WO2022233320A1 - 与Fc受体结合改变的Fc突变体 - Google Patents

与Fc受体结合改变的Fc突变体 Download PDF

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WO2022233320A1
WO2022233320A1 PCT/CN2022/091187 CN2022091187W WO2022233320A1 WO 2022233320 A1 WO2022233320 A1 WO 2022233320A1 CN 2022091187 W CN2022091187 W CN 2022091187W WO 2022233320 A1 WO2022233320 A1 WO 2022233320A1
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PCT/CN2022/091187
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English (en)
French (fr)
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付凤根
周帅祥
吴志海
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信达生物制药(苏州)有限公司
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Priority to CA3218187A priority Critical patent/CA3218187A1/en
Priority to JP2023568549A priority patent/JP2024516320A/ja
Priority to US18/558,655 priority patent/US20240228579A1/en
Priority to AU2022269145A priority patent/AU2022269145A1/en
Priority to EP22798660.1A priority patent/EP4335868A1/en
Priority to CN202280033488.0A priority patent/CN117295760A/zh
Publication of WO2022233320A1 publication Critical patent/WO2022233320A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention generally relates to the fields of immunology and antibody engineering, and in particular, the present invention relates to variants of IgG immunoglobulins, methods for their preparation and uses thereof.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CDC complement-dependent cytotoxicity
  • ADCC ADCC
  • ADCP ADCP
  • CDC effector functions are often required, This is especially true for immune checkpoint inhibitors that require low depletion of target cells because antibodies targeting cell surface antigens can induce unwanted immune stimulation of immune cells and associated effector functions and complement activation, with adverse consequences. Antibodies are especially important.
  • IgG1 Human IgG subclasses (IgG1, IgG2, IgG3 and IgG4) have different immune functions. For example, IgG antibodies of different subclasses have different ADCC activities. Compared with other subclasses, IgG1 and IgG3 have stronger ADCC activities; IgG1 , IgG2, IgG3 and IgG4 have antibody-dependent cellular phagocytosis (ADCP).
  • ADCP antibody-dependent cellular phagocytosis
  • partial amino acid mutations in the Fc region may have the effect of reducing or eliminating ADCC and/or ADCP activity, and reducing or eliminating CDC activity.
  • mutations that can completely eliminate the ADCC and CDC effects are relatively rare.
  • the wild-type Fc amino acid sequence is directionally modified to obtain a completely eliminated ADCC and CDC effects. Fc mutant molecules.
  • the present invention provides mutant molecules of modified immunoglobulin constant regions (Fc regions) that can be used to engineer antibodies or antibody-based therapeutics.
  • Fc regions modified immunoglobulin constant regions
  • antibodies, antibody-based therapeutics, and other molecules comprising the mutated Fc region that comprise the mutated Fc region of the present application can be compared with molecules comprising the wild-type Fc region. Binding of FcyR or C1q is greatly reduced, thereby significantly reducing undesired ADCC and/or ADCP and/or CDC effector function in vivo. Further, the Fc mutation disclosed in the present application does not affect the binding ability of antibodies, antibody therapeutics and other molecules comprising the mutant Fc region to FcRn, and thus does not affect the half-life of the corresponding molecules.
  • the present application provides Fc region polypeptide molecules carrying specific mutations, having reduced or eliminated ADCC and/or ADCP and/or CDC effector function, but still retaining the ability to bind FcRn, comprising the mutant Fc regions described above antibody molecules or similar structural molecules.
  • the present application provides Fc mutants with different modifications, the Fc mutants exhibit reduced affinity for Fc receptors and/or C1q compared to the wild-type Fc region, thereby reducing or eliminating the Fc mutants Induced ADCC, CDC and ADCP effector functions.
  • the ADCC, CDC and ADCP effector functions induced by the Fc mutant are reduced to at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or complete elimination.
  • the Fc mutants provided herein comprise one or more amino acid deletions.
  • the Fc mutants provided herein comprise a deletion of amino acid 329 ( ⁇ 329) according to the EU index numbering in Kabat.
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by a deletion at position 329 ( ⁇ 329) according to the EU index numbering in Kabat.
  • the Fc mutants provided herein comprise deletions of amino acids 329 and 330 according to the EU index numbering in Kabat ( ⁇ 329 and ⁇ 330).
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by the deletion of amino acids 329 and 330 according to the EU index numbering in Kabat ( ⁇ 329 and ⁇ 330).
  • the Fc mutants provided herein comprise one or more amino acid substitutions.
  • the Fc mutants provided by the present application comprise substitutions of amino acids at positions 234 and/or 235 according to the EU index numbering in Kabat, wherein A, V, L, and I are used to replace position 234, respectively L, V, F and/or L at position 235.
  • the Fc mutant comprises L234A+L235A, V234A or F234A+L235A.
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by L234A+L235A, V234A or F234A+L235A.
  • the Fc mutants provided herein comprise one or more amino acid deletions and one or more amino acid substitutions.
  • the Fc mutants provided herein comprise a deletion of amino acid 329 and a substitution of amino acid 330 according to the EU index numbering in Kabat, wherein the substitution at position 330 is made with G, D or Q.
  • the Fc mutant comprises A329+A330G or A329+S330G.
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by A329+A330G or A329+S330G.
  • the Fc mutant provided herein comprises a deletion of amino acid 329 and a substitution of amino acid 234 and 235 according to the EU index numbering in Kabat, wherein A, V, L, I are employed Substitute L, V, F at position 234 and/or L at position 235, respectively.
  • the Fc mutant comprises L234A+L235A+ ⁇ 329, V234A+ ⁇ 329 or F234A+L235A+ ⁇ 329.
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by L234A+L235A+ ⁇ 329, V234A+ ⁇ 329, or F234A+L235A+ ⁇ 329.
  • the Fc mutants provided herein comprise deletions of amino acids 329 and 330 and substitutions of amino acids 234 and 235 according to the EU index numbering in Kabat, wherein A, V, L, I replace L, V, F at position 234 and/or L at position 235, respectively.
  • the Fc mutant comprises L234A+L235A+ ⁇ 329+ ⁇ 330, V234A+ ⁇ 329+ ⁇ 330 or F234A+L235A+ ⁇ 329+ ⁇ 330.
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by L234A+L235A+ ⁇ 329+ ⁇ 330, V234A+ ⁇ 329+ ⁇ 330, or F234A+L235A+ ⁇ 329+ ⁇ 330.
  • the Fc mutant provided by the application comprises the deletion of amino acid 329 and the substitution of amino acid 234, 235 and 330 according to the EU index numbering in Kabat, wherein A, V, L and I respectively replace L, V, F at position 234 and/or L at position 235, and use G, D or Q to replace position 330.
  • the Fc mutant comprises L234A+L235A+A330G+ ⁇ 329, L234A+L235A+S330G+ ⁇ 329, V234A+L235A+A330G+ ⁇ 329, V234A+L235A+S330G+ ⁇ 329, F234A+L235A+ S330G+ ⁇ 329 or F234A+L235A+A330G+ ⁇ 329.
  • the Fc mutants provided herein differ from the corresponding wild-type Fc by L234A+L235A+A330G+ ⁇ 329, L234A+L235A+S330G+ ⁇ 329, V234A+L235A+A330G+ ⁇ 329, V234A+L235A+ S330G+ ⁇ 329, F234A+L235A+S330G+ ⁇ 329 or F234A+L235A+A330G+ ⁇ 329.
  • the Fc mutants provided by the present invention are IgG1-type Fc mutants that have reduced or even abolished ability to bind Fc ⁇ R compared to the corresponding wild-type Fc region.
  • the Fc mutants provided by the present invention are IgG2, IgG3, IgG4 type Fc mutants, which have reduced or even abolished ability to bind Fc ⁇ R compared to the corresponding wild-type Fc region.
  • the Fc mutant provided by the present invention retains the ability to bind to FcRn.
  • the Fc mutants disclosed in the present application can be used as a platform building block to be applied in any scenario where ADCC/ADCP/CDC effector functions need to be reduced or even eliminated, for example, any type of ADCC/ADCP/CDC effector functions that are expected to be reduced or eliminated Among the antibody molecules, molecules with an antibody-like structure.
  • the Fc mutant is essentially based on IgG sequences, in a preferred embodiment the Fc mutant is essentially based on human IgG sequences, in another embodiment the Fc mutant is essentially based Based on the human IgG1 sequence.
  • the Fc mutant may also comprise other modifications, such as those known in the art to reduce immunogenicity, increase stability, solubility, function, and clinical benefit.
  • the Fc mutant comprises the following sequence:
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 2, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 3, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 5, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 7, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to EU index in SEQ ID NO: 11, or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 13, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 14, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 15, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • SEQ ID NO: 16 according to the sequence of amino acids 221-447 of the EU index, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 17, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • SEQ ID NO: 19 according to the sequence of amino acids 221-447 of the EU index, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 20, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 23, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 24, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 25, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence of amino acids 221-447 according to the EU index in SEQ ID NO: 27, or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • SEQ ID NO: 29 according to the sequence of amino acids 221-447 of the EU index, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • SEQ ID NO: 31 according to the sequence of amino acids 221-447 of the EU index, or have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, an amino acid sequence of 98%, 99%, or even higher identity, or consists of such a sequence, or
  • sequence according to the 221-447th amino acid of EU index in SEQ ID NO: 33 or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, Amino acid sequences of 98%, 99%, or even greater identity, or consist of such sequences.
  • the present invention provides a polypeptide comprising the Fc mutant of the first aspect, said polypeptide having reduced or eliminated ADCC and/or ADCP and/or CDC effects compared to a polypeptide comprising a wild-type Fc region Sub-function, preferably, the polypeptide does not cause ADCC and/or ADCP and/or CDC effects.
  • the polypeptide also has an extended half-life.
  • the ADCC, CDC and ADCP effector functions induced by the polypeptide comprising the Fc mutant of the first aspect are reduced to at least 80% of the ADCC, CDC and ADCP effector functions induced by the polypeptide comprising the corresponding wild-type Fc %, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1% or complete elimination.
  • the polypeptide is an antibody molecule, preferably, the antibody molecule is an IgG class antibody molecule.
  • the antibody molecule is a multispecific antibody (eg, a bispecific antibody), a humanized antibody, a chimeric antibody, an antibody fusion.
  • the ability of the antibody molecule comprising the Fc mutant obtained in the present application to interact with Fc ⁇ R is reduced or even eliminated, thus having reduced or eliminated ADCC and/or ADCP and/or CDC effector functions .
  • the polypeptide is a fusion protein comprising one or more fusion partners operably linked to the Fc mutant, which may generally be any protein or small molecule, such as any antibody variant regions, target binding regions of receptors, adhesion molecules, ligands, enzymes, cytokines, chemokines, or some other protein or protein domain.
  • the fusion protein is, for example, an immunoadhesin.
  • the application provides an IgG antibody comprising an Fc mutant comprising a deletion of amino acid 329 ( ⁇ 329) according to the EU index numbering in Kabat. In another specific embodiment, the application provides an IgG antibody comprising an Fc mutant comprising deletions ( ⁇ 329 and ⁇ 330) of amino acids 329 and 330 according to the EU index numbering in Kabat.
  • the application provides an IgG antibody comprising an Fc mutant comprising substitutions of amino acids at positions 234 and/or 235 according to the EU index numbering in Kabat, wherein A, V, L, I are used, respectively Substitute L, V, F at position 234 and/or L at position 235.
  • the IgG antibody comprises L234A+L235A, V234A or F234A+L235A.
  • the application provides an IgG antibody comprising an Fc mutant comprising a deletion of amino acid 329 and a substitution of amino acid 330 according to the EU index numbering in Kabat, wherein G, D or Q is used for position 330 bits are substituted.
  • the IgG antibody comprises A329+A330G or A329+S330G.
  • the application provides an IgG antibody comprising an Fc mutant comprising the deletion of amino acid 329 and the substitution of amino acid 234 and 235 according to the EU index numbering in Kabat, wherein A, V , L and I replace L, V, F at position 234 and/or L at position 235, respectively.
  • the IgG antibody comprises L234A+L235A+ ⁇ 329, V234A+ ⁇ 329 or F234A+L235A+ ⁇ 329.
  • the application provides an IgG antibody comprising an Fc mutant comprising deletions of amino acids 329 and 330 and substitutions of amino acids 234 and 235 according to the EU index numbering in Kabat, wherein A, V, L, I are used to replace L, V, F at position 234 and/or L at position 235, respectively.
  • the IgG antibody comprises L234A+L235A+ ⁇ 329+ ⁇ 330, V234A+ ⁇ 329+ ⁇ 330 or F234A+L235A+ ⁇ 329+ ⁇ 330.
  • the application provides an IgG antibody comprising an Fc mutant comprising a deletion of amino acid 329 and a substitution of amino acid 234, 235, 330 according to the EU index numbering in Kabat, wherein A , V, L and I respectively replace L, V, F and/or L at the 234th position, and G, D or Q are used to replace the 330th position.
  • the IgG antibody comprises L234A+L235A+A330G+ ⁇ 329, L234A+L235A+S330G+ ⁇ 329, V234A+L235A+A330G+ ⁇ 329, V234A+L235A+S330G+ ⁇ 329, F234A+L235A+S330G + ⁇ 329 or F234A+L235A+A330G+ ⁇ 329.
  • the IgG antibodies provided herein are of the IgG1 type, which have reduced or even abolished ability to bind Fc[gamma]Rs compared to corresponding wild-type antibodies.
  • the IgG antibodies provided by the present invention are IgG2, IgG3, IgG4 type antibodies, which have reduced or even eliminated ability to bind Fc ⁇ Rs compared to the corresponding wild-type antibodies.
  • the antibodies provided by the present invention retain the ability to bind to FcRn.
  • the antibody molecule is an IgG antibody against claudin18.2.
  • the IgG antibody comprising the Fc mutant provided by the present invention comprises the following heavy and light chains:
  • polypeptides (eg antibodies) comprising the Fc mutants provided herein may further comprise other modifications in the Fc region, such as known in the art for reducing the immunogens of polypeptides (eg antibodies) Other modifications to improve stability, solubility, function, and clinical benefit.
  • the antibody comprising the Fc mutant provided herein has a heavy chain variable region and a light chain variable region known in the art for different target antigens.
  • One of skill in the art can readily graft the heavy and light chain variable regions known in the art to the Fc mutants disclosed herein to obtain ADCC with desired properties such as reduced or eliminated ADCC and/or ADCP and/or CDC effector function).
  • the present invention provides a pharmaceutical composition comprising the polypeptide described in the second aspect and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises an antibody molecule carrying the Fc mutant of the present invention.
  • the pharmaceutical composition comprises an IgG antibody carrying the Fc mutant of the present invention.
  • the pharmaceutical composition comprises an IgG1-type antibody, an IgG2-type antibody, an IgG3-type antibody or an IgG4-type antibody carrying the Fc mutant of the present invention.
  • the present invention provides methods for reducing or eliminating ADCC/ADCP/CDC effector functions of an antibody, while retaining or even increasing half-life, according to the Fc region modifications described herein.
  • the ADCC/ADCP/CDC effect of the antibody is reduced or eliminated by making one or more modifications as disclosed herein to the Fc region of the antibody that is desired to reduce or eliminate ADCC/ADCP/CDC effector function Subfunction.
  • the present invention provides the use of the Fc mutant in the preparation of a medicine.
  • the medicament is used for immunotherapy, adjunctive immunotherapy.
  • the drug has reduced or eliminated ADCC/ADCP/CDC effector function.
  • the drug is a fusion protein comprising an Fc mutant of the present application, eg, an IL-2 Fc fusion protein.
  • the drug is an antibody targeting an immune cell surface molecule comprising an Fc mutant of the present application, which has reduced or eliminated ADCC/ADCP/CDC effector function.
  • the medicament is used to treat a tumor in a subject, eg, in one embodiment, the medicament activates the patient's immune cells without activating ADCC/ADCP/CDC effects and prolonging the half-life , so as to achieve anti-tumor effect.
  • the present invention provides a method for treating a disease in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition for the corresponding disease comprising the Fc mutant described in the present application.
  • the disease is a disease requiring immunotherapy or immune adjuvant therapy.
  • the present invention provides a method of treating a disease in a subject comprising administering to the subject an effective amount of an antibody directed against an antigen on a corresponding immune cell comprising an Fc mutant described herein.
  • the present invention provides a kit comprising the Fc mutant disclosed in the present application, a polypeptide comprising the Fc mutant, or an immunoglobulin molecule comprising the Fc mutant.
  • the present invention provides a detection kit, which can not only improve the stability of the fusion protein by fusing functional molecules (such as enzymes, antigens, receptors, ligands, cytokines, etc.) with Fc, but also It can also be applied to non-clinical fields such as flow cytometry, immunohistochemistry, in vitro activity detection and protein microarray detection.
  • the antibody molecule carrying the specific Fc mutation provided in this application has reduced or eliminated ADCC, ADCP and CDC effector functions, but still retains the ability to bind FcRn, so it can be used as an improved antibody mutant, which not only improves the therapeutic effect of the antibody, The safety, stability and low immunogenicity of the antibody are also ensured. It was surprisingly found that deletion of the proline residue at position 329 of the Fc region significantly reduces the binding of the Fc region to the receptors FcyRIII, FcyRII, FcyRI, C1q, thereby significantly reducing or eliminating ADCC, ADCP and CDC activities.
  • a combined mutation of Pro329 of the Fc region with, for example, one or more selected from the group consisting of A330 deletion, A330G, L234A and L235A results in significantly reduced binding to receptors Fc ⁇ RIII, Fc ⁇ RII, Fc ⁇ RI, C1q, and thereby significantly reduced or eliminated ADCC, ADCP and CDC activities.
  • Figure 1 Crystal structures of human IgGl Fc and human CD16A (PDB: 3SGJ).
  • Figure 2 shows the binding of Fc mutants of the present invention to various Fc ⁇ Rs
  • Figure 2a Binding curves of Fc mutants to CD16A (F176) (pH 7.4)
  • Figure 2b Binding of Fc mutants to CD16A (V176) Binding curves (pH 7.4)
  • Figure 2c Binding curves of Fc mutants to CD16B (NA1) (pH 7.4)
  • Figure 2d Binding curves of Fc mutants to CD16B (NA2) (pH 7.4)
  • Figure 2e Fc mutations Binding curve of Fc mutant to CD32A (H167) (pH 7.4)
  • Figure 2f Binding curve of Fc mutant to CD32A (R167) (pH 7.4)
  • Figure 2g Binding curve of Fc mutant to CD32B (pH 7.4)
  • 2h Binding curve of Fc mutant to CD64 (pH 7.4)
  • Figure 2i Binding curve of Fc mutant to FcRn at pH 6.0
  • Figure 3 shows the binding of Fc mutants to various Fc ⁇ Rs based on TLC assays with 200 nM of antibody in solution.
  • Figure 3a Binding curve of Fc mutant to CD16A (V176) (pH 7.4)
  • Figure 3b Binding curve of Fc mutant to CD16A (F176) (pH 7.4)
  • Figure 3c Binding curve of Fc mutant to CD16B (NA1) Binding curves (pH 7.4)
  • Figure 3d Binding curves of Fc mutants to CD16B (NA2) (pH 7.4)
  • Figure 3e Binding curves of Fc mutants to CD32A (H167) (pH 7.4)
  • Figure 3f Fc mutations Binding curves of Fc mutants to CD32A (R167) (pH 7.4)
  • Figure 3g Binding curves of Fc mutants to CD32B (pH 7.4)
  • Figure 3h Binding curves of Fc mutants to CD64 (pH 7.4)
  • Figure 4 shows ADCC activity of antibodies.
  • antibody is used herein in the broadest sense and encompasses a variety of antibody structures including, but not limited to, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (eg, , bispecific antibodies), single chain antibodies, whole antibodies or antibody fragments thereof exhibiting the desired antigen-binding activity.
  • An intact antibody will generally contain at least two full-length heavy chains and two full-length light chains, but in some cases may contain fewer chains, eg, antibodies naturally occurring in camels may contain only heavy chains.
  • binding and “specific binding” mean that the binding of the antibody is selective for the antigen and can be distinguished from unwanted or nonspecific interactions.
  • the ability of an antibody to bind to a specific antigen can be determined by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) or optical interferometry of biofilm layers (ForteBio) or other conventional binding assays known in the art.
  • ELISA enzyme-linked immunosorbent assay
  • SPR surface plasmon resonance
  • FormeBio optical interferometry of biofilm layers
  • antibodies with a KD of about 1x10-7 or less, a KD of about 1x10-8 or less, a KD of about 1x10-9 or less, a KD of about 1x10-10 or A lower KD, about 1 x 10-11 or lower KD binds to BCMA or CD3, then the antibody is an antibody that "specifically binds to BCMA or CD3."
  • antibodies that specifically bind human BCMA or CD3 may be cross-reactive with BCMA or CD3 proteins from other species.
  • antibodies specific for human BCMA or CD3, in some embodiments, can cross-react with cynomolgus BCMA or CD3.
  • Methods for determining cross-reactivity include those described in the Examples as well as standard assays known in the art, eg, by using bio-light interference, or flow cytometry techniques.
  • variable domain residue numbering according to Kabat refers to the variable domain or light chain for an antibody heavy chain according to Kabat et al. Numbering system for variable domain editing (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).
  • the Kabat numbering system is generally applied to residues in the variable domains of antibodies (approximately light chain residues 1-107 and heavy chain residues 1-113).
  • EU numbering system As in Kabat or “EU index” generally apply to residues in the constant region of an immunoglobulin heavy chain (see eg Kabat et al, supra). Unless otherwise indicated herein, residue numbering in antibody variable domains herein is according to the Kabat numbering system; residue numbering in antibody constant domains is according to the EU numbering system.
  • Antibody effector function refers to the biological activity attributable to an antibody Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region) and which varies with antibody isotype.
  • antibody effector functions include: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), cell surface receptors such as B cells receptor) downregulation, B cell activation, etc.
  • effector cell refers to a cell that expresses one or more FcRs and performs effector function, such as a cell that expresses Fc ⁇ RIIIA and performs ADCC effector function, in one embodiment, a cell that mediates ADCC function, such as NK cells, Peripheral blood mononuclear cells, monocytes, cytotoxic T cells, neutrophils. Effector cells can be derived from the natural environment, such as blood.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • NK natural killer cells
  • macrophages, neutrophils, and eosinophils can also mediate ADCC effects.
  • eosinophils can kill certain parasites through ADCC.
  • ADCP antibody-dependent cellular phagocytosis
  • the complement system is part of the innate immune system composed of a series of proteins.
  • the proteins of the complement system are called "complement” and are represented by the abbreviations C1, C2, C3, etc., which are a group of heat-labile proteins that exist in human or vertebrate serum and tissue fluid and have enzymatic activity after activation.
  • C1q the first component of the complement-dependent cytotoxicity (CDC) pathway, is capable of binding six antibodies, but binding to two IgGs is sufficient to activate the complement cascade.
  • complement-dependent cytotoxicity refers to complement-involved cytotoxicity in which the Fc effector domain of an antibody that binds the target activates a series of complement cascades that form holes in the target cell membrane, leading to die.
  • Fc region refers to the C-terminal region of an immunoglobulin heavy chain, including native sequence Fc regions and variant Fc regions.
  • the Fc region of a human IgG heavy chain is generally defined as the segment from its amino acid residue at position Cys226 or Pro230 to the carboxyl terminus, the lysine residue at C-terminal position 447 of the Fc region (according to the EU numbering system) may be present or absent .
  • a complete antibody composition may include a population of antibodies with all K447 residues eliminated, a population of antibodies with no K447 residues eliminated, or a population of antibodies that mix antibodies with and without K447 residues.
  • the Fc region of an immunoglobulin comprises two constant domains, CH2 and CH3, and in other embodiments, the Fc region of an immunoglobulin comprises three constant domains, CH2, CH3 and CH4.
  • Binding of IgG to Fc ⁇ receptors or C1q is dependent on residues located in the hinge and CH2 domains. Two regions of the CH2 domain are critical for FcyR and complement C1q binding and have unique sequences in IgG2 and IgG4. Substitution of residues 233-236 in human IgG1 and IgG2 and substitution of residues 327, 330, and 331 in human IgG4 have been shown to substantially reduce ADCC and CDC activity (Armour et al., Eur.J.Immunol.29 (8). ), 1999, 2613-2624; Shields et al., J. Biol. Chem. 276(9), 2001, 6591-6604). Furthermore, Idusogie et al. showed that alanine substitutions at various positions including K322 significantly reduced complement activation (Idusogie EE et al, J. Immunol 164(8), 2000, 4178-84).
  • Functional Fc region and “functional Fc region” and similar terms are used interchangeably to refer to an Fc region having the effector functions of a wild-type Fc region.
  • Variant Fc regions “Fc mutants”, “Fc regions bearing mutations”, “mutated Fc regions”, “Fc region variants”, “Fc variants”, “variant Fc regions” and “mutated Fc regions” “region” and similar terms are used interchangeably to refer to an Fc region comprising at least one amino acid modification that distinguishes it from a native sequence Fc region/wild-type Fc region.
  • the variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region by one or more amino acid substitutions, deletions, or additions. In some embodiments, the variant Fc region has at least one amino acid deletion compared to the Fc region of a wild-type IgG. In some embodiments, the variant Fc region has at least one amino acid substitution compared to the Fc region of a wild-type IgG. In some embodiments, the variant Fc region has one or more amino acid substitutions and one or more amino acid deletions in the Fc region of a wild-type antibody. In some embodiments, the variant Fc region has at least one or two deletions of amino acids from the Fc region described herein.
  • the variant Fc region has at least one, two, three, or more amino acid substitutions in the Fc region described herein. In some embodiments, the variant Fc region has at least one, two, three or more amino acid substitutions in the Fc region described herein and at least one or two deletions in the Fc region described herein. In some embodiments, the variant Fc region has at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or more homology to the wild-type Fc region and/or the parental Fc region .
  • Fc receptor refers to a molecule that binds the Fc region of an antibody.
  • the FcR is a native human FcR.
  • an FcR is a receptor that binds an IgG antibody, ie, an FcyR, including three receptors, FcyRI (CD64), FcyRII (CD32), and FcyRIII (CD16), and allelic variants and variants of these receptors splicing form.
  • FcyRII receptors include FcyRIIA and FcyRIIB
  • FcyRIII receptors include FcyRIIIA and FcyRIIIB.
  • Fc ⁇ R can be divided into activating receptors (Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIC, Fc ⁇ RIIIA, Fc ⁇ RIIIB, also known as CD64, CD32A, CD32C, CD16A, CD16B) and inhibitory receptors (Fc ⁇ RIIB, also known as CD32B) ).
  • Activating receptors contain an immunoreceptor tyrosine-based activation motif (Immunoreceptor T ⁇ rosine-based Activation Motif, or ITAM) in its cytoplasmic domain, which transmits activation signals, promotes cell activation, and inhibits
  • Type receptors contain an immunoreceptor tyrosine-based inhibitory motif (Immunoreceptor T ⁇ rosine-based Inhibitor ⁇ Motif, or ITIM) in its cytoplasmic domain, which acts to inhibit cell activation.
  • the effector functions of activating Fc ⁇ Rs mainly include ADCC, ADCP and antigen presentation; while the effector functions of inhibitory Fc ⁇ Rs mainly include functions such as inhibition and sweeping.
  • Fc ⁇ RIIB is the only inhibitory Fc ⁇ R expressed in humans and mice, and in antibodies that directly target tumors, Fc ⁇ RIIB expression is associated with reduced antibody efficacy.
  • Fc ⁇ RIIIA CD16A
  • NK cells natural killer cells
  • the Fc ⁇ RIIIA receptor is the only receptor expressed on NK cells that can mediate ADCC function.
  • FcR also includes the neonatal receptor (FcRn), an IgG antibody receptor located on the surface of cell membranes.
  • FcRn is responsible for the transfer of maternal IgG to the fetus and regulates immunoglobulin homeostasis in vivo.
  • FcRn can bind to the Fc part of IgG to prevent IgG molecules from being cleaved by lysosomes, which can increase the half-life of IgG in vivo and participate in the in vivo transport, maintenance and distribution of IgG metabolism.
  • IgG1-IgG4 subclasses differ in their ability to bind Fc receptors.
  • IgG1 and IgG3 are universal ligands that bind to all Fc ⁇ Rs and have strong ADCC effects.
  • IgG2 or IgG4 also commonly referred to as "indolent" IgG subclasses, are used to avoid immune activating effects.
  • many mAbs choose to use IgG4 as the backbone of the antibody to avoid the ADCC effect.
  • IgG2 and IgG4 are not completely "inert" and can bind to activated forms of Fc ⁇ RIIa-H131 and Fc ⁇ RI, respectively, to initiate neutrophil activation.
  • the "LALA” (L234A+L235A) mutation of IgG1 is widely used, which can reduce the binding affinity of the Fc region of the antibody to Fc ⁇ R. 100 times.
  • the Fc mutant obtained by the present application has a lower binding affinity to Fc ⁇ R than the "LALA" mutation, thus reducing the effect of ADCC more strongly.
  • the expressions "reduced ADCC and/or ADCP and/or CDC effector function” or “reduced ADCC/ADCP/CDC effector function” and similar expressions refer to the corresponding wild-type
  • the numerical value of the subfunction has a sufficiently high reduction that a person skilled in the art would consider the reduction to be statistically significant within the corresponding biological context.
  • the reduction in both values is, for example, greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70% %, greater than about 80%, greater than about 90%, or greater than about 100% or even higher.
  • amino acid substitution refers to the replacement of at least one amino acid residue present in a predetermined amino acid sequence with another, different “substituted” amino acid residue.
  • substitutions refers to the substitution of one amino acid by another amino acid within the same class, e.g., substitution of an acidic amino acid by another acidic amino acid, substitution of a basic amino acid by another basic amino acid, or substitution of a neutral amino acid by another Neutral amino acid substitutions. Exemplary substitutions are shown in the following table:
  • amino acid deletion refers to the removal of at least one amino acid residue from a predetermined amino acid sequence.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the original primary transformed cell and progeny derived therefrom, regardless of the number of passages. Progeny may not be identical in nucleic acid content to the parent cell, but may contain mutations. Included herein are mutant progeny screened or selected for the same function or biological activity in the originally transformed cell.
  • the sequences are aligned for optimal comparison purposes (e.g., between the first and second amino acid sequences or nucleic acid sequences for optimal alignment. Gaps are introduced in one or both or non-homologous sequences can be discarded for comparison purposes).
  • the length of the reference sequences aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.
  • Sequence comparisons and calculation of percent identity between two sequences can be accomplished using mathematical algorithms.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (at http://www.gcg.com) is used that has been integrated into the GAP program of the GCG software package available), using the Blossum 62 matrix or the PAM250 matrix and gap weights 16, 14, 12, 10, 8, 6, or 4 and length weights 1, 2, 3, 4, 5, or 6, to determine the distance between two amino acid sequences percent identity.
  • the GAP program in the GCG software package (available at http://www.gcg.com) is used, using the NWSgapdna.CMP matrix and gap weights 40, 50, 60, 70 or 80 and A length weight of 1, 2, 3, 4, 5, or 6 determines the percent identity between two nucleotide sequences.
  • a particularly preferred set of parameters (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • Antibodies comprising Fc mutants provided herein can target any antigen, including but not limited to proteins, subunits, domains, motifs, and/or epitopes belonging to the following target antigens, such as Cytokines, membrane-bound factors, enzymes, receptors, ligands, pathogens and their toxins, virus particles, tumor-related factors, signaling pathway member molecules, etc.
  • target antigens such as Cytokines, membrane-bound factors, enzymes, receptors, ligands, pathogens and their toxins, virus particles, tumor-related factors, signaling pathway member molecules, etc.
  • Suitable antigens depend on the desired application. For anticancer therapy, it is desirable to have targets whose expression is restricted to cancer cells. Some targets that have proven particularly suitable for antibody therapy are those with signaling functions. Other therapeutic antibodies exert their effects by blocking the receptor's signaling by inhibiting the binding between the receptor and its conjugated ligand.
  • variable regions of these antibodies can be integrated with the Fc mutants disclosed herein to form products with improved superior properties.
  • the Fc mutants disclosed herein can be incorporated into humanized antibodies, affinity matured antibodies, engineered antibodies, eg, fused to their heavy chain variable regions.
  • Fc mutants, fusion proteins (eg antibodies) comprising Fc mutants, etc. can be further modified using various methods already disclosed in the prior art, for example, to reduce immunogenicity and improve stability , solubility, function, and other modifications of clinical benefit.
  • modifications include, but are not limited to, modifications such as those at positions 252, 254 and 256 that can extend serum half-life.
  • ADCC effector function of the Fc region
  • CDC effector function of the Fc region
  • IgG1 subclass antibodies ADCC function is achieved mainly through the Fc region binding to Fc ⁇ RIIIA.
  • IgG1 subclass antibodies that need to reduce ADCC and CDC functions their Fc regions need to be modified to reduce their binding to Fc ⁇ receptors.
  • the present example explores the key sites of interaction between human IgG1Fc and Fc ⁇ R, and designs corresponding Fc mutants to reduce its interaction with Fc ⁇ R.
  • the crystal structure (PDB: 3SGJ) of human IgG1 Fc and human CD16A (Fc ⁇ RIII) is shown in Figure 1, in which the two segments of Fc, P232-V240 and N325-E333, constitute the binding site for the CD16A epitope, wherein the The ⁇ -bond interaction formed between amino acid P329 and amino acids W90 and W113 of CD16A is the key amino acid on their mutual binding interface.
  • the inventors designed the Fc mutants shown in Table 1 based on the intermolecular interaction interface.
  • an IgG1 monoclonal antibody (Fcmut-01) against human claudin18.2 protein obtained by internal screening of Innovent Pharmaceuticals was used as an example to carry out related design and research. That is, based on Fcmut-01 as parent and control, a series of mutants (Fcmut-02 to Fcmut-024) with different mutations in Fc shown in Table 1 were obtained.
  • Fcmut-25 is Herceptin (Genentech)
  • Fcmut-26 ⁇ Fcmut32 are different modifications of Herceptin's Fc.
  • Plasmid construction According to conventional experimental methods, the nucleotide sequences of the above heavy chain Fc mutation and light chain were obtained, and cloned into the pcDNA3.1 vector to obtain each plasmid.
  • Purified product Centrifuge the cultured cell culture solution at 4000rpm for 50min, collect the supernatant, and purify the supernatant with a prepacked column Hitrap Mabselect Sure (GE, 11-0034-95).
  • the specific operations are as follows: equilibrate the packed column with 5 column volumes of equilibration solution (20mM Tris, 150mM NaCl, pH7.2) before purification; pass the collected supernatant through the column, and then wash the packed column with 10 column volumes of equilibration solution , to eliminate non-specifically bound proteins; rinse the packing with 5 column volumes of elution buffer (100 mM sodium citrate, pH 3.5), and collect the eluate. The eluate was adjusted to pH 6.0 with 2M Tris, and the concentration was determined to obtain the purified antibody product.
  • the collected antibody products were concentrated and exchanged into PBS (Gibco, 70011-044) by ultrafiltration, and then further separated and purified with superdex200 increase (GE, 10/300GL, 10245605), and the elution peaks of the monomer were collected, and the column was equilibrated and washed. Debuffering was PBS (Gibco, 70011-044).
  • SPR Surface plasmon resonance
  • a layer of biomolecular recognition film is first fixed on the surface of the sensor chip, and then the sample to be tested flows over the chip surface. If there are molecules in the sample that can interact with the biomolecular recognition film on the chip surface, the gold film will be caused. The change of the surface refractive index eventually leads to the change of the SPR angle. By detecting the change of the SPR angle, information such as the affinity and kinetic constant of the analyte can be obtained.
  • Biacore (C ⁇ tiva, T200) was used to measure the KD of the antibody carrying the mutation in the Fc region and human Fc ⁇ R obtained in Example 2.
  • the specific method is as follows: each Fc ⁇ R and FcRn protein containing histidine tags (the Fc receptors of each Fc receptor) See Table 2 below for information) capture on the chip surface coupled with anti-histidine antibody, and then obtain affinity and kinetic constants by detecting the binding and dissociation between the chip surface protein and the antibody in the mobile phase.
  • the method includes chip preparation and affinity detection.
  • the assay process used 10 ⁇ HBS-EP+ (BR-1006-69, C ⁇ tiva) diluted 10 times as the experimental buffer.
  • Chip preparation Using amino coupling kit (BR-1006-33, C ⁇ tiva) and histidine capture kit (28995056, C ⁇ tiva), the anti-histidine antibody in the histidine capture kit was coupled to the CM5 chip (29-1496-03, C ⁇ tiva) surface, after coupling, 1 M ethanolamine was injected to block the remaining active sites.
  • Each cycle includes capturing the receptor, binding to a certain concentration of the antibody carrying the mutation in the Fc region of the present application, and chip regeneration.
  • Each antibody mutant solution after gradient dilution (when combined with Fc ⁇ Rs, the dilution gradient is 0, 12.5, 25, 50, 100, 200, 400nM; when combined with FcRn, the dilution gradient is 0, 50, 100, 200, 400, 800, 1600nM), to get from The sequence of low concentration to high concentration completes each affinity detection cycle separately.
  • the antibody mutant solution was flowed over the chip surface at a flow rate of 30 ⁇ l/min for a binding time of 60 s and a dissociation time of 60 s.
  • the chip was finally regenerated using 10 mM Gl ⁇ cine pH 1.5 (BR-1003-54, C ⁇ tiva).
  • the data obtained were analyzed using Biacore T200 analysis software (version number 3.1) using analytical 1:1 binding or steady-state analytical models to obtain corresponding results.
  • Table 3 presents the affinity data of each antibody mutant and each Fc receptor in this study, and Figure 2 shows the fitting curve of the corresponding molecule.
  • the IgG1 wild-type Fcmut-01 antibody has a high affinity for CD64 with a K D value of 1.07E-09; it binds to both subtypes H167 and R167 of CD32A with a K D value of 1.07E-09.
  • the values were 2.01E-07 and 4.60E-08, respectively; the affinities for the two isoforms of CD16A, F176 and V176, were 1.57E-07 and 2.44E-08, respectively; and for CD16B (NA1), CD16B (NA2) and CD32B for weak binding.
  • Fcmut-26 ⁇ Fcmut-32 also showed similar results.
  • Fcmut-29 other antibody molecules carrying Fc mutations were not compatible with CD16A(F176), CD16A(V176), CD16B (NA1), CD16B (NA2), CD32A (H167), CD32A (R167) and CD32B binding.
  • F176 and V176 are the main genotypes of CD16A, and according to the crystal structure, amino acid 176 of CD16A is located in the binding region of Fc, so it has an important influence on the affinity of CD16A and Fc.
  • the Fc mutants obtained in the present application do not substantially interact with the genotypes F176 and V176 of CD16A, so it is clear that the Fc mutants obtained in the present application will not substantially interact with all functional CD16A, thereby not triggering ADCC effects .
  • Protein molecules (eg, antibody) molecules comprising the Fc mutants of the present application thus have substantially no ADCC effector function.
  • FcRn is expressed on a variety of cells in vivo, and the binding of FcRn to the Fc region is pH-dependent, only at a weakly acidic pH of 6.0, but not at neutral pH, thereby prolonging the half-life of the corresponding antibody.
  • the experimental data of the present application show that the mutation of the Fc region carried by the antibody does not affect the binding of the antibody molecule to FcRn. Therefore, FcRn can still prolong the in vivo half-life of the antibody mutant of the present application.
  • the antibody mutant obtained in the present application does not bind to CD32B, indicating that the therapeutic effect of the antibody can be enhanced by mutating the Fc region of the antibody.
  • the "LALA” mutation (Fcmut-05) widely used in the prior art reduces the binding affinity of the antibody Fc region to Fc ⁇ R by 100-fold.
  • the Fc mutant obtained by the present application has a lower binding affinity to Fc ⁇ R than the "LALA” mutation, thus reducing the effect of ADCC more strongly.
  • Wild-type IgG2 and IgG4 have weak or no ADCC effect, so many antibody molecules that are expected to avoid ADCC effect select the Fc region of IgG2 subtype or IgG4 subtype in production. In some cases, however, it is still desirable to further reduce the ADCC effector function of the IgG2 subtype or the IgG4 subtype.
  • the applicant further explored the binding of various FcRs after corresponding mutations in the corresponding sites in the Fc region of IgG2 and IgG4 subtypes (see Table 1). The data show that IgG2 subtype or IgG4 subtype Fc mutants containing the corresponding mutations of the present application have reduced binding to Fc ⁇ R relative to wild-type Fc.
  • Biofilm thin-layer interferometry was used to determine the affinity (KD) of the Fc mutants of the present invention for binding to human Fc receptors.
  • Fc-25 is an antibody that binds to HER2, and the constant region of the antibody is the wild-type IgG1 sequence; it can be seen from the results that deletion of P329 can reduce the binding of the molecule to Fc gamma receptor, but the binding to FcRn (pH6.0) is not affected. influences.
  • the affinity of the antibody Fc and C1q directly determines whether the antibody has the CDC effector function. Therefore, in this example, the affinity of each Fc mutant to C1q is detected to determine whether the Fc mutant also has the CDC effector function.
  • Biofilm thin-layer interferometry was used to determine the affinity (KD) of the antibody of the present invention for binding to human C1q.
  • the BLI method affinity determination was performed according to the existing method (Estep, P et al., High throughput solution Based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2): pp. 270-8).
  • Affinity detection was performed using the same BLI method as described in Example 4, except for the following.
  • FcyR antibody-dependent cell-mediated cytotoxicity
  • CD16A FcyRIIIA
  • ADCC effector cell the Jurkat-ADCCNF-AT luciferase effector cell line (hereinafter referred to as ADCC effector cell) of Promega Company was used, and the ADCC activity of the antibody was detected by detecting the activation of NF-AT signal.
  • the specific experimental process is as follows:
  • DANG-18.2 CCL Cell Lines Service
  • ADCC effector cells overexpressing human claudin18.2 on the surface the supernatant of cells DANG-18.2 and ADCC effector cells were eliminated by centrifugation, and the cells were washed twice with PBS solution, then Cells were resuspended in assay medium (1640 medium (Gibco) with 5% low IgG serum), the concentration of ADCC effector cells was adjusted to 6 ⁇ 10 6 cells/mL, and the concentration of DANG-18.2 cells was adjusted to 1 ⁇ 10 6 /ml.
  • assay medium 1640 medium (Gibco) with 5% low IgG serum
  • ADCC effector cells were added to each well plate, 25uL per well.
  • the test results are shown in Figure 4.
  • the control antibody Fcmut-01 has the Fc region of the wild-type IgG1 monoclonal antibody, which can effectively activate the NF-AT signal of ADCC effector cells by binding to the antigen on the target cell (DANG-18.2).
  • DANG-18.2 the downstream signaling pathway of ADCC is activated, indicating that the antibody has excellent ADCC killing ability.
  • other antibodies carrying mutations in the Fc region obtained in this application showed very weak or almost no ADCC effect, specifically: the mutant Fcmut-05 (carrying L234A & L235A mutations) was 10 times greater than the control Fcmut-01.
  • LALA mutation in the Fc region can significantly reduce the ADCC effect of the antibody.
  • the results of this example show that the Fc mutant obtained in the present application has a greater reduction effect on the ADCC effect than the LALA mutation.
  • the antibody mutants of the present application obtained by mutating one or more amino acids at positions 329, 330, 234 or 235 of the Fc region of the antibody substantially eliminate the ADCC effector function, and are obtained by mutation modification (eg, deletion, substitution) of one or more amino acids in the Fc region of the antibody. This suggests that the above amino acid positions are critical for the ADCC/ADCP effector function of the antibody.
  • those skilled in the art will be able to select one or more of the Fc region positions 329, 330, 234 or 235 according to the content disclosed in the present application Amino acids are modified accordingly, resulting in the actual technical effect.
  • deletion of amino acid 329 deletion of amino acids 329-330, deletion of amino acid 329, and substitution of amino acid 320 in the Fc region of the antibody, and combination of the above modifications with LALA modification (L234A+L235A) can be considered , thereby obtaining antibody molecules that eliminate ADCC/ADCP effector function.

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Abstract

涉及经过修饰的具有降低的与FcγR或C1q结合的免疫球蛋白恒定区(Fc)突变分子，该分子可以在机体内显著降低不期望的ADCC和/或ADCP和/或CDC效应子功能。此外，还涉及上述Fc突变分子的用途、包含所述突变分子的融合蛋白以及其用途。

Description

与Fc受体结合改变的Fc突变体 技术领域
本发明总体上涉及免疫学和抗体工程领域，具体而言，本发明涉及IgG免疫球蛋白的变体，其制备方法及其应用。
背景技术
随着近年来治疗性抗体的开发，研究人员一方面不断探索寻找新的靶标，一方面对成药性抗体进行不断的升级改造，以努力提高或增强其有益效果，其中研究的焦点之一集中在对抗体Fc区的改造。
众所周知，虽然抗体的Fc区无抗原结合活性，但是却是抗体与细胞表面Fc受体相互作用的部位，因此对抗体的效应子功能发挥至关重要的作用，Fc区通过与不同的Fc受体的相互作用，使得抗体可以发挥多种效应子功能，例如抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、补体依赖性细胞毒性(CDC)。关于ADCC、ADCP、CDC效应子功能，对于识别表达在肿瘤细胞或病原体上的抗原并以清除恶性细胞为目标的抗体而言，有效甚至强化的ADCC、ADCP、CDC效应子功能有利于抗体的治疗效果，然而对于针对例如表达在T细胞上的抗原以阻断细胞表面受体/细胞因子或者免疫调节为目标的抗体而言，则往往需要降低的甚至消除的ADCC、ADCP、CDC效应子功能，因为靶向细胞表面抗原的抗体可对免疫细胞诱发不必要的免疫剌激以及相关的效应子功能及补体活化，从而产生不利后果，这一点特别是对于需要目标细胞低损耗的免疫检查点抑制剂抗体，尤其重要。
人IgG亚类(IgGl、IgG2、IgG3和IgG4)具有不同的免疫功能，例如不同亚类的IgG抗体具有不同的ADCC活性，相比较于其他亚类，IgG1和IgG3具有较强的ADCC活性；IgGl、IgG2、IgG3和IgG4具有抗体依赖细胞吞噬作用(ADCP)。由于治疗性抗体和Fc融合构建体往往需要靶向和活化或中和靶配体功能区，但又不能破坏或损害所需的局部细胞或组织，因此在实际生 产应用中，一直存在渴望降低或者消除Fc效应子功能(例如降低或者消除ADCC和/或ADCP活性、降低或者消除CDC活性)的Fc突变体的实际需求，本申请满足了这种需求。
据已有文献报道，Fc区的部分氨基酸突变可能产生降低或者消除ADCC和/或ADCP活性、降低或者消除CDC活性的效果，例如，XENCOR的专利家族US8734791B2、US10183999B2、US8883147B2，Roche的专利家族CN103476795B等，多数能够达到降低ADCC、CDC活性的效果，然而，能够达到完全消除ADCC和CDC效应的突变较为少见，本申请通过对野生型Fc氨基酸序列进行定向改造，获得了能够完全消除ADCC和CDC效应的Fc突变分子。
发明概述
本发明提供了经过修饰的免疫球蛋白恒定区(Fc区)的突变分子，其可用于工程化改造抗体或抗体类治疗剂。
通过在Fc区包含本申请公开的突变和/或其组合，使得与包含野生型Fc区的分子相比，包含本申请突变Fc区的抗体、抗体类治疗剂以及包含突变Fc区的其他分子与FcγR或C1q的结合作用极大降低，从而在机体内显著降低不期望的ADCC和/或ADCP和/或CDC效应子功能。进一步地，本申请公开的Fc突变并没有影响包含本申请突变Fc区的抗体、抗体类治疗剂以及包含突变Fc区的其他分子与FcRn的结合能力，因而并不影响相应分子的半衰期。因此，本申请提供了携带特定突变的Fc区多肽分子、具有降低或者消除的ADCC和/或ADCP和/或CDC效应子功能，但仍保留了结合FcRn的能力的包含上文所述突变Fc区的抗体分子或类似结构分子。
第一方面，本申请提供了具有不同修饰的Fc突变体，与野生型Fc区相比，该Fc突变体显示降低的与Fc受体和/或C1q的亲和力，进而降低或者消除该Fc突变体诱导的ADCC、CDC和ADCP效应子功能。在一个实施方案中，Fc突变体诱导的ADCC、CDC和ADCP效应子功能降低至相应野生型Fc所诱导的ADCC、CDC和ADCP效应子功能的至少80％、70％、60％、50％、40％、30％、20％、10％、5％、2％、1％或完全消除。
在一个实施方案中，本申请提供的Fc突变体包含一个或者多个氨基酸缺失。在一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第329位氨基酸的缺失(Δ329)。在一个具体实施方案中，本申请提供的Fc突变体与相应野生型Fc的区别在于依据Kabat中的EU索引编号的第329位的缺失(Δ329)。在另一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第329位和第330位氨基酸的缺失(Δ329和Δ330)。在一个具体实施方案中，本申请提供的Fc突变体与相应野生型Fc的区别在于依据Kabat中的EU索引编号的第329位和第330位氨基酸的缺失(Δ329和Δ330)。
在一个实施方案中，本申请提供的Fc突变体包含一个或者多个氨基酸取代。在一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第234位和/或第235位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L。在一个优选的实施方案中，所述Fc突变体包含L234A+L235A、V234A或F234A+L235A。在一个具体实施方案中，本申请提供的Fc突变体与相应野生型Fc的区别在于L234A+L235A、V234A或F234A+L235A。
在一个实施方案中，本申请提供的Fc突变体包含一个或者多个氨基酸缺失和一个或多个氨基酸取代。在一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第329位氨基酸的缺失和第330位的取代，其中采用G、D或者Q对第330位进行取代。在一个优选的实施方案中，所述Fc突变体包含Δ329+A330G或Δ329+S330G。在一个具体实施方案中，本申请提供的Fc突变体与相应野生型Fc的区别在于Δ329+A330G或Δ329+S330G。
在一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第329位氨基酸的缺失和第234位和第235位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L。在一个优选的实施方案中，所述Fc突变体包含L234A+L235A+Δ329、V234A+Δ329或F234A+L235A+Δ329。在一个具体实施方案中，本申请提供的Fc 突变体与相应野生型Fc的区别在于L234A+L235A+Δ329、V234A+Δ329或F234A+L235A+Δ329。
在另一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第329位和第330位氨基酸的缺失以及第234位和第235位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L。在一个优选的实施方案中，所述Fc突变体包含L234A+L235A+Δ329+Δ330、V234A+Δ329+Δ330或F234A+L235A+Δ329+Δ330。在一个具体实施方案中，本申请提供的Fc突变体与相应野生型Fc的区别在于L234A+L235A+Δ329+Δ330、V234A+Δ329+Δ330或F234A+L235A+Δ329+Δ330。
在另一个具体实施方案中，本申请提供的Fc突变体包含依据Kabat中的EU索引编号的第329位氨基酸的缺失和第234位、235位、330位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L，采用G、D或者Q对第330位进行取代。在一个优选的实施方案中，所述Fc突变体包含L234A+L235A+A330G+Δ329、L234A+L235A+S330G+Δ329、V234A+L235A+A330G+Δ329、V234A+L235A+S330G+Δ329、F234A+L235A+S330G+Δ329或F234A+L235A+A330G+Δ329。在一个具体实施方案中，本申请提供的Fc突变体与相应野生型Fc的区别在于L234A+L235A+A330G+Δ329、L234A+L235A+S330G+Δ329、V234A+L235A+A330G+Δ329、V234A+L235A+S330G+Δ329、F234A+L235A+S330G+Δ329或F234A+L235A+A330G+Δ329。
在一些实施方案中，本发明提供的Fc突变体为IgG1型Fc突变体，与相应的野生型Fc区相比，所述突变体具有降低甚至消除的结合FcγR的能力。在另一些实施方案中，本发明提供的Fc突变体为IgG2、IgG3、IgG4型Fc突变体，与相应的野生型Fc区相比，所述突变体具有降低甚至消除的结合FcγR的能力。优选地，本发明提供的Fc突变体保留了与FcRn的结合能力。
本申请公开的Fc突变体可以作为一个平台构件，应用于任何需要降低甚至消除ADCC/ADCP/CDC效应子功能的场景中，例如应用于任何类型的期望降低甚至消除ADCC/ADCP/CDC效应子功能的抗体分子中，具有类似抗体结构的分子中。
在一个实施方案中，所述Fc突变体基本上基于IgG序列，在一个优选的 实施方案中，所述Fc突变体基本上基于人IgG序列，在另一个实施方案中，所述Fc突变体基本上基于人IgG1序列。
在另一个实施方案中，所述Fc突变体还可以包含其他修饰，例如现有技术中已知的用于降低免疫原性、提高稳定性、溶解性、功能和临床益处的其他修饰。
在一个具体的实施方案中，Fc突变体包含如下序列：
1)SEQ ID NO:2中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
2)SEQ ID NO:3中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
3)SEQ ID NO:4中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
4)SEQ ID NO:5中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
5)SEQ ID NO:6中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
6)SEQ ID NO:7中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
7)SEQ ID NO:8中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
8)SEQ ID NO:11中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
9)SEQ ID NO:12中按照EU索引第221-447位氨基酸的序列，或者与 该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
10)SEQ ID NO:13中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
11)SEQ ID NO:14中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
12)SEQ ID NO:15中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
13)SEQ ID NO:16中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
14)SEQ ID NO:17中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
15)SEQ ID NO:19中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
16)SEQ ID NO:20中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
17)SEQ ID NO:21中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
18)SEQ ID NO:22中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
19)SEQ ID NO:23中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、 99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
20)SEQ ID NO:24中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
21)SEQ ID NO:25中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
22)SEQ ID NO:27中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
23)SEQ ID NO:28中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
24)SEQ ID NO:29中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
25)SEQ ID NO:30中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
26)SEQ ID NO:31中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
27)SEQ ID NO:32中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
28)SEQ ID NO:33中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成。
第二个方面，本发明提供了包含第一方面所述Fc突变体的多肽，与包含野生型Fc区的多肽相比，所述多肽具有降低或消除的ADCC和/或ADCP和 /或CDC效应子功能，优选地，所述多肽不引起ADCC和/或ADCP和/或CDC效应。在一个具体实施方案中，所述多肽同时具有延长的半衰期。在一个实施方案中，包含第一方面所述Fc突变体的多肽诱导的ADCC、CDC和ADCP效应子功能降低至包含相应野生型Fc的多肽所诱导的ADCC、CDC和ADCP效应子功能的至少80％、70％、60％、50％、40％、30％、20％、10％、5％、2％、1％或完全消除。
在一些实施方案中，所述多肽是抗体分子，优选地，所述抗体分子是IgG类抗体分子。在另一个实施方案中，所述抗体分子是多特异性抗体(例如，双特异性抗体)、人源化抗体、嵌合抗体、抗体融合体。与相应的野生型抗体分子相比，本申请获得的包含所述Fc突变体的抗体分子与FcγR的能力得到降低甚至消除，因此具有降低或消除的ADCC和/或ADCP和/或CDC效应子功能。
在一些实施方案中，所述多肽是融合蛋白，其包含与Fc突变体有效连接的一个或者多个融合配偶体，所述融合配偶体通常可以为任何蛋白质或小分子，例如任何抗体的可变区、受体的靶结合区、粘附分子、配体、酶、细胞因子、趋化因子、或一些其他的蛋白或蛋白结构域。在一个实施方案中，所述融合蛋白例如是免疫粘附素。
在一个实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第329位氨基酸的缺失(Δ329)。在另一个具体实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第329位和第330位氨基酸的缺失(Δ329和Δ330)。
在一个实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第234位和/或第235位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L。在一个优选的实施方案中，所述IgG抗体包含L234A+L235A、V234A或F234A+L235A。
在一个实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第329位氨基酸的缺失和第330位的取代，其中采用G、D或者Q对第330位进行取代。在一个优选的实施方案中，所述IgG抗体包含Δ329+A330G或Δ329+S330G。
在一个实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第329位氨基酸的缺失和第234位和第235位氨基 酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L。在一个优选的实施方案中，所述IgG抗体包含L234A+L235A+Δ329、V234A+Δ329或F234A+L235A+Δ329。
在一个实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第329位和第330位氨基酸的缺失以及第234位和第235位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L。在一个优选的实施方案中，所述IgG抗体包含L234A+L235A+Δ329+Δ330、V234A+Δ329+Δ330或F234A+L235A+Δ329+Δ330。
在一个实施方案中，本申请提供包含Fc突变体的IgG抗体，其包含依据Kabat中的EU索引编号的第329位氨基酸的缺失和第234位、235位、330位氨基酸的取代，其中采用A、V、L、I分别取代第234位的L、V、F和/或第235位的L，采用G、D或者Q对第330位进行取代。在一个优选的实施方案中，所述IgG抗体包含L234A+L235A+A330G+Δ329、L234A+L235A+S330G+Δ329、V234A+L235A+A330G+Δ329、V234A+L235A+S330G+Δ329、F234A+L235A+S330G+Δ329或F234A+L235A+A330G+Δ329。
在一些实施方案中，本发明提供的IgG抗体为IgG1型抗体，与相应的野生型抗体相比，所述抗体具有降低甚至消除的结合FcγR的能力。在另一些实施方案中，本发明提供的IgG抗体为IgG2、IgG3、IgG4型抗体，与相应的野生型抗体相比，所述抗体具有降低甚至消除的结合FcγR的能力。优选地，本发明提供的抗体保留了与FcRn的结合能力。
在一些实施方案中，所述抗体分子是抗claudin18.2的IgG抗体。
在一个具体的实施方案中，本发明提供的包含Fc突变体的IgG抗体包含如下的重链和轻链：
1)包含SEQ ID NO:2所示的重链和SEQ ID NO:9所示的轻链；或
2)包含SEQ ID NO:3所示的重链和SEQ ID NO:9所示的轻链；或
3)包含SEQ ID NO:4所示的重链和SEQ ID NO:9所示的轻链；或
4)包含SEQ ID NO:5所示的重链和SEQ ID NO:9所示的轻链；或
5)包含SEQ ID NO:6所示的重链和SEQ ID NO:9所示的轻链；或
6)包含SEQ ID NO:7所示的重链和SEQ ID NO:9所示的轻链；或
7)包含SEQ ID NO:8所示的重链和SEQ ID NO:9所示的轻链；或
8)包含SEQ ID NO:11所示的重链和SEQ ID NO:9所示的轻链；或
9)包含SEQ ID NO:12所示的重链和SEQ ID NO:9所示的轻链；或
10)包含SEQ ID NO:13所示的重链和SEQ ID NO:9所示的轻链；或
11)包含SEQ ID NO:14所示的重链和SEQ ID NO:9所示的轻链；或
12)包含SEQ ID NO:15所示的重链和SEQ ID NO:9所示的轻链；或
13)包含SEQ ID NO:16所示的重链和SEQ ID NO:9所示的轻链；或
14)包含SEQ ID NO:17所示的重链和SEQ ID NO:9所示的轻链；或
15)包含SEQ ID NO:19所示的重链和SEQ ID NO:9所示的轻链；或
16)包含SEQ ID NO:20所示的重链和SEQ ID NO:9所示的轻链；或
17)包含SEQ ID NO:21所示的重链和SEQ ID NO:9所示的轻链；或
18)包含SEQ ID NO:22所示的重链和SEQ ID NO:9所示的轻链；或
19)包含SEQ ID NO:23所示的重链和SEQ ID NO:9所示的轻链；或
20)包含SEQ ID NO:24所示的重链和SEQ ID NO:9所示的轻链；或
21)包含SEQ ID NO:25所示的重链和SEQ ID NO:9所示的轻链；或
22)包含SEQ ID NO:27所示的重链和SEQ ID NO:34所示的轻链；或
23)包含SEQ ID NO:28所示的重链和SEQ ID NO:34所示的轻链；或
24)包含SEQ ID NO:29所示的重链和SEQ ID NO:34所示的轻链；或
25)包含SEQ ID NO:30所示的重链和SEQ ID NO:34所示的轻链；或
26)包含SEQ ID NO:31所示的重链和SEQ ID NO:34所示的轻链；或
27)包含SEQ ID NO:32所示的重链和SEQ ID NO:34所示的轻链；或
28)包含SEQ ID NO:33所示的重链和SEQ ID NO:34所示的轻链。
在另一个实施方案中，本申请提供的包含所述Fc突变体的多肽(例如抗体)在Fc区还可以包含其他修饰，例如现有技术中已知的用于降低多肽(例如抗体)免疫原性、增加半衰期、提高稳定性、溶解性、功能和临床益处的其他修饰。
在另一个实施方案中，本申请提供的包含所述Fc突变体的抗体具有现有技术已知的针对不同靶抗原的重链可变区和轻链可变区。本领域技术人员可以容易地将现有技术中已知的重链可变区和轻链可变区接枝到本申请公开的Fc突变体上，从而获得具有期望特性(例如降低或消除的ADCC和/或ADCP 和/或CDC效应子功能)的抗体变体。
第三方面，本发明提供了一种药物组合物，其包含第二方面所述的多肽及可药用载体。在一个实施方案中，所述药物组合物包含携带本发明所述Fc突变体的抗体分子。在一个实施方案中，所述药物组合物包含携带本发明所述Fc突变体的IgG抗体。在另一个实施方案中，所述药物组合物包含携带本发明所述Fc突变体的IgG1型抗体、IgG2型抗体、IgG3型抗体或IgG4型抗体。
第四方面，本发明提供了根据本申请所述Fc区修饰减少或者消除抗体的ADCC/ADCP/CDC效应子功能，且保留甚至提高半衰期的方法。在一个实施方案中，通过对需要减少或者消除ADCC/ADCP/CDC效应子功能的抗体Fc区进行如本申请所公开的一种或者多种修饰，从而减少或者消除抗体的ADCC/ADCP/CDC效应子功能。
第五方面，本发明提供了Fc突变体在制备药物中的用途。在一个实施方案中，所述药物用于免疫治疗，辅助免疫治疗。在一个实施方案中，所述药物具有降低或消除的ADCC/ADCP/CDC效应子功能作用。在一个具体的实施方案中，例如所述药物是包含本申请Fc突变体的融合蛋白，例如是IL-2Fc融合蛋白。在另一个具体的实施方案中，例如所述药物是包含本申请Fc突变体的靶向免疫细胞表面分子的抗体，其具有降低或消除的ADCC/ADCP/CDC效应子功能作用。在另一个实施方案中，所述药物用于治疗受试者中的肿瘤，例如在一个实施方案中，所述药物在不激活ADCC/ADCP/CDC作用且半衰期延长的情况下激活患者的免疫细胞，从而达到抗肿瘤的效果。
第六方面，本发明提供了治疗受试者疾病的方法，包括将有效量的包含本申请所述Fc突变体的针对相应疾病的药物组合物施用给受试者。在一个实施方案中，所述疾病是需要免疫治疗或者免疫辅助治疗的疾病。对于靶向细胞表面分子的抗体，特别是免疫细胞上的抗体，消除效应子功能是有利的。在一个实施方案中，本发明提供了治疗受试者疾病的方法，包括将有效量的包含本申请所述Fc突变体的针对相应免疫细胞上的抗原的抗体施用给受试 者。
第七方面，本发明提供了一种试剂盒，其包含本申请公开的Fc突变体、包含所述Fc突变体的多肽或者包含所述Fc突变体的免疫球蛋白分子。在一个实施方案中，本发明提供了一种检测试剂盒，通过将功能分子(如酶、抗原、受体、配体、细胞因子等)与Fc融合后，不仅可以提高融合蛋白的稳定性，还可以应用于流式细胞、免疫组化、体外活性检测和蛋白质微阵列检测等非临床领域。
本申请提供的携带具体Fc突变的抗体分子具有降低或者消除的ADCC、ADCP和CDC效应子功能，但仍保留了结合FcRn的能力，因此可以作为改良型抗体突变体，不仅提高了抗体的疗效，还确保了抗体的安全性，稳定性和低免疫原性。令人惊奇地发现，将Fc区的第329位脯氨酸残基缺失将显著降低Fc区与受体FcγRIII、FcγRII、FcγRI、C1q的结合、由此显著降低或消除ADCC、ADCP和CDC活性。此外，Fc区的Pro329与例如选自A330缺失、A330G、L234A和L235A中一种或者多种的组合突变导致显著降低与受体FcγRIII、FcγRII、FcγRI、C1q的结合，并由此显著降低或消除ADCC、ADCP和CDC活性。
附图说明：
图1：人IgG1Fc与人CD16A的晶体结构(PDB:3SGJ)。
图2：显示了本发明的Fc突变体与各种FcγR结合的情况，图2a：Fc突变体与CD16A(F176)的结合曲线(pH 7.4)；图2b：Fc突变体与CD16A(V176)的结合曲线(pH 7.4)；图2c：Fc突变体与CD16B(NA1)的结合曲线(pH 7.4)；图2d：Fc突变体与CD16B(NA2)的结合曲线(pH 7.4)；图2e：Fc突变体与CD32A(H167)的结合曲线(pH 7.4)；图2f：Fc突变体与CD32A(R167)的结合曲线(pH 7.4)；图2g：Fc突变体与CD32B的结合曲线(pH 7.4)；图2h：Fc突变体与CD64的结合曲线(pH 7.4)；图2i：Fc突变体与FcRn在pH6.0的结合曲线；图2J：Fc突变体与FcRn在pH 7.0的结合曲线；图2k.Fc突变体与C1q的结合与解离曲线。
图3显示了基于生物膜薄层干涉技术测定的Fc突变体与各种FcγR结合的情况，其中溶液中包含200nM的抗体。图3a：Fc突变体与CD16A(V176) 的结合曲线(pH 7.4)；图3b：Fc突变体与CD16A(F176)的结合曲线(pH 7.4)；图3c：Fc突变体与CD16B(NA1)的结合曲线(pH 7.4)；图3d：Fc突变体与CD16B(NA2)的结合曲线(pH 7.4)；图3e：Fc突变体与CD32A(H167)的结合曲线(pH 7.4)；图3f：Fc突变体与CD32A(R167)的结合曲线(pH 7.4)；图3g：Fc突变体与CD32B的结合曲线(pH 7.4)；图3h：Fc突变体与CD64的结合曲线(pH 7.4)；图3i：Fc突变体与FcRn在pH6.0的结合曲线。
图4显示了抗体的ADCC活性。
发明详述
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。这些方法的描述可以参见，例如，Sambrook等人，Molecular Cloning:A Laboratory Manual(第3版,2001)；Sambrook等人，Molecular Cloning:A Laboratory Manual(第2版,1989)；Maniatis等人，Molecular Cloning:A Laboratory Manual(1982)；Ausubel等人，Current Protocols in Molecular Biology(John Wiley和Sons,2008年7月更新)；Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,GreenePub.Associates和Wiley-Interscience；Glover,DNACloning:A Practical Approach,vol.I&II(IRL Press,Oxford,1985)；Anand,Techniques for the Analysis of Complex Genomes,(Academic Press,New York,1992)；Transcription and Translation(B.Hames&S.Higgins,Eds.,1984)；Perbal,A Practical Guide to Molecular Cloning(1984)；Harlow和Lane,Antibodies,(Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y.,1998)Current Protocols in Immunology Q.E.Coligan,A.M.Kruisbeek,D.H.Margulies,E.M.Shevach和W.Strober,eds.,1991)；Annual Review of Immunology；以及期刊专著如Advances in Immunology。
定义
在下文详细描述本发明前，应理解本发明不限于本文中描述的特定方法学、方案和试剂，因为这些可以变化。还应理解本文中使用的术语仅为了描述具体实施方案，而并不意图限制本发明的范围，其仅会由所附权利要求书 限制。除非另外定义，本文中使用的所有技术和科学术语与本发明所属领域中普通技术人员通常的理解具有相同的含义。
为了解释本说明书，将使用以下定义，并且只要适当，以单数形式使用的术语也可以包括复数，并且反之亦然。要理解，本文所用的术语仅是为了描述具体的实施方案，并且不意欲是限制性的。
术语“约”在与数字数值联合使用时意为涵盖具有比指定数字数值小5％的下限和比指定数字数值大5％的上限的范围内的数字数值。
术语“和/或”意指当用于连接两个或多个可选项时，应理解为意指可选项中的任一项或可选项中的任意两项或更多项。
术语“包含”或“包括”意指包括所述的要素、整数或步骤，但是不排除任意其它要素、整数或步骤。在本文中，当使用术语“包含”或“包括”时，除非另有指明，否则也涵盖由所述及的要素、整数或步骤组合的情形。例如，当提及“包含”某个具体序列的抗体可变区时，也旨在涵盖由该具体序列组成的抗体可变区。
术语“抗体”在本文中以最广意义使用并且涵盖多种抗体结构物，包括但不限于单克隆抗体、多克隆抗体、重组抗体、人源化抗体、嵌合抗体、多特异性抗体(例如，双特异性抗体)、单链抗体、完整抗体或其显示出所需的抗原结合活性的抗体片段。完整抗体通常将包含至少两条全长重链和两条全长轻链，但在某些情况下可包括较少的链，例如骆驼中天然存在的抗体可仅包含重链。
如本文所用，术语“结合”和“特异性结合”意指抗体的结合作用对抗原是选择性的，并且可以与不想要的或非特异的相互作用区别开。抗体与特定抗原结合的能力可以通过酶联免疫吸附测定法(ELISA)、表面等离子共振法(SPR)或生物膜层光学干涉技术(ForteBio)或本领域已知的其它常规结合测定法测定。例如在SPR中，抗体以大约1×10 -7或更低的KD，大约1×10 -8或更低的KD，大约1×10 -9或更低的KD，大约1×10 -10或更低的KD、大约1×10 -11或更低的KD与BCMA或CD3结合，则该抗体是“与BCMA或CD3特异性结合”的抗体。然而，特异性结合人BCMA或CD3的抗体可以与来自其它物种的BCMA或CD3蛋白具有交叉反应性。例如，特异于人BCMA或CD3的抗体，在一些实施方案中，可以与食蟹猴BCMA或CD3发生交叉反应。测定交叉反应性的方法包括实施例中所述的方法以及本领域已知的标准测定法，例如通过使用生物光干涉，或流式细胞术技术。
术语“Kabat的可变结构域残基编号方式”、“Kabat编号***”或“根 据Kabat的氨基酸位置编号方式”及其变化形式指根据Kabat等人的用于抗体重链可变域或轻链可变域编辑的编号***(参见Kabat等人,Sequences of Proteins of Immunological Interest,第5版,Public Health Service,National Institutes of Health,Bethesda,Md.(1991))。Kabat编号***一般适用于抗体的可变域中的残基(大约是第1-107位的轻链残基和第1-113位的重链残基)。
术语“EU编号***”、“如Kabat中的EU索引”或“EU索引”一般适用于免疫球蛋白重链恒定区中的残基(例如参见Kabat等人，参见上文)。除非本文中另有说明，否则本文的抗体可变结构域中的残基编号是根据Kabat编号***的残基编号；抗体恒定结构域中的残基编号根据EU编号***的残基编号方式。
抗体“效应子功能”指归因于抗体Fc区(天然序列Fc区或氨基酸序列变体Fc区)且随抗体同种型而变化的生物学活性。抗体效应子功能的例子包括:补体依赖的细胞毒性作用(CDC)、抗体依赖的细胞介导的细胞毒性作用(ADCC)、抗体依赖的细胞吞噬作用(ADCP)、细胞表面受体(例如B细胞受体)下调、B细胞活化等。
术语“效应细胞”指表达一种或者多种FcR并行使效应子功能的细胞，例如表达FcγRIIIA并行使ADCC效应子功能的细胞，在一个实施方案中，介导ADCC功能的细胞例如是NK细胞、外周血单个核细胞、单核细胞、细胞毒T细胞、嗜中性粒细胞。效应细胞可以来源于天然环境，例如血液。
术语“抗体依赖性细胞介导的细胞毒性”或“ADCC”指是细胞介导的免疫反应，其中某些细胞毒性细胞表面上存在的Fc受体识别靶细胞上结合的抗体，使得细胞毒性细胞可以特异性结合携带抗原的靶细胞，并激活免疫***的效应细胞从而裂解靶细胞的作用。经典的ADCC作用由自然杀伤细胞(NK)介导，巨噬细胞、中性粒细胞和嗜酸性粒细胞(嗜酸性球)也能介导ADCC作用。比如嗜酸性粒细胞(嗜酸性球)能通过ADCC作用杀死某些特定的寄生虫。
术语“抗体依赖性细胞吞噬”或“ADCP”指一种细胞反应，其中通过结合靶细胞的抗体与巨噬细胞表面的FcγRIIIa结合，诱导激活巨噬细胞，从而使靶细胞内化和被吞噬体酸化降解。ADCP也可由FcγRIIa和FcγRI介导，但是占比较小。
补体***是由一系列蛋白质组成的先天免疫***的一部分。补体***的 蛋白质称为“补体”，以缩写符号C1、C2、C3等表示，其是存在于人或脊椎动物血清、组织液中的一组不耐热的，经活化后具有酶活性的蛋白质。C1q是依赖补体的细胞毒性(CDC)途径的第一成分，其能够结合六个抗体，但与两个IgG结合就足以活化补体级联。
术语“依赖补体的细胞毒性”或“CDC”指补体参与的细胞毒性作用，其中结合靶标的抗体的Fc效应子结构域活化一系列补体级联反应，在靶细胞膜中形成孔洞，从而导致靶细胞死亡。
术语“Fc区”指免疫球蛋白重链的C端区域，包括天然序列Fc区和变异Fc区。人IgG重链Fc区通常定义为自其Cys226或Pro230位置的氨基酸残基至竣基末端的区段，Fc区的C末端447位的赖氨酸残基(依照EU编号***)可以存在或者缺失。因而，完整抗体组合物可以包括所有K447残基都被消除的抗体群、无K447残基被消除的抗体群、或者混合了有K447残基的抗体和没有K447残基的抗体的抗体群。
在某些实施方案中，免疫球蛋白的Fc区包含两个恒定结构域，即CH2和CH3，在另一些实施方案中，免疫球蛋白的Fc区包含三个恒定结构域，即CH2、CH3和CH4。
IgG与Fcγ受体或C1q的结合依赖于定位在铰链区和CH2结构域中的残基。CH2结构域的两个区域对FcγR和补体C1q结合至关重要，并且在IgG2和IgG4中具有唯一的序列。已显示取代人IgG1和IgG2中233-236位的残基和取代人IgG4中327、330和331位的残基可大幅降低ADCC和CDC活性(Armour等人,Eur.J.Immunol.29(8)，1999，2613-2624；Shields等人,J.Biol.Chem.276(9)，2001，6591-6604)。此外，Idusogie等人表明在包括K322在内的不同位置上的丙氨酸取代显著降低了补体激活(Idusogie EE等人,J.Immunol 164(8)，2000，4178-84)。
“功能性Fc区”与“有功能Fc区”等类似术语可以互换使用，指具有野生型Fc区的效应子功能的Fc区。
“变异Fc区”、“Fc突变体”、“携带突变的Fc区”、“突变Fc区”、“Fc区变体”、“Fc变体”、“变体Fc区”和“突变的Fc区”等类似术语可以互换使用，指包含至少一处氨基酸修饰而区别于天然序列Fc区/野生型Fc区的Fc区。
在一些实施方案中，变异Fc区包含与天然序列Fc区的氨基酸序列相差一处或多处氨基酸取代、缺失或添加的氨基酸序列。在一些实施方案中，变异Fc区与野生型IgG的Fc区相比具有至少一处氨基酸缺失。在一些实施方案中，变异Fc区与野生型IgG的Fc区相比具有至少一处氨基酸取代。在一些实施方案中，变异Fc区在野生型抗体的Fc区中具有一个或多个氨基酸取代以及一个或者多个氨基酸缺失。在一些实施方案中，变异Fc区具有至少一处或两处本文所述Fc区氨基酸缺失。在一些实施方案中，变异Fc区具有至少一处、两处、三处或更多处本文所述Fc区氨基酸取代。在一些实施方案中，变异Fc区具有至少一处、两处、三处或更多处本文所述Fc区氨基酸取代和至少一处、两处本文所述Fc区缺失。在一些实施方案中，变异Fc区与野生型Fc区和/或亲本Fc区具有至少约80％、90％、95％、96％、97％、98％、99％或更高的同源性。
“Fc受体”或“FcR”指结合抗体Fc区的分子。在一些实施方案中，FcR是天然人FcR。在一些实施方案中，FcR是结合IgG抗体的受体，即FcγR，包括FcγRI(CD64)、FcγRII(CD32)和FcγRIII(CD16)三种受体，以及这些受体的等位变体和可变剪接形式。FcγRII受体包括FcγRIIA和FcγRIIB，FcγRIII受体包括FcγRIIIA和FcγRIIIB。
根据受体功能，可以将FcγR分为激活型受体(FcγRI，FcγRIIA，FcγRIIC，FcγRIIIA，FcγRIIIB，又称为CD64，CD32A，CD32C，CD16A，CD16B)和抑制型受体(FcγRIIB，又称为CD32B)。激活型受体在其胞质结构域中包含免疫受体基于酪氨酸的活化基序(Immunoreceptor Tγrosine-based Activation Motif，或ITAM)，该基序起传递活化信号，促进细胞激活的作用，抑制型受体在其胞质结构域中包含免疫受体基于酪氨酸的抑制基序(Immunoreceptor Tγrosine-based InhibitorγMotif，或ITIM)，该基序起抑制细胞激活的作用。激活性FcγR的效应功能主要有ADCC、ADCP和抗原呈递；而抑制性FcγR的效应功能主要有抑制、清扫等功能。FcγRIIB是人类和小鼠表达的唯一抑制性FcγR，在直接靶向肿瘤的抗体中，FcγRIIB的表达与抗体疗效降低有关。
不同的FcγR具有不同的细胞表达谱，例如FcγRIIIA(CD16A)在巨噬细胞、单核细胞、自然杀伤细胞(NK细胞)等细胞上均有表达。FcγRIIIA受体是NK细胞上仅表达唯一受体，可以介导ADCC功能。
术语“FcR”还包括新生儿受体(FcRn)，FcRn是一种位于细胞膜表面的IgG抗体受体。FcRn负责将母体IgG转移给胎儿，并调节免疫球蛋白在体内的稳态。FcRn可以和IgG的Fc部分结合，阻止IgG分子被溶酶体裂解，可以起到增长IgG体内半衰期的作用，参与到IgG的体内转运、维持和分布代谢过程中。
IgG1-IgG4亚类结合Fc受体的能力不尽相同，针对不同的FcγR，IgG1和IgG3是通用的配体，它们结合所有的FcγR，具有很强的ADCC作用。IgG2或IgG4，通常又被称为“惰性”IgG亚类，采用这两种亚类可以避免免疫激活效应。事实上，许多单抗都选择以IgG4作为抗体的主干，以避免ADCC效应。然而IgG2和IgG4并非完全“惰性”，其分别可以结合激活型的FcγRIIa-H131和FcγRI，从而启动中性粒细胞激活。
此外，实际情况中，存在降低IgG1亚类引起的ADCC效应的需求，现有技术中，广泛采用IgG1的“LALA”(L234A+L235A)突变，该突变可以将抗体Fc区与FcγR的结合亲和力降低100倍。而通过本申请获得的Fc突变体，相比较于“LALA”突变，与FcγR的结合亲和力更低，因此更强地降低了ADCC的作用。
在涉及ADCC/ADCP/CDC的语境中，表述“降低的ADCC和/或ADCP和/或CDC效应子功能”或“降低的ADCC/ADCP/CDC效应子功能”以及类似表述指与相应的野生型分子引起的ADCC和/或ADCP和/或CDC效应子功能的数值相比，本申请所述的Fc突变体、包含Fc突变体的多肽等所引起的ADCC和/或ADCP和/或CDC效应子功能的数值具有足够高的降低，使得本领域技术人员将认为所述降低在相应的生物学背景内具有统计学显著性。例如，在某些实施方案中，所述两个数值的降低例如大于约10％、大于约20％、大于约30％、大于约40％、大于约50％、大于约60％、大于约70％、大于约80％、大于约90％、或者大于约100％甚至更高。
“氨基酸取代”指，将预定的氨基酸序列中存在的至少一个氨基酸残基替代为另外的不同“取代”氨基酸残基。
术语“保守取代”是指一个氨基酸经相同类别内的另一氨基酸取代，例如一个酸性氨基酸经另一酸性氨基酸取代，一个碱性氨基酸经另一碱性氨基酸取代，或一个中性氨基酸经另一中性氨基酸取代。示例性的取代如下表所示：
原始残基 示例性取代 优选保守取代
Ala(A) Val；Leu；Ile Val
Arg(R) Lys；Gln；Asn Lys
Asn(N) Gln；His；Asp,Lys；Arg Gln
Asp(D) Glu；Asn Glu
Cys(C) Ser；Ala Ser
Gln(Q) Asn；Glu Asn
Glu(E) Asp；Gln Asp
Gly(G) Ala Ala
His(H) Asn；Gln；Lys；Arg Arg
Ile(I) Leu；Val；Met；Ala；Phe；正亮氨酸 Leu
Leu(L) 正亮氨酸；Ile；Val；Met；Ala；Phe Ile
Lys(K) Arg；Gln；Asn Arg
Met(M) Leu；Phe；Ile Leu
Phe(F) Trp；Leu；Val；Ile；Ala；Tyr Tyr
Pro(P) Ala Ala
Ser(S) Thr Thr
Thr(T) Val；Ser Ser
Trp(W) Tyr；Phe Tyr
Tyr(Y) Trp；Phe；Thr；Ser Phe
Val(V) Ile；Leu；Met；Phe；Ala；正亮氨酸 Leu
按照共同的侧链性质分组：(1)疏水性：Ile、Met、Ala、Val、Leu、Ile；(2)中性亲水性：Cys、Ser、Thr、Asn、Gln；(3)酸性：Asp、Glu；(4)碱性：His、Lys、Arg；(5)影响链取向的残基：Gly、Pro；(6)芳香族：Trp、Tyr、Phe。非保守取代将需要将这些种类之一的成员换为另一种类。
“氨基酸缺失”指从预定的氨基酸序列去除至少一个氨基酸残基。
术语“宿主细胞”、“宿主细胞系”和“宿主细胞培养物”可交换地使用且是指其中引入外源核酸的细胞，包括这种细胞的后代。宿主细胞包括“转 化体”和“转化的细胞”，其包括最初原代转化的细胞和来源于其的后代，而不考虑传代的数目。后代在核酸内容上可能与亲本细胞不完全相同，而是可以包含突变。本文中包括在最初转化的细胞中筛选或选择的具有相同功能或生物学活性的突变体后代。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核苷酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核苷酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4)，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS,4:11-17)确定两个氨基酸序列或核苷酸序列之间的同一性百分数。
抗体的靶抗原
本申请所提供的包含Fc突变体的抗体可以靶向任何抗原，包括但不限于属于下列靶抗原中的蛋白、亚基、结构域、基序、和/或表位，所述靶抗原例如是细胞因子、膜结合因子、酶、受体、配体、病原体及其毒素、病毒颗粒、 肿瘤相关因子、信号通路成员分子等等。合适的抗原取决于所需的应用。对于抗癌治疗，期望具有表达限制在癌细胞内的靶。已证明特别适于抗体治疗的一些靶是具有信号传导功能的那些。其他治疗抗体通过抑制受体与其轭合配体之间的结合阻断受体的信号传导来发挥其作用。
已经被批准用于临床试验或正在开发的许多抗体可获益于本发明的Fc突变体。因此，可以将这些抗体的可变区与本申请公开的Fc突变体整合形成具有改进的优良性质的产品。在一个实施方式中，可以将本申请公开的Fc突变体整合到人源化抗体、亲和力成熟的抗体、工程化的抗体中，例如与其重链可变区融合。
Fc区的其他修饰
可以利用现有技术中已经公开的多种方法，对本申请提供的Fc突变体、包含Fc突变体的融合蛋白(例如抗体)等进行进一步的修饰，例如以用于降低免疫原性、提高稳定性、溶解性、功能和临床益处的其他修饰。此类修饰包括但不限于下列修饰，例如可以延长血清半衰期的在位置252，254和256处的修饰。
实施例
以下实施例进一步说明本发明，然而，应理解实施例以说明而非限定的方式来描述，并且本领域技术人员可以进行多种修改。
除非明确指明相反，否则本发明的实施将采用本领域技术内的常规化学、生物化学、有机化学、分子生物学、微生物学、重组DNA技术、遗传学、免疫学和细胞生物学的方法。
实施例1.Fc突变体设计
鉴于抗体Fc区与Fc受体的相互作用可能产生不利影响，在单克隆抗体应用中有时需要消除Fc区的效应子功能(例如ADCC、CDC)。例如对于IgG1亚类抗体，其主要通过Fc区结合FcγRIIIA而实现ADCC功能。针对需要降低ADCC、CDC功能的IgG1亚类抗体，则需要对其Fc区加以改造，以降低其与Fcγ受体的结合。因此，本实施例基于人IgG1Fc与FcγR的共结晶结构，探讨了人IgG1Fc与FcγR相互作用的关键位点，并设计了相应的Fc突变体， 以降低其与FcγR的相互作用。
人IgG1Fc与人CD16A(FcγRIII)的晶体结构(PDB:3SGJ)如图1所示，其中Fc的两个区段P232-V240和N325-E333构成了与CD16A表位结合的位点，其中Fc的氨基酸P329与CD16A的氨基酸W90和W113之间形成的π键相互作用，是它们相互结合界面上的关键氨基酸。基于该关键区段和氨基酸，发明人基于分子间作用界面设计了如表1所示的Fc突变体。
在本实施例中，以信达制药内部筛选获得的针对人claudin18.2蛋白的一个IgG1单克隆抗体(Fcmut-01)作为示例，进行相关设计和研究。即，基于作为亲本和对照的Fcmut-01，获得了表1所示的一系列具有Fc不同突变的突变体(Fcmut-02～Fcmut-024)。Fcmut-25是Herceptin(Genentech),Fcmut-26～Fcmut32是对Herceptin的Fc进行的不同改造。
表1.Fc突变体设计
Figure PCTCN2022091187-appb-000001
Figure PCTCN2022091187-appb-000002
实施例2：携带Fc突变的抗体的表达和纯化
质粒构建：根据常规实验方法，获得上述重链Fc突变和轻链的核苷酸序列，并分别克隆到pcDNA3.1载体中获得各个质粒。
蛋白质表达及纯化
瞬时转染质粒准备：取1/10(以转染体积计算)的减血清培养基Opti-MEM TM(Gibco，货号31985-070)，加入质粒混合物(50ug/50mL，其中重链和轻链质量比1：1)，将含有质粒的Opti-MEM TM培养基过滤至新的50ml离心管中，并向该离心管中加入已过滤的PEI(1g/L,Polγsciences)(质量比(质粒：PEI)＝1:3)，混匀静置20min。
细胞转染：将上述获得的DNA/PEI混合物轻柔倒入Expi293细胞(Gibco)并混匀，在37℃，8％CO 2的条件下转染14h，随后分别按转染细胞体积计，补加0.1％丙戊酸钠盐(VPA)(2.2M,Sigma)，补加2.5％的葡萄糖(200g/L，Sigma)以及2.5％的Feed溶液(1g/L Phγtone Peptone+1g/L Difco Select Phγtone)，再次将细胞置于37℃，8％CO 2的条件培养7d。
纯化产物：将培养后的细胞培养液以4000rpm离心50min，收集上清，用预装柱Hitrap Mabselect Sure(GE,11-0034-95)纯化上清液。具体操作如下：纯化前用5倍柱体积的平衡液(20mM Tris,150mM NaCl,pH7.2)平衡填料柱；将收集的上清液通过柱子，再用10倍柱体积的平衡液清洗填料柱，消除非特异性结合蛋白；用5倍柱体积的洗脱缓冲液(100mM柠檬酸钠，pH3.5)冲洗填料，收集洗脱液。将洗脱液用2M Tris调节pH至6.0，并测定浓度，获得纯化的抗体产物。
将收集的抗体产物超滤浓缩交换到PBS(Gibco，70011-044)中，然后用superdex200 increase(GE,10/300GL,10245605)进一步分离纯化，收集单体的洗脱峰，柱子的平衡和洗脱缓冲液为PBS(Gibco，70011-044)。
实施例3.Fc突变体与Fc受体的亲和力测定
一、SPR法测定携带Fc突变体的抗体的亲和力
采用表面等离子共振法(SPR)测定本发明获得的携带Fc突变体的抗体与人FcγR结合的情况，其中具体检测了各个抗体突变体与FcγRI,FcγRIIa,FcγRIIb,FcγRIIIa,FcγRIIIb及FcRn的平衡解离常数(KD)。基于SPR原理，当一束偏振光以一定的角度入射到棱镜端面，在棱镜与金膜的界面将产生表面等离子波，引起金属膜内自由电子产生共振，即表面等离子共振。分析时，先在传感芯片表面固定一层生物分子识别膜，然后将待测样品流过芯片表面，若样品中有能够与芯片表面的生物分子识别膜相互作用的分子，则会引起金膜表面折射率变化，最终导致SPR角度变化，通过检测SPR角度变化，获得被分析物的亲和力、动力学常数等信息。
本实施例通过Biacore(Cγtiva，T200)测定实施例2获得的携带Fc区突变的抗体与人FcγR的KD，具体方法如下：将含有组氨酸标签的各个FcγR及FcRn蛋白(各个Fc受体的信息参见下文的表2)捕获到偶联有抗组氨酸抗体的芯片表面，然后通过检测芯片表面蛋白与流动相中的抗体之间的结合与解离获得亲和力及动力学常数。该方法包括芯片制备和亲和力检测。测定过程使用10倍稀释后的10×HBS-EP+(BR-1006-69,Cγtiva)作为实验缓冲液。
表2检测用受体信息
受体名称 标签 货号 厂商
人FcγRIIIB/CD16B(NA1) his CDB-H5227 Acro
人FcγRIIIB/CD16B(NA2) his CDB-H5222 Acro
人FcRn/FCGRT&B2M异二聚体蛋白 His，Strep II FCM-H5286 Acro
人FcγRIIIA/CD16A(V176)蛋白 his CD8-H52H4 Acro
人FcγRIIIA/CD16A(F176)蛋白 his CDA-H5220 Acro
人CD32A/FCGR2A蛋白(167his) his 10374-H27H1 SINO
人CD32A/FCGR2A蛋白(167Arg) his 10374-H27H SINO
人CD32B/FCGR2B蛋白 his 10259-H08H SINO
人CD64/FCGR1A蛋白 his 10256-H08S-B SINO
根据厂商说明，具体方法如下：
芯片制备：使用氨基偶联试剂盒(BR-1006-33,Cγtiva)和组氨酸捕获试剂盒(28995056，Cγtiva)，将组氨酸捕获试剂盒中的抗组氨酸抗体偶联在CM5芯片(29-1496-03,Cγtiva)表面，偶联后注入1M乙醇胺，对剩余的活化位点进行封闭。
亲和力检测：每个循环包括捕获受体、结合一定浓度的本申请携带Fc区突变的抗体及芯片再生。将梯度稀释后的各个抗体突变体溶液(与FcγRs结合时，稀释梯度为0，12.5，25，50，100，200，400nM；与FcRn结合时，稀释梯度为0,50,100,200,400,800,1600nM)，以从低浓度到高浓度的顺序分别完成各个亲和力检测循环。在每个循环中，以30μl/min的流速使抗体突变体溶液流过芯片表面，结合时间60s，解离时间60s。最后使用10mM Glγcine pH 1.5(BR-1003-54,Cγtiva)对芯片进行再生。获得的数据使用Biacore T200分析软件(版本号3.1)，采用分析1:1结合或稳态分析模型进行分析，获得相应结果。
下表3给出了本研究各个抗体突变体与各个Fc受体的亲和力数据，图2示出了相应的分子的拟合曲线。
从结果可知：作为本研究的对照抗体，IgG1野生型Fcmut-01抗体与CD64有很高的亲和力，K D值为1.07E-09；与CD32A的两个亚型H167和R167都结合，K D值分别为2.01E-07和4.60E-08；与CD16A的两个亚型F176和V176的亲和力分别为1.57E-07和2.44E-08；而对CD16B(NA1)，CD16B(NA2)和CD32B为弱结合。
除了突变体Fcmut-05与CD16A(V176)有较弱的亲和力(3.06E-07)，其他携带Fc突变的抗体分子都不与CD16A(F176)、CD16A(V176)、CD16B(NA1)、CD16B(NA2)、CD32A(H167)、CD32A(R167)和CD32B结合。关于与野生型IgG1有强结合的CD64，突变体Fcmut-06/07/08都不与CD64结合，而Fcmut-02/03/04/05对CD64的结合也得到大幅度降低，与对照Fcmut-01相比，分别减弱了8.7倍，12.9倍，14.02倍和50.75倍。此外，Fcmut-26～Fcmut-32也显示出相似的结果，与Fcmut-25相比，除Fcmut-29外，其他携带Fc突变的抗体分子都不与CD16A(F176)、CD16A(V176)、CD16B(NA1)、CD16B(NA2)、CD32A(H167)、CD32A(R167)和CD32B结合。
F176和V176是CD16A主要的基因型，并且根据晶体结构可知CD16A的176位氨基酸位于与Fc的结合区中，因此对CD16A与Fc的亲和力有重要影响。本申请获得的Fc突变体基本上不与CD16A的基因型F176和V176相互作用，因此可以明确本申请获得的Fc突变体将基本上不与所有的功能性CD16A相互作用，由此不引发ADCC效应。包含本申请Fc突变体的蛋白质分子(例如抗体)分子因此基本上不具有ADCC效应子功能。
已知抗体的Fc区与FcRn在pH中性条件下不结合，仅在酸性条件下结合，这是抗体分子具体较长半衰期的主要机制。经检测可知，对照抗体Fcmut-01在pH6.0与FcRn的亲和力是3.86E-07，而在pH7.0条件下不与FcRn结合，本研究获得的携带Fc突变的抗体分子在相同条件下与FcRn的结合与对照抗体Fcmut-01一致(图2i，2j)，说明本研究获得的Fc突变体不影响与FcRn的结合。
FcRn在体内多种细胞上表达，且FcRn与Fc区的结合是pH依赖型的，只在弱酸性pH6.0左右结合，而在中性pH无结合，由此可以延长相应抗体的半衰期。本申请的实验数据表明抗体所携带的Fc区突变并没有影响抗体分子与FcRn的结合，因此，FcRn依然能够延长本申请抗体突变体的体内半衰期。
本申请获得的抗体突变体不与CD32B结合，表明通过对抗体的Fc区进行突变，可以增强抗体的疗效。
现有技术中广泛采用的“LALA”突变(Fcmut-05)将抗体Fc区与FcγR的结合亲和力降低100倍。而通过本申请获得的Fc突变体，相比较于“LALA”突变，与FcγR的结合亲和力更低，因此更强地降低了ADCC的作用。
野生型IgG2和IgG4具有弱的ADCC效应或者没有ADCC效应，因此许多期望避免ADCC效应的抗体分子在生产中选择IgG2亚型或者IgG4亚型的Fc区。然而在有些情况下，仍然希望进一步降低IgG2亚型或者IgG4亚型的ADCC效应功能。为此，申请人进一步探讨了对IgG2和IgG4亚型的Fc区相应位点(参见表1)进行相应突变后，其与各种FcR结合的情况。数据表明，包含本申请相应突变的IgG2亚型或者IgG4亚型Fc突变体相对于野生型的Fc，减弱了对FcγR的结合。
表3.Fc突变体与Fc受体的结合常数
Figure PCTCN2022091187-appb-000003
N.B.：不结合
二、生物膜薄层干涉技术测定本发明Fc突变体与Fc受体的结合
采用生物膜薄层干涉测定技术(BLI)测定本发明Fc突变体结合人Fc受体的亲和力(KD)。
实验开始前半个小时，根据样品数量，取合适数量的HIS1K传感器(18-5120，Sartorius,)浸泡于SD缓冲液(1x PBS，0.1％BSA，0.05％Tween-20)中，与FcRn结合时，实验所用缓冲液更改为10mM HEPS,150mM NaCl,3mM EDTA,0.05％P20,pH 6.0。将抗体稀释至200nM，Fc受体(样品信息见表2)稀释至100nM。
分别将200μl的SD缓冲液、200ul标记的抗体溶液、200ul Fc受体加入到96孔黑色聚苯乙烯微孔板(Greiner，655209)中。使用Fortebio Octet Red96e进行检测，根据样品位置布板，选择传感器位置。仪器设置参数如下：运行步骤：平衡基线120s、固化Fc受体100s、平衡基线120s、结合抗体60s和解离60s，转速为1000rpm，温度为30℃。实验完成后，使用ForteBio Octet分析软件分析KD值。
结果见下表4和图3。与Fcmut-25相比，除Fcmut-29外，其他携带Fc突变的抗体分子都不与CD16A(F176)、CD16A(V176)、CD16B(NA1)、CD16B(NA2)结合。突变体Fcmut-26/27/28/29/30/31/32突变体都不与CD32A H167、CD32A R167、CD32B结合。突变体Fcmut-30/31/32都不与CD64结合，而Fcmut-/26/27/28/29对CD64的结合也得到大幅度降低。
Fc-25是结合HER2的抗体，其抗体的恒定区为野生型的IgG1序列；从结果可知，删除P329可以降低分子对Fc gamma受体的结合，但对FcRn(pH6.0)的结合不受影响。
表4a突变体与HIS1K传感器上的CD16A V176结合
Figure PCTCN2022091187-appb-000004
表4b突变体与HIS1K传感器上的CD16A F176结合
Figure PCTCN2022091187-appb-000005
表4c突变体与HIS1K传感器上的CD16B NA1结合
Figure PCTCN2022091187-appb-000006
表4d突变体与HIS1K传感器上的CD16B NA2结合
Figure PCTCN2022091187-appb-000007
表4e突变体与HIS1K传感器上的CD32A H167结合
Figure PCTCN2022091187-appb-000008
Figure PCTCN2022091187-appb-000009
表4f突变体与HIS1K传感器上的CD32A R167结合
Figure PCTCN2022091187-appb-000010
表4g突变体与HIS1K传感器上的CD32B结合
Figure PCTCN2022091187-appb-000011
表4h突变体与HIS1K传感器上的CD64结合
Figure PCTCN2022091187-appb-000012
表4i突变体与HIS1K传感器上的FcRn结合
Sample ID Response KD(M) kon(1/Ms) kdis(1/s)
Fc-25 0.2392 2.84E-08 4.30E+05 1.22E-02
Fc-26 0.3488 5.09E-08 4.19E+05 2.13E-02
Fc-27 0.2855 6.23E-08 4.57E+05 2.84E-02
Fc-28 0.3545 4.55E-08 3.80E+05 1.73E-02
Fc-29 0.6114 3.28E-08 4.26E+05 1.40E-02
Fc-30 0.4914 2.21E-08 4.49E+05 9.93E-03
Fc-31 0.5716 2.58E-08 4.34E+05 1.12E-02
Fc-32 0.4803 2.39E-08 4.44E+05 1.06E-02
实验例4.生物膜薄层干涉技术测定本发明Fc突变体与C1q的亲和力
抗体Fc与C1q的亲和力的强弱直接决定了抗体是否具有CDC效应子功能，因此本实施例通过检测各个Fc突变体与C1q的亲和力来判断该Fc突变体是否还具有CDC效应子功能。
采用生物膜薄层干涉测定技术(BLI)测定本发明抗体结合人C1q的亲和力(KD)。BLI法亲和力测定按照现有的方法(Estep,P等人，High throughput solution Based measurement of antibody-antigen affinity and epitope binning.MAbs,2013.5(2):第270-8页)进行。
首先使用EZ-LinkTM Sulfo-NHS-LC-Biotin(21327，Thermo Scientific)生物素与抗体按摩尔比3：1混合后静置进行生物素标记，离心去除未标记的生物素，并将抗体等体积置换至PBS溶液中。
实验开始前半个小时，根据样品数量，取合适数量的SA传感器(Foretbio,18-5019)浸泡于SD缓冲液(1x PBS，0.1％BSA，0.05％Tween-20)中。将抗体稀释至约100nM，C1q(A099,Complement Technology)稀释至40nM。
分别将200μl的SD缓冲液、200ul标记的抗体溶液、200ulC1q抗原加入到96孔黑色聚苯乙烯微孔板(Greiner，655209)中。使用Fortebio Octet Red96e进行检测，根据样品位置布板，选择传感器位置。仪器设置参数如下：运行步骤：基线、加样～3nm、基线、缔合(Kon)和解离(Kdis)；各个步骤运行时间取决于样品结合和解离速度，转速为1000rpm，温度为30℃。实验完成后，使用ForteBio Octet分析软件分析KD值，导出坐标轴数据使用Graphpad Prism 8软件作图。
结果如表5和图2k所示，只有Fcmut-01可与C1q结合，亲和力为2.02E-08，Fcmut-2～Fcmut-8都不与C1q结合。说明本研究的Fc突变体可以减弱或阻止Fc与C1q的结合，因而可以降低或消除抗体的CDC效应子功能。与相应的野生型对照相比，Fcmut-10～Fcmut-16、Fcmut-18～Fcmut-24同样不与C1q结合。此外，与Fcmut-25相比，Fcmut-26～Fcmut-32同样不与C1q结合。
表5.Fc突变体与C1q的亲和力
Figure PCTCN2022091187-appb-000013
实验例5.生物膜薄层干涉技术测定本发明的抗体与抗原的亲和力
除以下内容，采用与实施例4所述的方法相同的BLI法进行亲和力的检测。
实验开始前半个小时，根据样品数量，取合适数量的AHC(Foretbio,18-5060)传感器浸泡于SD缓冲液(1x PBS，0.1％BSA，0.05％Tween-20)中。将抗体和Claudin18.2(cp0007,Genscript)分别稀释至100nM。
取200μl的SD缓冲液、200μl的抗体、200μl的Claudin18.2抗原分别加入到96孔黑色聚苯乙烯微孔板(Greiner，655209)中。使用Fortebio Octet Red96e进行检测，根据样品位置布板，选择传感器位置。仪器设置参数如下：运行步骤：基线、加样、基线、缔合(Kon)和解离(Kdis)；各个步骤运行时间取决于样品结合和解离速度，转速为1000rpm，温度为30℃。实验完成后，使用ForteBio Octet分析软件分析KD值。
结果见表6，Fcmut-02～08和抗原的KD值与Fcmut-01和抗原的KD值处于相同的数量级，因此说明本发明公开的在Fc区的具体突变不会影响相应抗体与其抗原的亲和力，进而表明包含本发明Fc突变体的抗体将保留其自有的抗原亲和力。
表6.抗体与抗原的亲和力
Figure PCTCN2022091187-appb-000014
Figure PCTCN2022091187-appb-000015
实施例6.抗体介导的ADCC效应
FcγR介导的主要效应子功能之一为抗体依赖的细胞介导的细胞毒性作用(ADCC)，其由NK细胞和巨噬细胞上的FcγRIIIA(CD16A)介导。在前面实施例研究了本申请获得的携带Fc突变的抗体与Fc受体的亲和力之后，本实施例继续探讨所述抗体突变体的ADCC效应子功能。
本实施例使用Promega公司的Jurkat-ADCCNF-AT luciferase效应细胞株(以下简称ADCC效应细胞)，通过检测NF-AT信号的激活情况，从而检测抗体的ADCC活性。具体实验过程如下：
1)细胞准备
对表面过表达人claudin18.2的细胞DANG-18.2(CLS Cell Lines Service)和ADCC效应细胞进行细胞计数：离心消除细胞DANG-18.2和ADCC效应细胞的上清，并用PBS溶液洗涤细胞两次，然后用检测培养基(含5％低IgG血清的1640培养基(Gibco))重悬细胞，将ADCC效应细胞的浓度调整为6×10 6个/mL，将DANG-18.2细胞的浓度调整为1×10 6个/ml。
2)铺板：将靶细胞DANG-18.2铺96孔板，每孔25uL。
3)加入系列稀释的本发明的抗体：每个携带Fc区突变的抗体样品及对照样品的起始浓度如表7所列，然后进行三倍稀释，共获得10个稀释梯度，分别依次加入孔板中，每孔25uL。
表7.抗体的起始浓度
Figure PCTCN2022091187-appb-000016
4)将ADCC效应细胞加入各个孔板中，每孔25uL。
5)37℃培养箱中孵育12小时。
6)取出96孔板，室温放置10分钟，每孔加入解冻的Luciferase测试试剂(Bio-Glo TM Luciferase assaγreagent)75ul。用酶标仪检测，用GraphPad 软件拟合浓度依赖的曲线。
检测结果如图4所示，对照抗体Fcmut-01具有野生型IgG1单克隆抗体的Fc区，其通过结合靶细胞(DANG-18.2)上的抗原，可以有效激活ADCC效应细胞的NF-AT信号，从而启动ADCC的下游信号通路，表明该抗体具有优良的ADCC杀伤能力。然而，本申请获得的携带Fc区突变的其他抗体则表现出非常弱或几近于无的ADCC效应，具体而言：突变体Fcmut-05(携带L234A&L235A突变)在10倍大于对照Fcmut-01的浓度下展示出非常弱的ADCC活性，而其他的突变体即便在10倍大于对照Fcmut-01的高浓度下也没有ADCC活性，表明本申请获得的携带Fc区突变的抗体分子的ADCC效应功能实质上被消除，该结果也与实施例3中各个抗体分子与CD16A的亲和力数据一致。
根据现有技术可知，通过对Fc区进行LALA突变，可以显著降低抗体的ADCC效应，本实施例的结果表明相比较于LALA突变，本申请获得的Fc突变体对ADCC效应的降低效果更大。
本申请通过对抗体Fc区第329位、330位、234位或235位的一个或者多个氨基酸进行突变修饰(例如，缺失、取代)获得的抗体突变体基本上消除了ADCC效应子功能，由此表明，上述氨基酸位置对于抗体的ADCC/ADCP效应子功能至关重要。当目标抗体在实际需求中需要避免ADCC/ADCP效应子功能时，本领域技术人员将可以根据本申请公开的内容选择对Fc区第329位、330位、234位或235位的一个或者多个氨基酸进行相应修饰，从而产生实际的技术效果。具体而言，可以考虑缺失抗体Fc区的第329位氨基酸、缺失第329-330位氨基酸、缺失第329位氨基酸以及取代第320位氨基酸，以及将上述修饰与LALA修饰(L234A+L235A)组合应用，从而获得消除ADCC/ADCP效应子功能的抗体分子。

Claims (15)

  1. 一种Fc突变体，其相对于人野生型Fc区包含一个或者多个氨基酸修饰，所述修饰选自依据Kabat中的EU索引编号的第329位、第234位、第235位或第330位氨基酸。
  2. 权利要求1的Fc突变体，其包含如下修饰：
    1)依据Kabat中的EU索引编号的第329位氨基酸的缺失(Δ329)；或
    2)依据Kabat中的EU索引编号的第329位和第330位氨基酸的缺失(Δ329和Δ330)；或
    4)依据Kabat中的EU索引编号的如下修饰：Δ329+A330G或者Δ329+S330G；或
    5)依据Kabat中的EU索引编号的如下修饰：L234A+L235A+Δ329、V234A+Δ329或F234A+L235A+Δ329；或
    6)依据Kabat中的EU索引编号的如下修饰：L234A+L235A+Δ329+Δ330、V234A+Δ329+Δ330或F234A+L235A+Δ329+Δ330；或
    7)依据Kabat中的EU索引编号的如下修饰：L234A+L235A+A330G+Δ329、L234A+L235A+S330G+Δ329、V234A+L235A+A330G+Δ329、V234A+L235A+S330G+Δ329、F234A+L235A+S330G+Δ329或F234A+L235A+A330G+Δ329。
  3. 权利要求1或2所述的Fc突变体，其为人IgG型Fc突变体，优选地其为IgG1、IgG2、IgG4型Fc突变体。
  4. 权利要求1-3中任一项所述Fc突变体，其包含如下序列：
    1)SEQ ID NO:2中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    2)SEQ ID NO:3中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、 甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    3)SEQ ID NO:4中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    4)SEQ ID NO:5中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    5)SEQ ID NO:6中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    6)SEQ ID NO:7中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    7)SEQ ID NO:8中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    8)SEQ ID NO:11中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    9)SEQ ID NO:12中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    10)SEQ ID NO:13中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    11)SEQ ID NO:14中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    12)SEQ ID NO:15中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    13)SEQ ID NO:16中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    14)SEQ ID NO:17中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    15)SEQ ID NO:19中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    16)SEQ ID NO:20中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    17)SEQ ID NO:21中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    18)SEQ ID NO:22中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    19)SEQ ID NO:23中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    20)SEQ ID NO:24中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    21)SEQ ID NO:25中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    22)SEQ ID NO:27中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    23)SEQ ID NO:28中按照EU索引第221-447位氨基酸的序列，或者与 该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    24)SEQ ID NO:29中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    25)SEQ ID NO:30中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    26)SEQ ID NO:31中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    27)SEQ ID NO:32中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成，或
    28)SEQ ID NO:33中按照EU索引第221-447位氨基酸的序列，或者与该序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、甚至更高的同一性的氨基酸序列，或者由该序列组成。
  5. 一种多肽，其包含权利要求1-4中任一项所述的Fc突变体。
  6. 权利要求5所述的多肽，其是抗体分子，优选地，所述抗体分子是IgG类抗体分子。
  7. 权利要求6所述的包含Fc突变体的抗体分子，其包含如下的重链和轻链：
    1)包含SEQ ID NO:2所示的重链和SEQ ID NO:9所示的轻链；或
    2)包含SEQ ID NO:3所示的重链和SEQ ID NO:9所示的轻链；或
    3)包含SEQ ID NO:4所示的重链和SEQ ID NO:9所示的轻链；或
    4)包含SEQ ID NO:5所示的重链和SEQ ID NO:9所示的轻链；或
    5)包含SEQ ID NO:6所示的重链和SEQ ID NO:9所示的轻链；或
    6)包含SEQ ID NO:7所示的重链和SEQ ID NO:9所示的轻链；或
    7)包含SEQ ID NO:8所示的重链和SEQ ID NO:9所示的轻链；或
    8)包含SEQ ID NO:11所示的重链和SEQ ID NO:9所示的轻链；或
    9)包含SEQ ID NO:12所示的重链和SEQ ID NO:9所示的轻链；或
    10)包含SEQ ID NO:13所示的重链和SEQ ID NO:9所示的轻链；或
    11)包含SEQ ID NO:14所示的重链和SEQ ID NO:9所示的轻链；或
    12)包含SEQ ID NO:15所示的重链和SEQ ID NO:9所示的轻链；或
    13)包含SEQ ID NO:16所示的重链和SEQ ID NO:9所示的轻链；或
    14)包含SEQ ID NO:17所示的重链和SEQ ID NO:9所示的轻链；或
    15)包含SEQ ID NO:19所示的重链和SEQ ID NO:9所示的轻链；或
    16)包含SEQ ID NO:20所示的重链和SEQ ID NO:9所示的轻链；或
    17)包含SEQ ID NO:21所示的重链和SEQ ID NO:9所示的轻链；或
    18)包含SEQ ID NO:22所示的重链和SEQ ID NO:9所示的轻链；或
    19)包含SEQ ID NO:23所示的重链和SEQ ID NO:9所示的轻链；或
    20)包含SEQ ID NO:24所示的重链和SEQ ID NO:9所示的轻链；或
    21)包含SEQ ID NO:25所示的重链和SEQ ID NO:9所示的轻链；或
    22)包含SEQ ID NO:27所示的重链和SEQ ID NO:34所示的轻链；或
    23)包含SEQ ID NO:28所示的重链和SEQ ID NO:34所示的轻链；或
    24)包含SEQ ID NO:29所示的重链和SEQ ID NO:34所示的轻链；或
    25)包含SEQ ID NO:30所示的重链和SEQ ID NO:34所示的轻链；或
    26)包含SEQ ID NO:31所示的重链和SEQ ID NO:34所示的轻链；或
    27)包含SEQ ID NO:32所示的重链和SEQ ID NO:34所示的轻链；或
    28)包含SEQ ID NO:33所示的重链和SEQ ID NO:34所示的轻链。
  8. 一种药物组合物，其包含权利要求1-4任一项所述的Fc突变体，或者包含权利要求5-7中任一项所述的多肽，及可药用载体。
  9. 权利要求1-4任一项所述的Fc突变体在减少或者消除ADCC、ADCP或CDC效应子功能中的用途。
  10. 编码权利要求1-4任一项所述的Fc突变体、或编码权利要求5-7中任一项所述的多肽的核酸分子。
  11. 一种载体，其包含权利要求10所述的核酸分子。
  12. 一种宿主细胞，其包含权利要求10所述的核酸分子或11所述的载体。
  13. 一种治疗受试者的肿瘤的方法，包括将有效量的权利要求8所述的药物组合物或权利要求1-4中任一项所述的Fc突变体，或者权利要求5-7中任一项所述的多肽施用给有需要的受试者。
  14. 一种试剂盒，其包含权利要求1-4任一项所述的Fc突变体、权利要求5-7中任一项所述的多肽。
  15. 一种制备如权利要求1-4中任一项所述的Fc突变体，或者制备如权利要求5-7中任一项所述的多肽的方法，包括在适当的条件下在如权利要求12所述的宿主细胞中表达如权利要求10所述的核酸分子或者表达如权利要求11所述的载体，任选地，所述方法还包括回收表达的Fc突变体或多肽。
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