326th, EU numbering 327th, EU number Ring 329th, EU numbering 330th, EU numbering 333th, EU numbering 335th, EU numbering 337th, EU numbering 360
- the modification of TS9-19, TS28-43, TS51, and TS56 in the amino acid modification site shown in the examples below will be modified to reduce the binding activity to Fc ⁇ R compared to the parent polypeptide. May be included.
- the modified sites of TS1 to TS67 are as described above.
- amino acid modification in a polypeptide having an Fc region of an antibody (for example, human IgG).
- the present invention provides a nucleic acid encoding a polypeptide in which at least one amino acid in a loop site of the Fc region is modified in a polypeptide having an Fc region of an antibody, and the stability is improved compared to the parent polypeptide.
- the nucleic acid of the present invention may be in any form such as DNA or RNA.
- the present invention provides a vector containing the nucleic acid of the present invention.
- the type of vector can be appropriately selected by those skilled in the art depending on the host cell into which the vector is introduced. For example, the above-described vectors can be used.
- the present invention relates to a host cell transformed with the vector of the present invention.
- the host cell can be appropriately selected by those skilled in the art.
- the above-described host cell can be used.
- the present invention provides a pharmaceutical composition comprising a polypeptide or Fc fusion protein molecule of the present invention.
- the pharmaceutical composition of the present invention can be formulated by a known method by introducing a pharmaceutically acceptable carrier in addition to the above-described polypeptide or Fc fusion protein molecule of the present invention.
- a pharmaceutically acceptable carrier for example, it can be used parenterally in the form of a sterile solution with water or other pharmaceutically acceptable liquid, or an injection of suspension.
- a pharmacologically acceptable carrier or medium specifically, sterilized water, physiological saline, vegetable oil, emulsifier, suspension, surfactant, stabilizer, flavoring agent, excipient, vehicle, preservative It is conceivable to formulate by combining with a binder or the like as appropriate and mixing in a unit dosage form generally required for pharmaceutical practice.
- silicic acid lactose, crystalline cellulose, mannitol, starch, carmellose calcium, carmellose sodium, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylacetal diethylaminoacetate, polyvinylpyrrolidone, gelatin, medium chain fatty acid triglyceride
- the carrier include polyoxyethylene hydrogenated castor oil 60, sucrose, carboxymethylcellulose, corn starch, inorganic salts, and the like. The amount of active ingredients in these preparations is such that an appropriate volume within the indicated range can be obtained.
- a sterile composition for injection can be formulated in accordance with normal pharmaceutical practice using a vehicle such as distilled water for injection.
- Aqueous solutions for injection include, for example, isotonic solutions containing physiological saline, glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride, and suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM) and HCO-50 may be used in combination.
- isotonic solutions containing physiological saline glucose and other adjuvants such as D-sorbitol, D-mannose, D-mannitol and sodium chloride
- suitable solubilizers such as Alcohols, specifically ethanol, polyalcohols such as propylene glycol, polyethylene glycol, nonionic surfactants such as polysorbate 80 (TM) and HCO-50 may be used in combination.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
- oily liquid examples include sesame oil and soybean oil, which may be used in combination with benzyl benzoate or benzyl alcohol as a solubilizing agent.
- buffer for example, phosphate buffer, sodium acetate buffer, a soothing agent, for example, procaine hydrochloride, stabilizer, for example, benzyl alcohol, phenol, antioxidant.
- the prepared injection solution is usually filled into a suitable ampoule.
- Administration is preferably parenteral administration, and specific examples of the dosage form include an injection dosage form, a nasal dosage form, a pulmonary dosage form, and a transdermal dosage form.
- Examples of administration in an injection form include intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, and the like, and can be administered systemically or locally.
- the administration method can be appropriately selected depending on the age and symptoms of the patient.
- the dose of the pharmaceutical composition containing the antibody or the polynucleotide encoding the antibody can be selected, for example, in the range of 0.0001 mg to 1000 mg per kg body weight. Alternatively, for example, the dose can be selected in the range of 0.001 to 100,000 mg / body per patient, but is not necessarily limited to these values.
- the dose and administration method vary depending on the weight, age, symptoms, etc. of the patient, but can be appropriately selected by those skilled in the art.
- the pharmaceutical composition containing the polypeptide of the present invention is useful as an active ingredient of, for example, a therapeutic or prophylactic agent for immunoinflammatory diseases, cancer and the like.
- Immunoinflammatory disease includes, but is not limited to: rheumatoid arthritis, autoimmune hepatitis, autoimmune thyroiditis, autoimmune blistering, autoimmune corticosteroids , Autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, megacytic anemia, autoimmune atrophic gastritis, autoimmune neutropenia, autoimmune orchitis, autoimmune encephalomyelitis , Autoimmune receptor disease, autoimmune infertility, chronic active hepatitis, glomerulonephritis, interstitial pulmonary fibrosis, multiple sclerosis, pageutism, osteoporosis, multiple myeloma, uveitis, acute And chronic spondylitis, gouty arthritis, inflammatory rheumatoi
- ischemic reperfusion trauma atherosclerosis, brain trauma, multiple sclerosis, cerebral malaria, sepsis, septic shock, toxic shock syndrome, fever, and staining due to malgias, aplastic anemia, hemolysis Anemia, idiopathic thrombocytopenia, Goodpasture syndrome, Guillain-Barre syndrome, Hashimoto disease, pemphigus, IgA nephropathy, hay fever, antiphospholipid antibody syndrome, polymyositis, Wegener's sarcoma, nodular arteritis, mixed Sexual connective tissue disease, fibromyalgia.
- cancer means or refers to a physiological state of a mammal that is typically characterized by uncontrolled cell growth.
- the type of cancer is not particularly limited, but includes the following. Cancers (epithelial cancers) include pancreatic cancer, prostate cancer, breast cancer, skin cancer, gastrointestinal cancer, lung cancer, hepatocellular carcinoma, cervical cancer, uterine body cancer, ovarian cancer, fallopian tube cancer, vaginal cancer, liver cancer , Bile duct cancer, bladder cancer, ureteral cancer, thyroid cancer, adrenal cancer, kidney cancer, or other glandular tissue cancer.
- Sarcomas include liposarcoma, leiomyosarcoma, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, fibrosarcoma, malignant peripheral nerve tumor, gastrointestinal stromal tumor, tendonoma, Ewing sarcoma, Examples include osteosarcoma, chondrosarcoma, leukemia, lymphoma, myeloma and other parenchymal tumors such as melanoma or brain tumor.
- amino acid sites modified in the Fc region were in accordance with the EU numbering system (see Sequences of proteins of immunological interest, NIH Publication No.91-3242).
- Example 1 Improvement of thermal stability by modification of CH2 domain loop region Generally, it is known that a ⁇ sheet structure is likely to undergo structural changes due to amino acid modification, and a thermal stability decrease due to modification. (Biochemistry 1994; 33: 5510-5517, Nature 1994; 367: 660-663). Therefore, in the present invention, an attempt was made to improve thermal stability by modifying the loop portion of the Fc region.
- GpH7- Fc variant / GpL16-k0 An antibody consisting of a variable region GpH7 (SEQ ID NO: 17) as an H chain, an Fc-variant as an Fc region, a variable region GpL16 (SEQ ID NO: 18) as an L chain, and a constant region k0 (SEQ ID NO: 19) (hereinafter GpH7- Fc variant / GpL16-k0) was prepared according to the method of Reference Example 1.
- Tm refers to the Tm of the CH2 region when a sample in the form of IgG is used for measurement.
- the obtained Tm data is shown in FIG. Tm of B3 (GpH7-B3 / GpL16-k0: GpH7 (SEQ ID NO: 17), B3 (SEQ ID NO: 16), GpL16 (SEQ ID NO: 18), k0 (SEQ ID NO: 19))) into which no mutation has been introduced
- the mutants having a Tm higher than this value at about 68 ° C. are summarized in Table 1-1 and Table 1-2.
- Table 1-2 is a continuation of Table 1-1.
- Example 2 Production and evaluation of novel Fc region IgG1 for improving thermal stability Based on the data and structural information of Example 1 (Nature 2000; 406: 267-273), the effect of improving thermal stability is particularly excellent. Some of the modifications considered to be selected were selected.
- the selected single modification or multiple modifications can be performed by anti-GPC3 antibody (GpH7-G1d / GpL16-k0: GpH7 (SEQ ID NO: 17), G1d (SEQ ID NO: 15), GpL16 (SEQ ID NO: 18), k0 (SEQ ID NO: 19), Anti-IL6R antibody (MH0-G1d / ML0-k0: GpH7 (SEQ ID NO: 17), G1d (SEQ ID NO: 15), ML0 (SEQ ID NO: 21), k0 (SEQ ID NO: 19) )
- TS1 to TS19 SEQ ID NOs: 26 to 44 shown in Table 2.
- Tm was compared by the method shown in Reference Example 2.
- the measurement results of Tm are shown in Table 3.
- Tm is further improved. From the above, it is possible to create a plurality of combinations of modifications that improve Tm based on the structural information other than TS1-3 and TS5-19 shown here.
- TSm01 and TSm02 evaluated at the same time expressed only the CH2 domain in the previous paper (J Biol Chem. 2009; 284: 14203-14210), and as a result of evaluating Tm, a Tm improvement effect of about 10-20 ° C was observed. It was done. However, when Tm was evaluated in the form of IgG in this study, TSm01 detected multiple heat denaturation points, and it was difficult to clearly grasp the heat denaturation of the CH2 domain. This suggests that TSm01 is likely to be structurally heterogeneous.
- TSm02 only increased Tm by about 4 ° C compared to IgG1. From this, it was clarified that the effect of improving Tm in the form of IgG by introducing disulfide bonds is small, and the modification found by the present inventors is more effective in improving Tm.
- the anti-GPC3 antibody used in this study has a Fab Tm of 74.7 ° C.
- TS13 to TS19 in which modifications were made to the anti-GPC3 heavy chain, the fluorescence transition curves of CH2 and Fab overlapped, suggesting that accurate Tm calculation was difficult. Therefore, it was considered unsuitable to use the anti-GPC3 heavy chain in evaluating the further Tm improvement effect by the combination of the modified residues, and the anti-IL6R heavy chain was used in the subsequent studies.
- Example 4 Production and evaluation of a novel Fc region combined with a modification that maintains the binding to hFcgRs.
- a cancer antibody whose effector function is important is used for hFcgRs. It is important to maintain the bond.
- the modifications of TS1 to TS8 maintaining the binding to hFcgRs were combined to newly produce the variants of TS20 to TS27 shown in Table 5 (SEQ ID NOs: 45 to 52). Expression and purification of each antibody were performed by the method described in Reference Example 1. The prepared antibody was evaluated for Tm by the method shown in Reference Example 2, and the results are shown in Table 6.
- Table 7 shows the binding of each antibody to each hFcgRs when the binding of G1 to each hFcgRs is defined as 100.
- Tm was improved while maintaining the binding ability to hFcgRs in all the modified TS20 to TS27.
- the variants with the greatest Tm improvement effect were TS20 and TS24, and the Tm was improved by about 3 ° C over G1.
- Example 5 Production and evaluation of a novel Fc region combined with a modification with reduced binding to hFcgRs
- the effector function of the neutralizing antibody should be as low as possible. desirable.
- TS9 to TS19 modifications with reduced binding to hFcgRs were combined to newly produce TS28 to TS43 variants shown in Table 8 (SEQ ID NOs: 53 to 68).
- Expression and purification of each antibody were performed by the method described in Reference Example 1. The prepared antibody was evaluated for Tm by the method shown in Reference Example 2, and the results are shown in Table 9.
- Table 10 shows the binding of each antibody to each hFcgRs when the binding of G1 to each hFcgRs is defined as 100.
- Tm increased by 13 ° C or more in all modified TS28 to TS43. Furthermore, the ability to bind to FcgRs was also significantly reduced by combining modifications.
- Example 6 Evaluation of aggregate content of novel Fc region IgG1 with improved thermal stability Since aggregates affect storage stability and immunogenicity, modifications that improve thermal stability can be made as much as possible. It is desirable not to increase the aggregate content. Therefore, the content of aggregates of TS20 to TS43 combined with multiple modifications, and TSm01 and TSm02 were evaluated by the method shown in Reference Example 4. The chromatogram of the measurement is shown in FIG. 2, and the aggregate content is shown in Table 11.
- Example 7 Production and evaluation of novel Fc region IgG1 that improves thermal stability It was revealed from the above that Tm is improved by introducing mutation into CH2 domain. Therefore, even in the region where the introduction of the modification was not examined in Example 1, it was examined whether there was a mutation that improves Tm, and a modified TS44 to TS67 shown in Table 12 was newly prepared (SEQ ID NO: : 69-92). Expression and purification of each antibody were carried out by the method described in Reference Example 1, and the results are shown in Table 13. Furthermore, the binding to hFcgRs was measured by the method described in Reference Example 3. The binding of each antibody to each hFcgRs when the binding of G1 to each hFcgRs is defined as 100 is shown in Table 14.
- the variant with the highest Tm improvement effect was TS48, which showed an improvement of about 5 ° C., and other variants also showed an improvement in Tm.
- TS51 and TS56 had a slight decrease in binding to hFcgRs, and other than TS51 and TS56, binding to hFcgRs was maintained.
- the antibody was purified from the obtained culture supernatant by a method known to those skilled in the art using rProtein A Sepharose TM Fast Flow (GE Healthcare).
- the purified antibody concentration was determined by measuring the absorbance at 280 nm using a spectrophotometer, and calculating the antibody concentration using the extinction coefficient calculated by the PACE method from the obtained value (Protein Science 1995; 4: 2411- 2423).
- Tm Thermal denaturation midpoint evaluation of modified antibodies by inspection scanning fluorescence quantification
- antibodies were detected using inspection scanning fluorescence quantification using Rotor-Gene Q (QIAGEN).
- the thermal stability was evaluated by measuring the heat denaturation midpoint (Tm).
- this technique shows a good correlation with Tm evaluation using a scanning scanning calorimeter widely known as an antibody thermal stability evaluation method (Journal of Pharmaceutical Science 2010; 4: 1707-1720).
- a sample for measurement was prepared by diluting 5000 times SYPRO orange (Molecular Probes) with PBS (Sigma) and then mixing with the antibody solution. 20 ⁇ L of each sample was set in a measuring tube and the temperature was raised from 30 ° C. to 99 ° C. After raising the temperature by 0.4 ° C. and resting for about 6 seconds, the fluorescence intensity was detected at 470 nm (excitation wavelength) / 555 nm (fluorescence wavelength). For data, the temperature at which fluorescence transition was observed was calculated using Rotor-Gene Q Series Software (QIAGEN), and this value was defined as Tm.
- hFc ⁇ R diluted with running buffer was allowed to interact with hFcgRIa for 5 minutes at a flow rate of 30 ⁇ L / min, and other hFc ⁇ R for 1 minute at a flow rate of 5 ⁇ L / min.
- the capture amount of each antibody is 200 RU (resonance unit) for hFcgRIa and 1000 RU (resonance unit) for other hFcgRs.
- the amount of binding was corrected.
- 10 mM glycine-HCl, pH 1.5 was reacted at a flow rate of 30 ⁇ L / min for 30 seconds to wash the antibody captured on the chip, and the chip was regenerated and used repeatedly.
- the antibody Fc region variants reported have decreased the stability of the Fc region. Further, it is important that the Fc portion of the antibody binds to an effector function, that is, Fc ⁇ R. According to the present invention, it is possible to provide a highly stable Fc that maintains the binding to Fc ⁇ R, or a highly stable Fc region variant that decreases the binding to Fc ⁇ R and has a high stability.
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Abstract
Description
つまり、これまでに見出された改変型Fc領域の多くは、機能増強に伴い抗体の利点のひとつである良好な安定性が失われている。
以上のように、活性と安定性の両面において優れたFc領域はこれまでに報告されていない。
本発明者らは鋭意研究を行った結果、抗体のFc領域を有するポリペプチドであって、該Fc領域のループ部位における少なくとも一つのアミノ酸を改変する事で、親ポリペプチドと比較して、安定性が向上したポリペプチドを取得することに成功した。
さらに、ループ部位のアミノ酸の改変を複数組み合わせることで、親ポリペプチドと比較して、熱安定性を向上させつつFcγRへの結合活性が維持又は増強したポリペプチド、及び熱安定性を向上させつつFcγRへの結合活性が減少したポリペプチドを取得することに成功した。さらには、熱安定性を向上させつつFcγRへの結合活性を調整するだけでなく、会合体含有量が減少したポリペプチドを取得することに成功した。
〔1〕 抗体のFc領域を有するポリペプチドであって、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチド。
〔2〕 前記安定性が熱変性中点(Tm)を指標として評価あるいは判断される、〔1〕に記載のポリペプチド。
〔3〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング236番目、EUナンバリング237番目、EUナンバリング238番目、EUナンバリング239番目、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング265番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング271番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、EUナンバリング327番目、EUナンバリング329番目、EUナンバリング330番目、EUナンバリング333番目、EUナンバリング335番目、EUナンバリング337番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕または〔2〕に記載のポリペプチド。
〔4〕 さらに、親ポリペプチドと比較してFcγRへの結合活性が維持又は増強した、〔1〕~〔3〕のいずれか一項に記載のポリペプチド。
〔5〕 さらに、親ポリペプチドと比較してFcγRへの結合活性が減少した、〔1〕~〔3〕のいずれか一項に記載のポリペプチド。
〔6〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔5〕のいずれか一項に記載のポリペプチド。
〔7〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング266番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔4〕、〔6〕のいずれか一項に記載のポリペプチド。
〔8〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のIleへの置換、EUナンバリング266番目のアミノ酸のIleへの置換、EUナンバリング268番目のアミノ酸のGlnへの置換、EUナンバリング269番目のアミノ酸のAspへの置換、EUナンバリング270番目のアミノ酸のGluへの置換、EUナンバリング295番目のアミノ酸のMet又はLeuへの置換、EUナンバリング300番目のアミノ酸のGluへの置換、EUナンバリング324番目のアミノ酸のHisへの置換、EUナンバリング326番目のアミノ酸のSer又はAlaへの置換、及びEUナンバリング330番目のアミノ酸のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔4〕、〔6〕、〔7〕のいずれか一項に記載のポリペプチド。
〔9〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕、〔6〕のいずれか一項に記載のポリペプチド。
〔10〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLys又はArgへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング267番目のアミノ酸のProへの置換、EUナンバリング268番目のアミノ酸のMet又はLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のGly、His又はMetのいずれかへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔6〕、〔9〕のいずれか一項に記載のポリペプチド。
〔11〕 前記Fc領域のループ部位における、EUナンバリング295番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔4〕のいずれか一項に記載のポリペプチド。
〔12〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング295番目のアミノ酸のMet又はLeuへの置換、EUナンバリング326番目のアミノ酸のSer又はAlaへの置換、及びEUナンバリング330番目のアミノ酸のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔4〕、〔11〕のいずれか一項に記載のポリペプチド。
〔13〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕のいずれか一項に記載のポリペプチド。
〔14〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLys又はSerへの置換、EUナンバリング268番目のアミノ酸のLys又はHisへの置換、EUナンバリング270番目のアミノ酸のPhe又はAspへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔13〕のいずれか一項に記載のポリペプチド。
〔15〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸配列において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕のいずれか一項に記載のポリペプチド。
〔16〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔15〕のいずれか一項に記載のポリペプチド。
〔17〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸配列において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕のいずれか一項に記載のポリペプチド。
〔18〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔17〕のいずれか一項に記載のポリペプチド。
〔19〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕のいずれか一項に記載のポリペプチド。
〔20〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔19〕のいずれか一項に記載のポリペプチド。
〔21〕 前記Fc領域のループ部位におけるアミノ酸改変部位が、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕のいずれか一項に記載のポリペプチド。
〔22〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔21〕のいずれか一項に記載のポリペプチド。
〔23〕 前記Fc領域のループ部位における、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔4〕のいずれか一項に記載のポリペプチド。
〔24〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング247番目のアミノ酸のValへの置換、EUナンバリング250番目のアミノ酸のPhe、Ile、Met、Val、Trp、又はTyrのいずれかへの置換、EUナンバリング307番目のアミノ酸のAla、Gln、又はProのいずれかへの置換、EUナンバリング309番目のアミノ酸のAla、Arg、又はProのいずれかへの置換、EUナンバリング315番目のアミノ酸のAlaへの置換、EUナンバリング360番目のアミノ酸のHisへの置換、EUナンバリング385番目のアミノ酸のAspへの置換、EUナンバリング386番目のアミノ酸のProへの置換、EUナンバリング387番目のアミノ酸のGluへの置換、EUナンバリング389番目のアミノ酸のSerへの置換、EUナンバリング428番目のアミノ酸のHis、Trp、Tyr、またはPheのいずれかへの置換、及びEUナンバリング433番目のアミノ酸のLysへの置換からなる群より選択される少なくも一つ以上のアミノ酸改変である、〔1〕~〔4〕、〔23〕のいずれか一項に記載のポリペプチド。
〔25〕 前記Fc領域のループ部位における、EUナンバリング298番目又はEUナンバリング309番目のアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、〔1〕~〔3〕、〔5〕のいずれか一項に記載のポリペプチド。
〔26〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング309番目のアミノ酸のAspへの置換からなる群より選択される、少なくとも一つ以上のアミノ酸改変である、〔1〕~〔3〕、〔5〕、〔25〕のいずれか一項に記載のポリペプチド。
〔27〕 抗体のFc領域を有するポリペプチドにおいて、該Fc領域のループ部位に少なくとも一つのアミノ酸改変を加えることにより、親ポリペプチドと比較して安定性を向上させる方法。
〔28〕 前記安定性を熱変性中点(Tm)を指標として評価あるいは判断する、〔27〕に記載の方法。
〔29〕 以下の工程を含む、抗体のFc領域を有し、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドの製造方法;
(a)抗体のFc領域を有するポリペプチドにおいて、該Fc領域のループ部位に少なくとも一つのアミノ酸改変を加える工程、
(b)前記工程(a)で改変されたポリペプチドの安定性を測定する工程、および
(c)親ポリペプチドと比較して、安定性が向上したポリペプチドを選択する工程。
〔30〕 以下の工程を含む、抗体のFc領域を有し、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドの製造方法;
(a)親ポリペプチドと比較して、安定性が向上するように、当該ポリペプチドをコードする核酸を改変する工程、
(b)宿主細胞に当該改変された核酸を導入し発現するように培養する工程、
(c)宿主細胞培養物から当該ポリペプチドを回収する工程。
〔31〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング236番目、EUナンバリング237番目、EUナンバリング238番目、EUナンバリング239番目、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング265番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング271番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、EUナンバリング327番目、EUナンバリング329番目、EUナンバリング330番目、EUナンバリング333番目、EUナンバリング335番目、EUナンバリング337番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔30〕のいずれか一項に記載の方法。
〔32〕 さらに親ポリペプチドと比較してFcγRへの結合活性を維持又は増強するように改変する工程を含む、〔27〕~〔31〕のいずれか一項に記載の方法。
〔33〕 さらに、親ポリペプチドと比較してFcγRへの結合活性が減少するように改変する工程を含む、〔27〕~〔31〕のいずれか一項に記載の方法。
〔34〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔33〕のいずれか一項に記載の方法。
〔35〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング266番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔32〕、〔34〕のいずれか一項に記載の方法。
〔36〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のIleへの置換、EUナンバリング266番目のアミノ酸のIleへの置換、EUナンバリング268番目のアミノ酸のGlnへの置換、EUナンバリング269番目のアミノ酸のAspへの置換、EUナンバリング270番目のアミノ酸のGluへの置換、EUナンバリング295番目のアミノ酸のMet又はLeuへの置換、EUナンバリング300番目のアミノ酸のGluへの置換、EUナンバリング324番目のアミノ酸のHisへの置換、EUナンバリング326番目のアミノ酸のSer又はAlaへの置換、及びEUナンバリング330番目のアミノ酸のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔32〕、〔34〕、〔35〕のいずれか一項に記載の方法。
〔37〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕、〔34〕のいずれか一項に記載の方法。
〔38〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLys又はArgへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング267番目のアミノ酸のProへの置換、EUナンバリング268番目のアミノ酸のMet又はLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のGly、His又はMetのいずれかへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔34〕、〔37〕のいずれか一項に記載の方法。
〔39〕 前記Fc領域のループ部位における、EUナンバリング295番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔32〕のいずれか一項に記載の方法。
〔40〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング295番目のMet又はLeuへの置換、EUナンバリング326番目のSer又はAlaへの置換、及びEUナンバリング330番目のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔32〕、〔39〕のいずれか一項に記載の方法。
〔41〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目又はEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕のいずれか一項に記載の方法。
〔42〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLys又はSerへの置換、EUナンバリング268番目のアミノ酸のLys又はHisへの置換、EUナンバリング270番目のアミノ酸のPhe又はAspへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔41〕のいずれか一項に記載の方法。
〔43〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕のいずれか一項に記載の方法。
〔44〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔43〕のいずれか一項に記載の方法。
〔45〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕のいずれか一項に記載の方法。
〔46〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔45〕のいずれか一項に記載の方法。
〔47〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕のいずれか一項に記載の方法。
〔48〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔47〕のいずれか一項に記載の方法。
〔49〕 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕のいずれか一項に記載の方法。
〔50〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔49〕のいずれか一項に記載の方法。
〔51〕 前記Fc領域のループ部位における、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔32〕のいずれか一項に記載の方法。
〔52〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング247番目のアミノ酸のValへの置換、EUナンバリング250番目のアミノ酸のPhe、Ile、Met、Val、Trp、又はTyrのいずれかへの置換、EUナンバリング307番目のアミノ酸のAla、Gln、又はProのいずれかへの置換、EUナンバリング309番目のアミノ酸のAla、Arg、又はProのいずれかへの置換、EUナンバリング315番目のアミノ酸のAlaへの置換、EUナンバリング360番目のアミノ酸のHisへの置換、EUナンバリング385番目のアミノ酸のAspへの置換、EUナンバリング386番目のアミノ酸のProへの置換、EUナンバリング387番目のアミノ酸のGluへの置換、EUナンバリング389番目のアミノ酸のSerへの置換、EUナンバリング428番目のアミノ酸のHis、Trp、Tyr、またはPheのいずれかへの置換、及びEUナンバリング433番目のアミノ酸のLysへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、〔27〕~〔32〕、〔51〕のいずれか一項に記載の方法。
〔53〕 前記Fc領域のループ部位における、EUナンバリング298番目又はEUナンバリング309番目のアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、〔27〕~〔31〕、〔33〕のいずれか一項に記載の方法。
〔54〕 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング309番目のアミノ酸のAspへの置換からなる群より選択される、少なくとも一つ以上のアミノ酸改変である、〔27〕~〔31〕、〔33〕、〔53〕のいずれか一項に記載の方法。
〔55〕 前記改変が、ヒトIgGのFc領域を有するポリペプチドにおける改変である、〔27〕~〔54〕のいずれか一項に記載の方法。
〔56〕 抗体のFc領域を有するポリペプチドであって、Fc領域のループ部位の少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドをコードする核酸。
〔57〕 〔56〕に記載の核酸を含むベクター。
〔58〕 〔57〕に記載のベクターにより形質転換された宿主細胞。
〔59〕 〔1〕~〔26〕のいずれかに記載のポリペプチド、または〔27〕~〔55〕のいずれかに記載の方法により作製されたポリペプチドを含有する医薬組成物。
〔60〕 〔59〕に記載の医薬組成物を含有する免疫炎症性疾患または癌の治療剤又は予防剤。
〔61〕 免疫炎症性疾患が関節リウマチ、自己免疫性肝炎、自己免疫性甲状腺炎、自己免疫性水疱症、自己免疫性副腎皮質炎、自己免疫性溶血性貧血、自己免疫性血小板減少性紫斑病、巨赤血球性貧血、自己免疫性萎縮性胃炎、自己免疫性好中球減少症、自己免疫性精巣炎、自己免疫性脳脊髄炎、自己免疫性レセプター病、自己免疫不妊、慢性活動型肝炎、糸球体腎炎、間質性肺腺維症、多発性硬化症、パジュット症、オステオポローシス、多発性骨髄腫、ブドウ膜炎、急性及び慢性脊椎炎、痛風性関節炎、炎症性腸疾患、成人呼吸促進症候群(ARDS)、乾癬、クローン病、バセドウ病、若年性糖尿病、アジソン病、重症筋無力症、水晶体性ブドウ膜炎、全身性エリテマトーデス、アレルギー性鼻炎、アレルギー性皮膚炎、潰瘍性大腸炎、過敏症、ぜん息、筋肉変性、悪液質、全身性強皮症、限局性強皮症、シェーグレン症候群、ベーチェット病、ライター症候群、I型及びII型糖尿病、骨吸収疾患、移植片vs.宿主反応、虚血性再灌流外傷、アテローム硬化症、脳トラウマ、多発生硬化症、大脳マラリア、敗血症、敗血性ショック、トキシックショック症候群、発熱、及び染色によるマルギアス(malgias)、再生不良性貧血、溶血性貧血、突発性血小板減少症、グッドパスチャー症候群、ギラン・バレー症候群、橋本病、天疱瘡、IgA腎症、花粉症、抗リン脂質抗体症候群、多発性筋炎、ウェゲナー肉腫、結節性動脈炎、混合性結合組織病、または線維筋痛症である、〔60〕に記載の治療剤又は予防剤。
〔62〕 癌が膵臓癌、前立腺癌、乳癌、皮膚癌、消化管の癌、肺癌、肝細胞癌、子宮頸癌、子宮体癌、卵巣癌、卵管癌、膣癌、肝臓癌、胆管癌、膀胱癌、尿管の癌、甲状腺癌、副腎癌、腎臓癌、その他の腺組織の癌、脂肪肉腫、平滑筋肉腫、黄紋筋肉腫、滑膜肉腫、血管肉腫、線維肉腫、悪性末梢神経腫瘍、消化管間質系腫瘍、類腱腫、ユーイング肉腫、骨肉腫、軟骨肉腫、白血病、リンパ腫、骨髄腫、又はその他の実質臓器の腫瘍である、〔60〕に記載の治療剤または予防剤。
〔63〕 〔1〕~〔26〕のいずれかに記載のポリペプチド、または〔27〕~〔55〕のいずれかに記載の方法により作製されたポリペプチドを対象に投与する工程を含む、免疫炎症性疾患または癌を治療又は予防する方法。
〔64〕 免疫炎症性疾患または癌の治療又は予防において使用するための、〔1〕~〔26〕のいずれかに記載のポリペプチド、または〔27〕~〔55〕のいずれかに記載の方法により作製されたポリペプチド。
〔65〕 免疫炎症性疾患または癌の治療剤又は予防剤の製造における、〔1〕~〔26〕のいずれかに記載のポリペプチド、または〔27〕~〔55〕のいずれかに記載の方法により作製されたポリペプチドの使用。
〔66〕 〔1〕~〔26〕のいずれかに記載のポリペプチド、または〔27〕~〔55〕のいずれかに記載の方法により作製されたポリペプチドを使用する工程を含む、免疫炎症性疾患または癌の治療剤又は予防剤を製造する方法。
さらに本発明は、抗体Fc領域のループ部位にアミノ酸置換を導入することで親ポリペプチドと比較して、該Fc領域を有するポリペプチドの安定性を向上させる方法を提供する。さらに本発明は、抗体Fc領域のループ部位にアミノ酸置換を導入することで親ポリペプチドと比較して安定性が向上した該Fc領域を有するポリペプチドの製造方法を提供する。
天然型IgGのFc領域とは、天然に見出されるIgGを起源とするFc領域と同一のアミノ酸配列を包含するFc領域を意味する。天然型IgGのFc領域は図3(配列番号:11~14)に示しているが、例えば天然型ヒトIgG1を起源とするFc領域、天然型ヒトIgG2を起源とするFc領域、天然型ヒトIgG3を起源とするFc領域、天然型ヒトIgG4を起源とするFc領域を意味する。天然型IgGのFc領域にはそれから自然に生じる変異体等も含まれる。
Fc領域は、IgG1、IgG2、IgG3、IgG4モノクローナル抗体等をペプシン等の蛋白質分解酵素にて部分消化した後に、プロテインAカラムに吸着された画分を再溶出することによって好適に取得され得る。かかる蛋白質分解酵素としてはpH等の酵素の反応条件を適切に設定することにより制限的にFabやF(ab')2を生じるように全長抗体を消化し得るものであれば特段の限定はされず、例えば、ペプシンやパパイン等が例示できる。
親ポリペプチドと比較してFcγRへの結合活性が維持または増強するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング266番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が維持または増強するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング295番目、EUナンバリング326番目及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が維持または増強するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドが挙げられる。
親ポリペプチドと比較してFcγRへの結合活性が減少するポリペプチドとしては、例えば前記Fc領域のループ部位における、EUナンバリング298番目、及びEUナンバリング309番目のアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されているポリペプチドを挙げることができる。
FcγIIAのポリヌクレオチド配列及びアミノ酸配列は、それぞれ配列番号:3(BC020823.1)及び4(AAH20823.1)に、
FcγIIBのポリヌクレオチド配列及びアミノ酸配列は、それぞれ配列番号:5(BC146678.1)及び6(AAI46679.1)に、
FcγIIIAのポリヌクレオチド配列及びアミノ酸配列は、それぞれ配列番号:7(BC033678.1)及び8(AAH33678.1)に、及び
FcγIIIBのポリヌクレオチド配列及びアミノ酸配列は、それぞれ配列番号:9(BC128562.1)及び10(AAI28563.1)に記載されている(カッコ内はRefSeq登録番号を示す)。
尚、FcγRIIaには、FcγRIIaの131番目のアミノ酸がヒスチジン(H型)あるいはアルギニン(R型)に置換された2種類の遺伝子多型が存在する(J. Exp. Med, 172, 19-25, 1990)。
例えば、上記の測定法で測定した結合量において、親ポリペプチドの各FcγRに対する結合量を100とした場合のポリペプチド変異体の各FcγRに対する結合量(以下、結合量比という)が、好ましくは120以上、150以上、200以上、300以上に向上しているものを言う。
例えば、上記の測定法で測定した結合量において、親ポリペプチドの各FcγRに対する結合量を100とした場合のポリペプチド変異体の各FcγRに対する結合量(以下、結合量比という)が、好ましくは80以上かつ120以下であるものを言う。
例えば、別のFc領域に対する改変として、ヒトIgG(IgG1、IgG2、IgG3、IgG4)に対して、EUナンバリング238番目のProをAspに置換する改変および/またEUナンバリング328番目のLeuをGluに置換する改変を挙げることができる。ヒトIgGにEUナンバリング238番目のProをAspに置換した改変および/またEUナンバリング328番目のLeuをGluに置換した改変を導入することにより、親ポリペプチドと比較して、FcγRI、FcγRIIIaおよびFcγRIIaのR型、H型のいずれの遺伝子多型に対しても結合活性が維持あるいは減少し、かつFcγRIIbに対する結合活性が増強したポリペプチドを提供することが可能である。
抗体の血漿中滞留性を制御するために、本発明のアミノ酸の改変と抗体の等電点の値(pI値)を改変させるためのアミノ酸の改変を組み合わせることが可能である。定常領域の改変については、例えば、公知の文献(J. Immunol. 2006, 176(1):346-356やNat. Biotechnol. 1997 15(7):637-640等)に記載のEUナンバリング250位や428位等のアミノ酸の改変が挙げられる。また、可変領域の改変としてWO2007/114319やWO2009/041643に記載のアミノ酸の改変が挙げられる。改変されるアミノ酸は、抗原結合活性を有するポリペプチドの表面に露出しているアミノ酸が好ましい。例えば、本発明のポリペプチドが重鎖定常領域を有する場合、重鎖定常領域のアミノ酸配列におけるEUナンバリング196位のアミノ酸の置換が挙げられる。重鎖定常領域がIgG4の場合、例えば196番目のリジンをグルタミンに置換することでpI値を低くし、血漿中滞留性を高めることが可能である。また、血漿中滞留性はFcRnに対する結合力を改変することによっても制御可能である。FcRnに対する結合力改変のためのアミノ酸改変としては、例えば、公知の文献(The Journal of Biological Chemistry vol.276, No.9 6591-6604, 2001やMolecular Cell, Vol.7, 867-877, 2001)に記載の抗体重鎖定常領域のアミノ酸の置換が挙げられる。
例えば、本発明のポリペプチドがIgG4重鎖定常領域を有する場合、酸性条件下におけるIgG4のhalf-molecule化を抑え安定な4鎖構造(H2L2構造)を維持することが好ましい。そのため、4鎖構造の維持に重要な役割を果たしているEUナンバリング409位のアミノ酸であるアルギニン(Immunology 2002, 105, 9-19)を、酸性条件下においても安定な4鎖構造を維持するIgG1タイプのリジンに置換することが好ましい。このような改変を本発明のアミノ酸の改変と組合わせて用いることができる。
本発明のアミノ酸の改変とWO2009/041613に記載の方法を組合わせることが可能である。具体的には、例えば本発明のポリペプチドがIgG1重鎖定常領域を有する場合、IgG1重鎖定常領域のC末端の2アミノ酸、すなわちEUナンバリング446番目のグリシン及び447番目のリジンを欠損させる改変を本発明の実施例に基づくアミノ酸の改変と組み合わせ、ヘテロジェニティーの低減を図ることが可能である。
本発明のアミノ酸の改変と脱アミド化反応の抑制のためのアミノ酸の改変を組合わせることが可能である。脱アミド化反応は、特にアスパラギン(N)とグリシン(G)が隣接した部位(・・・NG・・・)において起こりやすいことが報告されている(Geigerら J. Bio. Chem. 1987; 262:785-794)。本発明のポリペプチドにアスパラギンとグリシンが隣接した部位が存在する場合には、当該アミノ酸配列を改変することにより脱アミド化反応を抑制することができる。具体的には例えば、アスパラギンとグリシンどちらか一方若しくは両方のアミノ酸配列を他のアミノ酸に置換する。より具体的には例えば、アスパラギンをアスパラギン酸に置換する。
例えば以下の(a)~(c)のような点について改変する事を目的として、アミノ酸残基を別のアミノ酸残基に置換することができる;
(a) シート構造、若しくは、らせん構造の領域におけるポリペプチドの背骨構造、
(b) 標的部位における電荷若しくは疎水性、または
(c)側鎖の大きさ。
(1) 疎水性: ノルロイシン、met、ala、val、leu、ile;
(2) 中性親水性: cys、ser、thr、asn、gln;
(3) 酸性: asp、glu;
(4) 塩基性: his、lys、arg;
(5) 鎖の配向に影響する残基: gly、pro;及び
(6) 芳香族性: trp、tyr、phe。
・哺乳動物の抗体産生細胞
・抗体をコードするDNAを含む発現ベクターで形質転換された真核細胞
より具体的には、例えばFc領域の糖鎖にシアル酸を付加したものであってもよい(MAbs. 2010 Sep-Oct;2(5):519-27.)。
さらに、本発明は上述のいずれかに記載のアミノ酸配列が改変されたFc領域を有する抗体を提供する。
本発明の抗体は、抗原の種類、抗体の由来などは限定されず、いかなる抗体でもよい。抗体の由来としては、特に限定されないが、ヒト抗体、マウス抗体、ラット抗体、ウサギ抗体などを挙げることができる。
また、抗体遺伝子を取得する方法としてBernasconiら(Science (2002) 298, 2199-2202)またはWO2008/081008に記載のようなB細胞クローニング(それぞれの抗体のコード配列の同定およびクローニング、その単離、およびそれぞれの抗体(特に、IgG1、IgG2、IgG3またはIgG4)の作製のための発現ベクター構築のための使用等)の手法が、上記のほか適宜使用され得る。
他のタンパク質、生理活性ペプチドとしては、例えば受容体、接着分子、リガンド、酵素が挙げられるが、これらに限定されるものではない。
本発明の製造方法の一態様として、例えば抗体のFc領域を有し、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドの製造方法を提供する。
例えば以下の工程を含む製造方法を挙げることができる;
(a)抗体のFc領域を有するポリペプチドにおいて、該Fc領域のループ部位に少なくとも一つのアミノ酸改変を加える工程、
(b)前記工程(a)で改変されたポリペプチドの安定性を測定する工程、および
(c)親ポリペプチドと比較して、安定性が向上したポリペプチドを選択する工程。
(a)親ポリペプチドと比較して、安定性が向上するように、当該ポリペプチドをコードする核酸を改変する工程、
(b)宿主細胞に当該核酸を導入し発現するように培養する工程、
(c)宿主細胞培養物から当該ポリペプチドを回収する工程。
TS1~TS67の改変部位については上述の通りである。
本発明は、本発明のポリペプチド又はFc融合タンパク質分子を含有する医薬組成物を提供する。
本発明の医薬組成物は、本発明の上記ポリペプチド又はFc融合タンパク質分子に加えて医薬的に許容し得る担体を導入し、公知の方法で製剤化することが可能である。例えば、水もしくはそれ以外の薬学的に許容し得る液との無菌性溶液、又は懸濁液剤の注射剤の形で非経口的に使用できる。例えば、薬理学上許容される担体もしくは媒体、具体的には、滅菌水や生理食塩水、植物油、乳化剤、懸濁剤、界面活性剤、安定剤、香味剤、賦形剤、ベヒクル、防腐剤、結合剤などと適宜組み合わせて、一般に認められた製薬実施に要求される単位用量形態で混和することによって製剤化することが考えられる。具体的には、軽質無水ケイ酸、乳糖、結晶セルロース、マンニトール、デンプン、カルメロースカルシウム、カルメロースナトリウム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルアセタールジエチルアミノアセテート、ポリビニルピロリドン、ゼラチン、中鎖脂肪酸トリグリセライド、ポリオキシエチレン硬化ヒマシ油60、白糖、カルボキシメチルセルロース、コーンスターチ、無機塩類等を担体として挙げることができる。これら製剤における有効成分量は指示された範囲の適当な容量が得られるようにするものである。
アラニン:Ala:A
アルギニン:Arg:R
アスパラギン:Asn:N
アスパラギン酸:Asp:D
システイン:Cys:C
グルタミン:Gln:Q
グルタミン酸:Glu:E
グリシン:Gly:G
ヒスチジン:His:H
イソロイシン:Ile:I
ロイシン:Leu:L
リジン:Lys:K
メチオニン:Met:M
フェニルアラニン:Phe:F
プロリン:Pro:P
セリン:Ser:S
スレオニン:Thr:T
トリプトファン:Trp:W
チロシン:Tyr:Y
バリン:Val:V
一般的に、βシート構造はアミノ酸改変による構造変化が生じやすいこと、改変による熱安定性の低下が生じやすいことが知られている(Biochemistry 1994;33:5510-5517、Nature 1994;367:660-663)。そこで、本発明ではFc領域のループ部分を改変することによる熱安定性の向上を試みた。
変異を導入していないB3(GpH7-B3/GpL16-k0:GpH7(配列番号:17)、B3(配列番号:16)、GpL16(配列番号:18)、k0(配列番号:19))のTmは約68℃であり、この値よりも高いTmを示した変異体を表1-1および表1-2にまとめた。
実施例1のデータと構造情報(Nature 2000 ; 406 : 267-273)に基づいて熱安定性の向上する効果が特に優れていると考えられる改変を幾つか選択した。選択した単独の改変、または複数の改変を、Anti-GPC3抗体(GpH7-G1d/GpL16-k0:GpH7(配列番号:17)、G1d(配列番号:15)、GpL16(配列番号:18)、k0(配列番号:19)、Anti-IL6R抗体(MH0-G1d/ML0-k0:GpH7(配列番号:17)、G1d(配列番号:15)、ML0(配列番号:21)、k0(配列番号:19)のH鎖に対して導入し、表2に示したTS1~TS19(配列番号:26~44)を作製した。同時に、ジスルフィド結合導入によるIgGの形での熱安定性改善効果を評価するため、先行論文において報告されているm01ならびにm02の改変(J Biol Chem. 2009;284:14203-14210)をanti-IL6RのH鎖に導入し、TSm01ならびにTSm02の改変体を作製した(配列番号:24、25)。各抗体の発現と精製は参考例1で記した方法で実施した。
抗体のFc領域の改変には、エフェクター機能であるADCC活性などの機能を増強させたFc領域、またはエフェクター機能であるADCC活性などの機能を低下させたFc領域が報告されている(参考文献:current opinion,2009,20,685-691)。エフェクター機能を増強させた抗体は主にがん抗体に、エフェクター機能を低下させた抗体は中和抗体、Enbrel、OrencisなどのReceptor-Fc fusionなどに有用だと考えられ、それぞれの目的において使い分けることが重要である。
安定性向上を目的とした改変を導入するにあたり、エフェクター機能が重要であるがん抗体においては、hFcgRsに対する結合を維持していることは重要である。実施例3においてhFcgRsへの結合を維持しているTS1~TS8の改変を組み合わせ、表5に示したTS20~TS27の改変体を新たに作製した(配列番号:45~52)。各抗体の発現と精製は参考例1で記した方法で実施した。調製した抗体は参考例2に示した方法でTmを評価し、結果を表6に示した。
安定性向上を目的とした改変を導入するにあたり、中和抗体においてエフェクター機能はできる限り低下していたほうが望ましい。実施例3においてhFcgRsへの結合が低下したTS9~TS19の改変を組み合わせ、表8に示したTS28~TS43の改変体を新たに作製した(配列番号:53~68)。各抗体の発現と精製は参考例1で記した方法で実施した。調製した抗体は参考例2に示した方法でTmを評価し、結果を表9に示した。
会合体は保存安定性や免疫原性に影響を与えるため、熱安定性を向上させる改変は、できる限り会合体含有量を増加させないことが望ましい。そこで、複数改変を組み合わせたTS20~TS43、ならびに、TSm01とTSm02の会合体含有量を参考例4に示した方法で評価した。測定のクロマトグラムは図2に、会合体含有量は表11に示した。
CH2ドメインに変異を導入することにより、Tmが向上することが上記より明らかとなった。そこで、実施例1において改変導入の検討を実施していない領域に関しても、Tmが向上する変異があるかを検討し、表12に示したTS44~TS67の改変体を新たに作製した(配列番号:69~92)。各抗体の発現と精製は参考例1に記した方法で実施し、結果を表13に示した。さらに、hFcgRsに対する結合を参考例3に記した方法で測定した。G1の各hFcgRsに対する結合を100とした場合の各抗体の各hFcgRsに対する結合を算出して表14に示した。
アミノ酸置換の導入はQuikChange Site-Directed Mutagenesis Kit(Stratagene)、PCR、等またはIn fusion Advantage PCR cloning kit (TAKARA)等を用いて当業者公知の方法で行い、発現ベクターを構築した。得られた発現ベクターの塩基配列は当業者公知の方法で決定した。作製したプラスミドをヒト胎児腎癌細胞由来HEK293H株(Invitrogen)、またはFreeStyle293細胞(Invitrogen社)に、一過性に導入し、抗体の発現を行った。得られた培養上清から、rProtein A SepharoseTM Fast Flow(GEヘルスケア)を用いて当業者公知の方法で、抗体を精製した。精製抗体濃度は、分光光度計を用いて280 nmでの吸光度を測定し、得られた値からPACE法により算出された吸光係数を用いて抗体濃度を算出した(Protein Science 1995 ; 4 : 2411-2423)。
本検討では、Rotor-Gene Q(QIAGEN)を用いた示査走査型蛍光定量法を用いて抗体の熱変性中点(Tm)を測定することにより熱安定性を評価した。なお、本手法は、抗体の熱安定性評価法として広く知られている示査走査型熱量計を用いたTm評価と良好な相関を示すことが既に報告されている(Journal of Pharmaceutical Science 2010 ; 4 : 1707-1720)。
データはRotor-Gene Q Series Software(QIAGEN)を用いて蛍光遷移が認められた温度を算出し、この値をTmとした。
Biacore T100(GE Healthcare)を用いて、抗体とhFcgRs(hFcgRIa, hFcgRIIa(R), hFcgRIIa(H), hFcgRIIb, hFcgRIIIa(F), hFcgRIIIa(V))との相互作用解析を行った。ランニングバッファーにはHBS-EP+ (GE Healthcare)を用い、測定温度は25℃とした。アミンカップリング法によりProtein A (Invitrogen) を固定化し、そこへ目的の抗体をキャプチャーさせた。抗体をキャプチャーさせたところへ、ランニングバッファーで希釈したhFcγRを流速30μL/minでhFcgRIaは5分間、それ以外のhFcγRは流速5μL/minで1分間相互作用させ、抗体に対する結合量を測定し、抗体間で比較した。ただし、hFcγRの結合量はキャプチャーした抗体の量に依存するため、各抗体のキャプチャー量がhFcgRIaは200 RU (resonance unit)、それ以外のhFcgRsは1000 RU (resonance unit)となるように、FcgRの結合量を補正した。また、10 mM glycine-HCl、pH1.5を流速30μL/minで30秒間反応させることで、チップにキャプチャーした抗体を洗浄し、チップを再生して繰り返し用いた。
Alliance system(Waters)を用いたSEC分析により、精製抗体中の会合体含有量を評価した。移動相には300 mMの塩化ナトリウムを含む50 mMリン酸緩衝液, pH7.0(伊勢久)を、分析カラムにはG3000SWXL(TOSOH)を用い、215 nmの波長で測定を行った。Empower2(Waters)を用いてデータ解析を実施し、単量体よりも高分子量側に溶出した成分を一括して会合体としてその含有量を算出した。
Claims (62)
- 抗体のFc領域を有するポリペプチドであって、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチド。
- 前記安定性が熱変性中点(Tm)を指標として評価あるいは判断される、請求項1に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング236番目、EUナンバリング237番目、EUナンバリング238番目、EUナンバリング239番目、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング265番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング271番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、EUナンバリング327番目、EUナンバリング329番目、EUナンバリング330番目、EUナンバリング333番目、EUナンバリング335番目、EUナンバリング337番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1または2に記載のポリペプチド。
- さらに、親ポリペプチドと比較してFcγRへの結合活性が維持又は増強した、請求項1~3のいずれか一項に記載のポリペプチド。
- さらに、親ポリペプチドと比較してFcγRへの結合活性が減少した、請求項1~3のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング266番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~4、6のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のIleへの置換、EUナンバリング266番目のアミノ酸のIleへの置換、EUナンバリング268番目のアミノ酸のGlnへの置換、EUナンバリング269番目のアミノ酸のAspへの置換、EUナンバリング270番目のアミノ酸のGluへの置換、EUナンバリング295番目のアミノ酸のMet又はLeuへの置換、EUナンバリング300番目のアミノ酸のGluへの置換、EUナンバリング324番目のアミノ酸のHisへの置換、EUナンバリング326番目のアミノ酸のSer又はAlaへの置換、及びEUナンバリング330番目のアミノ酸のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~4、6、7のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5、6のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLys又はArgへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング267番目のアミノ酸のProへの置換、EUナンバリング268番目のアミノ酸のMet又はLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のGly、His又はMetのいずれかへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、6、9のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング295番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~4のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング295番目のアミノ酸のMet又はLeuへの置換、EUナンバリング326番目のアミノ酸のSer又はAlaへの置換、及びEUナンバリング330番目のアミノ酸のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~4、11のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLys又はSerへの置換、EUナンバリング268番目のアミノ酸のLys又はHisへの置換、EUナンバリング270番目のアミノ酸のPhe又はAspへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、13のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸配列において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、15のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸配列において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、17のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、19のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変部位が、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、21のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~4のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング247番目のアミノ酸のValへの置換、EUナンバリング250番目のアミノ酸のPhe、Ile、Met、Val、Trp、又はTyrのいずれかへの置換、EUナンバリング307番目のアミノ酸のAla、Gln、又はProのいずれかへの置換、EUナンバリング309番目のアミノ酸のAla、Arg、又はProのいずれかへの置換、EUナンバリング315番目のアミノ酸のAlaへの置換、EUナンバリング360番目のアミノ酸のHisへの置換、EUナンバリング385番目のアミノ酸のAspへの置換、EUナンバリング386番目のアミノ酸のProへの置換、EUナンバリング387番目のアミノ酸のGluへの置換、EUナンバリング389番目のアミノ酸のSerへの置換、EUナンバリング428番目のアミノ酸のHis、Trp、Tyr、またはPheのいずれかへの置換、及びEUナンバリング433番目のアミノ酸のLysへの置換からなる群より選択される少なくも一つ以上のアミノ酸改変である、請求項1~4、23のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位における、EUナンバリング298番目又はEUナンバリング309番目のアミノ酸部位において、少なくとも一つ以上のアミノ酸変異が導入されている、請求項1~3、5のいずれか一項に記載のポリペプチド。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング309番目のアミノ酸のAspへの置換からなる群より選択される、少なくとも一つ以上のアミノ酸改変である、請求項1~3、5、25のいずれか一項に記載のポリペプチド。
- 抗体のFc領域を有するポリペプチドにおいて、該Fc領域のループ部位に少なくとも一つのアミノ酸改変を加えることにより、親ポリペプチドと比較して安定性を向上させる方法。
- 前記安定性を熱変性中点(Tm)を指標として評価あるいは判断する、請求項27に記載の方法。
- 以下の工程を含む、抗体のFc領域を有し、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドの製造方法;
(a)抗体のFc領域を有するポリペプチドにおいて、該Fc領域のループ部位に少なくとも一つのアミノ酸改変を加える工程、
(b)前記工程(a)で改変されたポリペプチドの安定性を測定する工程、および
(c)親ポリペプチドと比較して、安定性が向上したポリペプチドを選択する工程。 - 以下の工程を含む、抗体のFc領域を有し、該Fc領域のループ部位における少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドの製造方法;
(a)親ポリペプチドと比較して、安定性が向上するように、当該ポリペプチドをコードする核酸を改変する工程、
(b)宿主細胞に当該改変された核酸を導入し発現するように培養する工程、
(c)宿主細胞培養物から当該ポリペプチドを回収する工程。 - 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング236番目、EUナンバリング237番目、EUナンバリング238番目、EUナンバリング239番目、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング265番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング271番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、EUナンバリング327番目、EUナンバリング329番目、EUナンバリング330番目、EUナンバリング333番目、EUナンバリング335番目、EUナンバリング337番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~30のいずれか一項に記載の方法。
- さらに親ポリペプチドと比較してFcγRへの結合活性を維持又は増強するように改変する工程を含む、請求項27~31のいずれか一項に記載の方法。
- さらに、親ポリペプチドと比較してFcγRへの結合活性が減少するように改変する工程を含む、請求項27~31のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング266番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング325番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング266番目、EUナンバリング268番目、EUナンバリング269番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング300番目、EUナンバリング324番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~32、34のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のIleへの置換、EUナンバリング266番目のアミノ酸のIleへの置換、EUナンバリング268番目のアミノ酸のGlnへの置換、EUナンバリング269番目のアミノ酸のAspへの置換、EUナンバリング270番目のアミノ酸のGluへの置換、EUナンバリング295番目のアミノ酸のMet又はLeuへの置換、EUナンバリング300番目のアミノ酸のGluへの置換、EUナンバリング324番目のアミノ酸のHisへの置換、EUナンバリング326番目のアミノ酸のSer又はAlaへの置換、及びEUナンバリング330番目のアミノ酸のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~32、34、35のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング267番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33、34のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLys又はArgへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング267番目のアミノ酸のProへの置換、EUナンバリング268番目のアミノ酸のMet又はLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のGly、His又はMetのいずれかへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、34、37のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング295番目、EUナンバリング326番目、及びEUナンバリング330番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~32のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング295番目のMet又はLeuへの置換、EUナンバリング326番目のSer又はAlaへの置換、及びEUナンバリング330番目のHis又はTyrへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~32、39のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLys又はSerへの置換、EUナンバリング268番目のアミノ酸のLys又はHisへの置換、EUナンバリング270番目のアミノ酸のPhe又はAspへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、41のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、43のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング239番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング239番目のアミノ酸のLysへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、45のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング268番目、EUナンバリング270番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング268番目のアミノ酸のLysへの置換、EUナンバリング270番目のアミノ酸のPheへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、47のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング234番目、EUナンバリング235番目、EUナンバリング295番目、EUナンバリング296番目、EUナンバリング298番目、及びEUナンバリング325番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング234番目のアミノ酸のLysへの置換、EUナンバリング235番目のアミノ酸のLys又はArgへの置換、EUナンバリング295番目のアミノ酸のMetへの置換、EUナンバリング296番目のアミノ酸のGlyへの置換、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング325番目のアミノ酸のHis又はGlyへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、49のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング247番目、EUナンバリング250番目、EUナンバリング307番目、EUナンバリング309番目、EUナンバリング315番目、EUナンバリング360番目、EUナンバリング385番目、EUナンバリング386番目、EUナンバリング387番目、EUナンバリング389番目、EUナンバリング428番目、及びEUナンバリング433番目からなる群より選択されるアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~32のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング247番目のアミノ酸のValへの置換、EUナンバリング250番目のアミノ酸のPhe、Ile、Met、Val、Trp、又はTyrのいずれかへの置換、307番目のアミノ酸のAla、Gln、又はProのいずれかへの置換、309番目のアミノ酸のAla、Arg、又はProのいずれかへの置換、315番目のアミノ酸のAlaへの置換、360番目のアミノ酸のHisへの置換、385番目のアミノ酸のAspへの置換、386番目のアミノ酸のProへの置換、387番目のアミノ酸のGluへの置換、389番目のアミノ酸のSerへの置換、428番目のアミノ酸のHis、Trp、Tyr、またはPheのいずれかへの置換、及び433番目のアミノ酸のLysへの置換からなる群より選択される少なくとも一つ以上のアミノ酸改変である、請求項27~32、51のいずれか一項に記載の方法。
- 前記Fc領域のループ部位における、EUナンバリング298番目又はEUナンバリング309番目のアミノ酸部位において、少なくとも一つ以上のアミノ酸変異を導入する、請求項27~31、33のいずれか一項に記載の方法。
- 前記Fc領域のループ部位におけるアミノ酸改変が、EUナンバリング298番目のアミノ酸のGlyへの置換、及びEUナンバリング309番目のアミノ酸のAspへの置換からなる群より選択される、少なくとも一つ以上のアミノ酸改変である、請求項27~31、33、53のいずれか一項に記載の方法。
- 前記改変が、ヒトIgGのFc領域を有するポリペプチドにおける改変である、請求項27~54のいずれか一項に記載の方法。
- 抗体のFc領域を有するポリペプチドであって、Fc領域のループ部位の少なくとも一つのアミノ酸が改変され、親ポリペプチドと比較して安定性が向上したポリペプチドをコードする核酸。
- 請求項56に記載の核酸を含むベクター。
- 請求項57に記載のベクターにより形質転換された宿主細胞。
- 請求項1~26のいずれかに記載のポリペプチド、または請求項27~55のいずれかに記載の方法により作製されたポリペプチドを含有する医薬組成物。
- 請求項59に記載の医薬組成物を含有する免疫炎症性疾患または癌の治療剤又は予防剤。
- 免疫炎症性疾患が関節リウマチ、自己免疫性肝炎、自己免疫性甲状腺炎、自己免疫性水疱症、自己免疫性副腎皮質炎、自己免疫性溶血性貧血、自己免疫性血小板減少性紫斑病、巨赤血球性貧血、自己免疫性萎縮性胃炎、自己免疫性好中球減少症、自己免疫性精巣炎、自己免疫性脳脊髄炎、自己免疫性レセプター病、自己免疫不妊、慢性活動型肝炎、糸球体腎炎、間質性肺腺維症、多発性硬化症、パジュット症、オステオポローシス、多発性骨髄腫、ブドウ膜炎、急性及び慢性脊椎炎、痛風性関節炎、炎症性腸疾患、成人呼吸促進症候群(ARDS)、乾癬、クローン病、バセドウ病、若年性糖尿病、アジソン病、重症筋無力症、水晶体性ブドウ膜炎、全身性エリテマトーデス、アレルギー性鼻炎、アレルギー性皮膚炎、潰瘍性大腸炎、過敏症、ぜん息、筋肉変性、悪液質、全身性強皮症、限局性強皮症、シェーグレン症候群、ベーチェット病、ライター症候群、I型及びII型糖尿病、骨吸収疾患、移植片vs.宿主反応、虚血性再灌流外傷、アテローム硬化症、脳トラウマ、多発生硬化症、大脳マラリア、敗血症、敗血性ショック、トキシックショック症候群、発熱、及び染色によるマルギアス(malgias)、再生不良性貧血、溶血性貧血、突発性血小板減少症、グッドパスチャー症候群、ギラン・バレー症候群、橋本病、天疱瘡、IgA腎症、花粉症、抗リン脂質抗体症候群、多発性筋炎、ウェゲナー肉腫、結節性動脈炎、混合性結合組織病、または線維筋痛症である、請求項60に記載の治療剤又は予防剤。
- 癌が膵臓癌、前立腺癌、乳癌、皮膚癌、消化管の癌、肺癌、肝細胞癌、子宮頸癌、子宮体癌、卵巣癌、卵管癌、膣癌、肝臓癌、胆管癌、膀胱癌、尿管の癌、甲状腺癌、副腎癌、腎臓癌、その他の腺組織の癌、脂肪肉腫、平滑筋肉腫、黄紋筋肉腫、滑膜肉腫、血管肉腫、線維肉腫、悪性末梢神経腫瘍、消化管間質系腫瘍、類腱腫、ユーイング肉腫、骨肉腫、軟骨肉腫、白血病、リンパ腫、骨髄腫、又はその他の実質臓器の腫瘍である、請求項60に記載の治療剤または予防剤。
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2013
- 2013-02-08 SG SG11201404751UA patent/SG11201404751UA/en unknown
- 2013-02-08 EP EP13746387.3A patent/EP2813568A4/en active Pending
- 2013-02-08 WO PCT/JP2013/053011 patent/WO2013118858A1/ja active Application Filing
- 2013-02-08 CN CN202110769343.4A patent/CN113480639A/zh active Pending
- 2013-02-08 SG SG10201704849PA patent/SG10201704849PA/en unknown
- 2013-02-08 JP JP2013557590A patent/JP6226752B2/ja active Active
- 2013-02-08 CN CN201380018995.8A patent/CN104204204A/zh active Pending
- 2013-02-08 CN CN202110768927.XA patent/CN113527469A/zh active Pending
- 2013-02-08 US US14/377,556 patent/US20150210763A1/en not_active Abandoned
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2015
- 2015-02-24 HK HK15101863.5A patent/HK1201296A1/xx unknown
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2017
- 2017-10-10 JP JP2017196496A patent/JP6820242B2/ja active Active
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2020
- 2020-02-20 US US16/795,676 patent/US20200181257A1/en active Pending
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2021
- 2021-01-04 JP JP2021000023A patent/JP7289325B2/ja active Active
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JP2021052797A (ja) | 2021-04-08 |
CN113527469A (zh) | 2021-10-22 |
EP2813568A1 (en) | 2014-12-17 |
US20150210763A1 (en) | 2015-07-30 |
SG10201704849PA (en) | 2017-07-28 |
CN104204204A (zh) | 2014-12-10 |
JP6226752B2 (ja) | 2017-11-08 |
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JP7289325B2 (ja) | 2023-06-09 |
JP2018050622A (ja) | 2018-04-05 |
JP2023101651A (ja) | 2023-07-21 |
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CN113480639A (zh) | 2021-10-08 |
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