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

Info

Publication number
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
Authority
WO
WIPO (PCT)
Prior art keywords
seq
sequence
chain shown
amino acid
index
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2022/091187
Other languages
English (en)
French (fr)
Chinese (zh)
Inventor
付凤根
周帅祥
吴志海
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Innovent Biologics Suzhou Co Ltd
Original Assignee
Innovent Biologics Suzhou Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Innovent Biologics Suzhou Co Ltd filed Critical Innovent Biologics Suzhou Co Ltd
Priority to CA3218187A priority Critical patent/CA3218187A1/en
Priority to EP22798660.1A priority patent/EP4335868A4/en
Priority to JP2023568549A priority patent/JP2024516320A/ja
Priority to US18/558,655 priority patent/US20240228579A1/en
Priority to CN202280033488.0A priority patent/CN117295760A/zh
Priority to AU2022269145A priority patent/AU2022269145A1/en
Publication of WO2022233320A1 publication Critical patent/WO2022233320A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], 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)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Mycology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Microbiology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
PCT/CN2022/091187 2021-05-07 2022-05-06 与Fc受体结合改变的Fc突变体 Ceased WO2022233320A1 (zh)

Priority Applications (6)

Application Number Priority Date Filing Date Title
CA3218187A CA3218187A1 (en) 2021-05-07 2022-05-06 Fc mutant with altered binding to fc receptor
EP22798660.1A EP4335868A4 (en) 2021-05-07 2022-05-06 FC MUTANT WITH ALTERED FC RECEPTOR BINDING
JP2023568549A JP2024516320A (ja) 2021-05-07 2022-05-06 Ｆｃ受容体への結合が改変されたＦｃ変異体
US18/558,655 US20240228579A1 (en) 2021-05-07 2022-05-06 Fc mutant with altered binding to fc receptor
CN202280033488.0A CN117295760A (zh) 2021-05-07 2022-05-06 与Fc受体结合改变的Fc突变体
AU2022269145A AU2022269145A1 (en) 2021-05-07 2022-05-06 Fc mutant with altered binding to fc receptor

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202110495363 2021-05-07
CN202110495363.7 2021-05-07
CN202210436873 2022-04-25
CN202210436873.1 2022-04-25

Publications (1)

Publication Number Publication Date
WO2022233320A1 true WO2022233320A1 (zh) 2022-11-10

Family

ID=83932014

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/091187 Ceased WO2022233320A1 (zh) 2021-05-07 2022-05-06 与Fc受体结合改变的Fc突变体

Country Status (8)

Country Link
US (1) US20240228579A1 (https=)
EP (1) EP4335868A4 (https=)
JP (1) JP2024516320A (https=)
CN (1) CN117295760A (https=)
AU (1) AU2022269145A1 (https=)
CA (1) CA3218187A1 (https=)
TW (1) TWI839729B (https=)
WO (1) WO2022233320A1 (https=)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025047847A1 (ja) * 2023-08-30 2025-03-06 アステラス製薬株式会社 Ｆｃ領域改変体

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004092219A2 (en) * 2003-04-10 2004-10-28 Protein Design Labs, Inc Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
CN101987870A (zh) * 2004-07-15 2011-03-23 赞科股份有限公司 优化的Fc变体
US8734791B2 (en) 2002-03-01 2014-05-27 Xencor, Inc. Optimized fc variants and methods for their generation
US8883147B2 (en) 2004-10-21 2014-11-11 Xencor, Inc. Immunoglobulins insertions, deletions, and substitutions
CN104395339A (zh) * 2012-06-27 2015-03-04 弗·哈夫曼-拉罗切有限公司 用于选择并产生含有至少两种不同结合实体的定制高度选择性和多特异性靶向实体的方法及其用途
CN103476795B (zh) 2011-03-29 2016-07-06 罗切格利卡特公司 抗体Fc变体
CN108713027A (zh) * 2015-12-17 2018-10-26 詹森生物科技公司 特异性结合hla-dr的抗体及其用途
US10183999B2 (en) 2002-09-27 2019-01-22 Xencor, Inc. Optimized Fc variants and methods for their generation
CN109963871A (zh) * 2016-08-05 2019-07-02 豪夫迈·罗氏有限公司 具有激动活性的多价及多表位抗体以及使用方法
WO2020039321A2 (en) * 2018-08-20 2020-02-27 Pfizer Inc. Anti-gdf15 antibodies, compositions and methods of use

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6737056B1 (en) * 1999-01-15 2004-05-18 Genentech, Inc. Polypeptide variants with altered effector function
US10053513B2 (en) * 2009-11-30 2018-08-21 Janssen Biotech, Inc. Antibody Fc mutants with ablated effector functions
TW201245227A (en) * 2011-03-29 2012-11-16 Roche Glycart Ag Antibody Fc variants
CN113603780B (zh) * 2017-03-28 2022-07-01 礼进生物医药科技（上海）有限公司 用于增强肿瘤微环境中免疫应答的治疗剂和方法

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8734791B2 (en) 2002-03-01 2014-05-27 Xencor, Inc. Optimized fc variants and methods for their generation
US10183999B2 (en) 2002-09-27 2019-01-22 Xencor, Inc. Optimized Fc variants and methods for their generation
WO2004092219A2 (en) * 2003-04-10 2004-10-28 Protein Design Labs, Inc Alteration of fcrn binding affinities or serum half-lives of antibodies by mutagenesis
CN101987870A (zh) * 2004-07-15 2011-03-23 赞科股份有限公司 优化的Fc变体
US8883147B2 (en) 2004-10-21 2014-11-11 Xencor, Inc. Immunoglobulins insertions, deletions, and substitutions
CN103476795B (zh) 2011-03-29 2016-07-06 罗切格利卡特公司 抗体Fc变体
CN104395339A (zh) * 2012-06-27 2015-03-04 弗·哈夫曼-拉罗切有限公司 用于选择并产生含有至少两种不同结合实体的定制高度选择性和多特异性靶向实体的方法及其用途
CN108713027A (zh) * 2015-12-17 2018-10-26 詹森生物科技公司 特异性结合hla-dr的抗体及其用途
CN109963871A (zh) * 2016-08-05 2019-07-02 豪夫迈·罗氏有限公司 具有激动活性的多价及多表位抗体以及使用方法
WO2020039321A2 (en) * 2018-08-20 2020-02-27 Pfizer Inc. Anti-gdf15 antibodies, compositions and methods of use

Non-Patent Citations (15)

* Cited by examiner, † Cited by third party
Title
ADVANCES IN IMMUNOLOGY
ANAND: "Techniques for the Analysis of Complex Genomes", 1992, ACADEMIC PRESS
ARMOUR ET AL., EUR. J. IMMUNOL., vol. 29, no. 8, 1999, pages 2613 - 2624
AUSUBEL: "Current Protocols in Molecular Biology", July 2008, JOHN WILEY AND SONS
E. MEYERSW MILLER, CABIOS, vol. 4, 1989, pages 11 - 17
ESTEP, P ET AL.: "High throughput solution based measurement of antibody-antigen affinity and epitope binding", MABS, vol. 5, no. 2, 2013, pages 270 - 8, XP055105281, DOI: 10.4161/mabs.23049
GLOVER: "Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology", vol. 1, 2, 1985, GREENE PUB. ASSOCIATES AND WILEY-INTERSCIENCE
HARLOWLANE: "Antibodies", 1998, COLD SPRING HARBOR LABORATORY PRESS
IDUSOGIE EE ET AL., J. IMMUNOL, vol. 164, no. 8, 2000, pages 4178 - 84
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, PUBLIC HEALTH SERVICE, NATIONAL INSTITUTES OF HEALTH
MANIATIS ET AL.: "Molecular Cloning: A Laboratory Manual", 1982
NEEDLEMAWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444 - 453
PERBAL: "A Practical Guide to Molecular Cloning", 1984
See also references of EP4335868A4
SHIELDS ET AL., J. BIOL. CHEM., vol. 276, no. 9, 2001, pages 6591 - 6604

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025047847A1 (ja) * 2023-08-30 2025-03-06 アステラス製薬株式会社 Ｆｃ領域改変体

Also Published As

Publication number Publication date
JP2024516320A (ja) 2024-04-12
EP4335868A1 (en) 2024-03-13
EP4335868A4 (en) 2025-08-13
US20240228579A1 (en) 2024-07-11
CN117295760A (zh) 2023-12-26
TW202246313A (zh) 2022-12-01
TWI839729B (zh) 2024-04-21
CA3218187A1 (en) 2022-11-10
AU2022269145A1 (en) 2023-12-21

Similar Documents

Publication Publication Date Title
JP2024029183A (ja) 細胞傷害誘導治療剤
JP2020508666A (ja) 認知障害を治療または予防するためのヒト化抗体、その製造プロセス、及びそれを用いた認知障害を治療または予防するための薬剤
CN111601821B (zh) 具有对FcRn的亲和力提高以及对至少一个Fc片段受体的亲和力提高的Fc片段的变体
JP7731359B2 (ja) ヘテロ二量体Ｆｃポリペプチド
JP2025098167A (ja) Ｆｃｒｎアンタゴニストで抗体媒介性障害を処置する方法
JP7842914B2 (ja) Ｃ５ａＲを標的とする抗体
JP2025517120A (ja) アルブミン結合ポリペプチド及びその使用
TWI839729B (zh) 與Fc受體結合改變的Fc突變體
CN115175736A (zh) 杂交抗体
CN116783220A (zh) 抗bcma抗体及其制备方法和应用
CN117715940A (zh) 抗trem-1抗体
TW202409086A (zh) FcRn-結合多肽及其用途
TW202607021A (zh) 結合ｔｓｌｐ之抗體及使用方法
WO2025185738A1 (zh) 靶向cxcr3的抗原结合蛋白及其应用
JP2026514143A (ja) Ｏｘ４０ｌに結合する抗体及びその使用方法
HK40061359A (en) Antibodies targeting c5ar
HK40061359B (zh) 靶向c5ar的抗体
Mårtensson Development of an Antigen-independent Affinity Assay to study the Binding of IgG to FcgammaR

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22798660

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 3218187

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 202280033488.0

Country of ref document: CN

Ref document number: 2023568549

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 2022269145

Country of ref document: AU

Ref document number: AU2022269145

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 2022798660

Country of ref document: EP

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2022798660

Country of ref document: EP

Effective date: 20231207

ENP Entry into the national phase

Ref document number: 2022269145

Country of ref document: AU

Date of ref document: 20220506

Kind code of ref document: A