WO2022265331A1 - Variants fc à mécanisme immunitaire contrôlé et demi-vie dans le sang accrue - Google Patents

Variants fc à mécanisme immunitaire contrôlé et demi-vie dans le sang accrue Download PDF

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WO2022265331A1
WO2022265331A1 PCT/KR2022/008336 KR2022008336W WO2022265331A1 WO 2022265331 A1 WO2022265331 A1 WO 2022265331A1 KR 2022008336 W KR2022008336 W KR 2022008336W WO 2022265331 A1 WO2022265331 A1 WO 2022265331A1
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antibody
polypeptide
domain
variant
amino acid
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Korean (ko)
Inventor
정상택
고상환
최소영
손명호
박소라
김은성
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Korea University Research and Business Foundation
Osong Medical Innovation Foundation
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Korea University Research and Business Foundation
Osong Medical Innovation Foundation
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Priority claimed from KR1020220070880A external-priority patent/KR102754724B1/ko
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    • 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
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present invention relates to Fc variants with reduced or silenced effector function and/or extended half-life in blood and a method for preparing the same.
  • Therapeutic antibodies are being developed for the treatment of a variety of diseases, the efficacy of some of which derives in part from the Fc region of the antibody mediating one or more effector functions. These effector functions result from the interaction of antibodies and antibody-antigen complexes with cells of the immune system to stimulate a variety of responses, including antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) (Daeron, Annu Rev. Immunol.15:203-234 (1997)).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • the effector functions mediated by the Fc region may cause undesirable adverse effects, and thus the development of antibodies with reduced or silenced effector functions is required.
  • the present inventors discover novel Fc variants that have the effect of extending half-life while effector functions of the Fc region, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), are reduced or silenced. made an earnest effort to do so.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • an object of the present invention is to provide a polypeptide comprising an antibody Fc variant, wherein the Fc variant has reduced binding ability to Fc ⁇ RIIIa compared to a wild type antibody Fc domain.
  • Another object of the present invention is to provide an antibody comprising the polypeptide.
  • Another object of the present invention is to provide a nucleic acid molecule encoding the polypeptide.
  • Another object of the present invention is to provide a vector containing the nucleic acid molecule.
  • Another object of the present invention is to provide a host cell containing the vector.
  • Another object of the present invention is to provide a method for producing a polypeptide comprising an Fc variant having a reduced effector function compared to a wild-type Fc domain.
  • Another object of the present invention is to provide a method for preparing an antibody having a reduced effector function compared to a wild type antibody.
  • Another object of the present invention is an effector function comprising administering a pharmaceutical composition comprising a nucleic acid molecule encoding the polypeptide or a vector containing the nucleic acid molecule to a subject in need thereof. function) to provide a method for delivering an Fc variant with reduced function.
  • the present invention is a polypeptide comprising an antibody Fc variant, wherein the Fc variant has reduced binding ability to Fc ⁇ RIIIa compared to a wild type antibody Fc domain, a polypeptide provides
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • An antibody is a protein that specifically binds to a particular antigen, and natural antibodies are usually heterodimeric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains.
  • IgA immunoglobulin A
  • IgD immunoglobulin D
  • IgE immunoglobulin D
  • IgG immunoglobulin G
  • IgM immunoglobulin M
  • Papain digestion of antibodies forms two Fab fragments and one Fc fragment, and in human IgG molecules, the Fc region is generated by papain digestion of the N-terminus of Cys 226 (Deisenhofer, Biochemistry 20: 2361-2370, 1981) .
  • the term "Fc region” is used herein to define the C-terminal region of an antibody.
  • the Fc region consists of two identical protein fragments derived from the second and third constant domains of the two heavy chains of an antibody: chain A and chain B.
  • the second and third constant domains are known as CH2 domains and CH3 domains, respectively.
  • the CH2 domain includes the CH2 domain sequence of chain A and the CH2 domain sequence of chain B.
  • the CH3 domain includes the CH3 domain sequence of chain A and the CH3 domain sequence of chain B.
  • the Fc region includes the hinge region defined below.
  • Fc region or “Fc domain” is used to define the C-terminal region of an immunoglobulin heavy chain.
  • An “Fc region sequence” may be a native Fc region sequence or a variant Fc region sequence. Although the boundaries of the Fc region sequences of immunoglobulin heavy chains can vary, human IgG heavy chain Fc region sequences are usually defined as extending from the amino acid residues at position Cys226 or Pro230 to their carboxyl termini.
  • CH2 domain sequence (also called “Cy2” domain sequence) of a human IgG Fc region sequence usually extends from about amino acid 231 to about amino acid 340.
  • a "CH3 domain sequence” includes the extension of the C-terminal residues from the Fc region sequence to the CH2 domain sequence (ie, from about amino acid residue 341 to about amino acid residue 447 of an IgG).
  • a “functional Fc region” retains the "effector function” or "effector function” of a wild-type Fc region.
  • exemplary “effector functions” include C1q binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (eg B cell receptor; BCR); and the like.
  • Such effector functions generally require an Fc region to be combined with a binding domain (eg an antibody variable domain) and can be assessed using, for example, various assays disclosed herein.
  • Wild-type Fc region sequence includes an amino acid sequence identical to that of an Fc region found in nature. Wild-type Fc regions include wild-type human IgG1 Fc regions (non-A and A allotypes); wild-type human IgG2 Fc region; wild-type human IgG3 Fc region; and wild-type human IgG4 Fc regions as well as naturally occurring variants thereof.
  • a “variant Fc region sequence” includes an amino acid sequence that differs from a wild-type Fc region sequence by “one or more amino acid modifications” as defined herein.
  • the variant Fc region sequence has at least one amino acid substitution compared to the wild-type Fc region sequence or the Fc region sequence of the parent polypeptide, for example, from about 1 to about 10 amino acid substitutions in the wild-type Fc region sequence or the Fc region sequence of the parent polypeptide; preferably from about 1 to about 5 amino acid substitutions.
  • a variant Fc region sequence herein has at least about 80% identity to, most preferably at least about 90% identity to, more preferably at least about 90% identity to, a wild-type Fc region sequence or an Fc region sequence of a parent polypeptide. have at least about 95% identity with
  • Fc receptor or “FcR” is used to describe a receptor that binds to the Fc region of an antibody.
  • Preferred FcRs are native human FcRs.
  • an FcR is one that binds an IgG antibody (gamma receptor, Fc ⁇ R), including allelic variants and alternatively spliced forms of these receptors, Fc ⁇ RI (CD64), Fc ⁇ RII (CD32), and receptors of the Fc ⁇ RIII (CD16) subclass.
  • FcR also includes the neonatal receptor, FcRn, which is involved in the transfer of maternal IgGs to the fetus (Guyer et al, J. Immunol. 1 17:587 (1976)).
  • Fc ⁇ R does not include FcRn.
  • Antibody-dependent cell-mediated cytotoxicity and “ADCC” refer to non-specific cytotoxic cells expressing FcRs (e.g., natural killer (NK) cells) that recognize antibodies bound on target cells and subsequently induce lysis of the target cells. cells, neutrophils, and macrophages).
  • FcRs e.g., natural killer (NK) cells
  • NK cells the main cells mediating ADCC, express Fc ⁇ RIII and low levels of Fc ⁇ RIIc
  • monocytes express Fc ⁇ Rl, Fc ⁇ RII and Fc ⁇ RIII.
  • a “human effector cell” is a leukocyte that expresses one or more FcRs and performs effector functions. Preferably, the cell expresses at least Fc ⁇ RIII and performs ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils; PBMCs and NK cells are preferred. Effector cells can be isolated from their natural source, eg blood or PBMCs.
  • PBMCs peripheral blood mononuclear cells
  • NK natural killer cells
  • Effector cells can be isolated from their natural source, eg blood or PBMCs.
  • Fc variants with “reduced”, “silenced”, “negligible” or “negligible” Fc ⁇ R binding affinity and C1q binding affinity are compared to the parent polypeptide or a polypeptide comprising a wild-type Fc region sequence. It has reduced Fc ⁇ R binding activity and C1q binding activity.
  • Fc variants with “reduced”, “silenced”, “negligible” or “removed” Fc ⁇ R binding affinity and C1q binding affinity also have a parent polypeptide or wild-type Fc region sequence.
  • the Fc variant has reduced ADCC compared to the wild-type antibody Fc domain.
  • the Fc variant has reduced CDC compared to the wild-type antibody Fc domain.
  • an Fc variant that “displays reduced or undetectable binding” to an Fc ⁇ R binds all Fc ⁇ Rs with lower affinity than the parent polypeptide. Such variants that exhibit reduced binding to Fc ⁇ R may retain little or no appreciable binding to Fc ⁇ R.
  • the variant exhibits 0-10% binding to an Fc ⁇ R relative to a native IgG Fc region, as determined, for example, in the Examples herein or as measured by change in equilibrium constant.
  • the variant exhibits 0-5% binding to an Fc ⁇ R compared to a native IgG Fc region.
  • the variant exhibits 0-3% binding to an Fc ⁇ R compared to a native IgG Fc region.
  • the variant exhibits 0-1% binding to an Fc ⁇ R relative to a native IgG Fc region.
  • Parent antibody refers to a construct that does not contain amino acid modifications to the hinge region.
  • the parent antibody is one that does not contain amino acid modifications to the hinge region and does contain modifications to the CH3 domain that promote formation of a heterodimeric Fc region.
  • amino acid modification refers to a change in the amino acid sequence of a predetermined amino acid sequence. Exemplary modifications include amino acid substitutions, insertions and/or deletions.
  • an amino acid modification herein is a substitution.
  • an "amino acid modification" at a specified position in an Fc region refers to a substitution or deletion of a specified residue, or insertion of at least one amino acid residue adjacent to the specified residue.
  • An insertion "adjacent to" a specified residue means an insertion within one or both of these residues.
  • the insertion is N-terminal or C-terminal to a specified residue.
  • amino acid substitution refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence with another different “alternative” amino acid residue.
  • the replacement residue or residues may be “naturally occurring amino acid residues” (ie those encoded by the genetic code) and may be selected from the group consisting of: alanine (Ala); arginine (Arg); asparagine (Asn); aspartic acid (Asp); cysteine (Cys); glutamine (Gln); glutamic acid (Glu); glycine (Gly); histidine (His); Isoleucine (Ile): Leucine (Leu); Lysine (Lys); methionine (Met); phenylalanine (Phe); Proline (Pro): Serine (Ser); threonine (Thr); tryptophan (Trp); Tyrosine (Tyr); and valine (Val).
  • the replacement moiety is not cysteine.
  • substitutions with one or more non-naturally occurring amino acid residues are also encompassed by the definition of amino acid substitutions herein.
  • a "non-naturally occurring amino acid residue” refers to a residue other than the naturally occurring amino acid residues listed above, which may covalently bind adjacent amino acid residue(s) in a polypeptide chain. Examples of non-naturally occurring amino acid residues include norleucine, ornithine, norvaline, homoserine and other amino acid residue analogs such as Ellman et al. Meth. Enzym. 202:301-336 (1991).
  • An antibody Fc domain can be an Fc domain of an IgA, IgM, IgE, IgD, or IgG antibody, or variants thereof.
  • the domain is an Fc domain of an IgG antibody (eg, an Fc domain of an IgG1, IgG2a, IgG2b, IgG3, or IgG4 antibody).
  • the Fc domain may be an IgG1 Fc domain (eg, trastuzumab or rituximab Fc domain) or an IgG4 Fc domain (eg, nivolumab Fc domain).
  • polypeptide comprising the Fc domain of the present invention
  • some or all of the polypeptide may be non-glycated or glycosylated.
  • the polypeptide may contain one or more regions from antibodies in addition to the Fc domain.
  • the polypeptide may include an antibody-derived antigen binding domain, and a plurality of polypeptides may form an antibody or antibody-like protein.
  • amino acid residue numbers of the antibody Fc domain herein follow the Kabat EU numbering system commonly used in the art (Kabat et al., in Sequences of Proteins of Immunological Interest 5th Ed., U.S. Department of EU index number as in Health and Human Services, NIH Publication No. 91-3242, 1991).
  • the substituted Fc variant of the present invention comprises an amino acid substitution at one or more positions selected from the group consisting of S228, L234, L235, T299 and P329 according to the Kabat EU numbering system.
  • the Fc variants of the present invention have reduced binding ability to Fc ⁇ RIIIa compared to the wild-type antibody Fc domain.
  • the term "avidity” or "binding affinity” herein generally refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg antibody) and its binding partner (eg Fc ⁇ R).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd or KD). Affinity can be measured by common methods known in the art, including those described herein.
  • the Fc variants of the present invention preferably have a KD of 10 nM or less, more preferably a KD of 5 nM or less, even more preferably a KD of 3 nM or less, and even more preferably a KD of 1 nM or less for Fc ⁇ RIIIa. , most preferably exhibits a reduced binding force to an unmeasurable level.
  • Fc-fusion refers to a protein in which one or more polypeptides are operably linked to an Fc region or derivative thereof.
  • Fc fusions combine the Fc region of an immunoglobulin with a fusion partner, which generally can be any protein or small molecule.
  • the role of the non-Fc portion of an Fc fusion, i.e. the fusion partner, is to mediate target binding and is therefore functionally similar to the variable region of an antibody.
  • Virtually any protein or small molecule can be linked to Fc to create an Fc fusion.
  • a protein fusion partner may include, but is not limited to, a target-binding region of a receptor, junction attachment, ligand, enzyme, cytokine, chemokine, or some other protein or protein domain.
  • Small molecule fusion partners can include any therapeutic agent that induces Fc fusion to a therapeutic target.
  • a target may be any molecule, preferably an extracellular receptor involved in a disease.
  • GPCRs G-protein coupled receptors
  • ion channels including K+, Na+, Ca+ channels.
  • an Fc protein described herein may, for example, bind to one or more of GABA receptors, purinergic receptors, adrenergic receptors, histamine receptors, opioid receptors, chemokine receptors, glutamate receptors, nicotinic receptors, 5HT (serotonin) receptors, and estrogen receptors.
  • a fusion partner can be a small molecule mimic of a protein that targets a therapeutically useful target.
  • Specific examples of specific drugs that can act as Fc fusion partners are [L. S. Goodman et al, Eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics (McGraw-Hill, New York, ed. 9, 1996).
  • linkers can be used to covalently link an Fc to a fusion partner to create an Fc fusion.
  • the Fc variant comprises one or more amino acid substitutions selected from the group consisting of S228P, L234A, L235A, L235E, T299L and P329G according to the Kabat EU numbering system in the Fc domain of the wild type antibody.
  • the Fc variant comprises the following amino acid substitutions according to the Kabat EU numbering system in the wild-type antibody Fc domain:
  • the Fc variant comprises an additional amino acid mutation for extending half-life compared to the wild-type antibody Fc domain.
  • the Fc variant may further include an amino acid substitution at one or more positions selected from the group consisting of M252, S254, T256, L309, Q311, M428 and N434 according to the Kabat EU numbering system in the Fc domain of the wild type antibody. there is.
  • said additional amino acid substitution may be selected from the group consisting of M252Y, S254T, T256E, L309G, Q311R, M428L and N434S.
  • the additional amino acid substitution may include the following amino acid substitution:
  • the additional amino acid substitution modulates binding and dissociation to the neonatal Fc receptor (FcRn).
  • FcRn neonatal Fc receptor
  • the Fc variant has a binding affinity to FcRn at pH 5.6 to 6.2 (preferably pH 5.8 to 6.0) of 10% or more, 20% or more compared to the wild-type Fc domain. ⁇ , ⁇ 30%, ⁇ 40%, ⁇ 50%, ⁇ 60%, ⁇ 70%, ⁇ 80%, ⁇ 90%, or ⁇ 100%, or at least 2-fold, 3-fold, or 4-fold greater than the wild-type Fc domain.
  • the degree of dissociation of the Fc variant from the neonatal Fc receptor (FcRn) at pH 7.0 to 7.8 is equal to or substantially greater than that of the wild-type Fc domain. may not change to
  • the substituted Fc variants of the present invention showed significantly improved binding affinity under slightly acidic conditions (e.g., pH 5.8 to 6.0) compared to wild-type Fc or other previously developed Fc variants. , showed the same or substantially equal or higher degree of dissociation under neutral conditions (eg, pH 7.0).
  • the substituted Fc variant of the present invention has an increased half-life compared to the wild-type antibody Fc domain.
  • the half-life of the substituted Fc variants of the present invention is 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more compared to the wild-type Fc domain. Increased by at least 100% or more, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold or at least 10-fold more than the wild-type Fc domain there is.
  • the substituted Fc variant of the present invention showed a significantly improved in vivo half-life compared to the wild type.
  • the present invention provides an antibody comprising the above polypeptide.
  • antibody herein is a polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, antibody mimetic, chimeric antibody, antibody conjugate, human antibody or humanized antibody, or means fragment.
  • the half-life of the Fc domain or a polypeptide including the Fc domain is maximized through optimization of the antibody Fc region, while reducing or silencing the effector function.
  • the present invention provides a nucleic acid molecule encoding the polypeptide, a vector containing the nucleic acid molecule, or a host cell containing the vector.
  • Nucleic acid molecules of the present invention may be isolated or recombinant, and include DNA and RNA in single-stranded and double-stranded form, as well as corresponding complementary sequences.
  • An isolated nucleic acid is a nucleic acid that has been separated from surrounding genetic sequences present in the genome of the individual from which the nucleic acid was isolated, in the case of a nucleic acid isolated from a naturally occurring source.
  • the nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule.
  • nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct.
  • Nucleic acids are operably linked when placed into a functional relationship with another nucleic acid sequence.
  • DNA of a full sequence or secretory leader is operably linked to DNA of a polypeptide when the polypeptide is expressed as a preprotein in its pre-secreted form, and a promoter or enhancer is the polypeptide sequence. is operably linked to a coding sequence when it affects transcription of, or when the ribosome binding site is positioned to facilitate translation.
  • operably linked means that the DNA sequences to be linked are contiguous, and in the case of a secretory leader, contiguous and in the same reading frame.
  • enhancers need not be contiguous.
  • Linkage is achieved by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.
  • vector refers to a carrier capable of inserting a nucleic acid sequence into a cell capable of replicating the nucleic acid sequence.
  • a nucleic acid sequence may be exogenous or heterologous.
  • Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, AAV, bacteriophage).
  • viruses eg, AAV, bacteriophage.
  • One skilled in the art can construct vectors by standard recombinant techniques (Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY, 1988; and Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994, etc.).
  • expression vector refers to a vector containing a nucleic acid sequence encoding at least a portion of a gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide.
  • Expression vectors may contain various regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.
  • the term host cell includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector.
  • the host cell may be transfected or transformed by the vector, which means a process in which an exogenous nucleic acid molecule is delivered or introduced into the host cell.
  • transformation is used as a meaning including the transfection (Transfected) and transduction (Transduced).
  • the (host) cells of the present invention are not limited, but preferably insect cells or mammalian cells, more preferably Sf9 in the case of insect cells, HEK293 cells in the case of mammalian cells, HeLa cells, ARPE-19 cells, RPE- 1 cell, HepG2 cell, Hep3B cell, Huh-7 cell, C8D1a cell, Neuro2A cell, CHO cell, MES13 cell, BHK-21 cell, COS7 cell, COP5 cell, A549 cell, MCF-7 cell, HC70 cell, HCC1428 cell , BT-549 cells, PC3 cells, LNCaP cells, Capan-1 cells, Panc-1 cells, MIA PaCa-2 cells, SW480 cells, HCT166 cells, LoVo cells, A172 cells, MKN-45 cells, MKN-74 cells, Kato-III cells, NCI-N87 cells, HT-144 cells, SK-MEL-2 cells, SH-SY5Y cells, C6 cells, HT-22 cells,
  • the host cell of the present invention is preferably an isolated host cell.
  • the present invention provides a method for producing a polypeptide comprising an Fc variant having a reduced effector function compared to a wild-type Fc domain, comprising the steps of:
  • the present invention provides a method for producing an antibody with reduced effector function compared to wild type, comprising the steps of:
  • antibody purification may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably Protein Affinity chromatography using A can be used.
  • the present invention provides a composition comprising the polypeptide, the antibody, the nucleic acid molecule or the vector comprising the Fc variant comprising the amino acid substitution.
  • the composition of the present invention is a pharmaceutical composition.
  • the present invention provides a pharmaceutical composition comprising a polypeptide comprising the Fc variant, a nucleic acid molecule encoding the polypeptide, or a vector comprising the nucleic acid molecule, to a subject in need thereof ( It provides a method for delivering an Fc variant with reduced effector function, comprising the step of administering to a subject.
  • the Fc variant of the present invention has a low Fc ⁇ RIIIa binding affinity and / or no ADCC (antibody dependent cellular cytotoxicity) to the degree of undetectable compared to the control group (eg, rituximab or nivolumab) and/or Complement-Dependent Cytotoxicity (CDC) While exhibiting activity, it has remarkable half-life increasing ability at the same time.
  • ADCC antibody dependent cellular cytotoxicity
  • the pharmaceutical composition of the present invention comprises (a) the polypeptide, a nucleic acid molecule encoding the polypeptide, or a vector containing the nucleic acid molecule; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention may be used for prevention or treatment of a specific disease (eg, tumor, viral or bacterial infection, etc.).
  • a specific disease eg, tumor, viral or bacterial infection, etc.
  • prevention refers to inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed with, but is likely to have, the disease or condition.
  • treatment refers to (a) inhibiting the development of a disease, disorder or condition; (b) alleviation of the disease, condition or symptom; or (c) eliminating the disease, disorder or condition.
  • the composition of the present invention may be a composition for treating viral infection by itself, or may be applied as a therapeutic adjuvant to improve its therapeutic response by being administered together with other pharmacological ingredients. Accordingly, the terms “treatment” or “therapeutic agent” herein include the meaning of “therapeutic aid” or "therapeutic agent”.
  • administration refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.
  • the term "pharmaceutically effective amount” or “therapeutically effective amount” refers to the content of the composition contained in a sufficient level to provide a therapeutic or preventive effect of the pharmacological component in the composition to the subject to whom the pharmaceutical composition of the present invention is to be administered. It means, and it is meant to include a “prophylactically effective amount” therein.
  • the term "subject” includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon or rhesus monkey. Specifically, the subject of the present invention is a human.
  • the pharmaceutical composition of the present invention contains a pharmaceutically acceptable carrier or excipient.
  • the pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container.
  • the pharmaceutical composition according to the present invention may be formulated and used in various forms according to conventional methods.
  • it can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions and syrups, and can be formulated and used in the form of external preparations, suppositories and sterile injection solutions.
  • composition of the present invention may contain one or more known active ingredients having anticancer activity, antiviral or antibacterial activity.
  • the pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenteral administration, such as intravenous injection, transdermal administration, subcutaneous injection, intramuscular injection, intravitreal injection, subretinal injection. Subretinal injection, suprachoroidal injection, eye drop administration, intracerebroventricular injection, intrathecal injection, intraamniotic injection, intra-arterial intraarterial injection, intraarticular injection, intracardiac injection, intracavernous injection, intracerebral injection, intracisternal injection, intracoronary injection (intracoronary injection), intracranial injection, intrathecal injection, epidural injection, intrahippocampal injection, intranasal injection, intraosseous injection injection), intraperitoneal injection, intrathoracic injection, intraspinal injection, intrathoracic injection, intrathymic injection, intrauterine injection , intravaginal injection ( It can be administered by intravaginal injection), intraventricular injection, intravesical injection, subconjunctival injection, intratumoral injection,
  • Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • injectable esters such as ethyl oleate
  • witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used as a base for the suppository.
  • the dosage of the pharmaceutical composition of the present invention may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type and degree of response to be achieved by administration of the pharmaceutical composition. , various factors, including the type of subject to be administered, age, weight, general health condition, symptoms or severity of disease, sex, diet, excretion, drugs used simultaneously or at the same time in the subject, and other components of the composition, and the like It can be varied according to similar factors well known in the medical field, and those skilled in the art can easily determine and prescribe an effective dosage for the desired treatment.
  • the administration route and administration method of the pharmaceutical composition of the present invention may be each independent, and are not particularly limited in the method, and any administration route and administration method as long as the pharmaceutical composition can reach the target site can follow
  • the present invention provides a polypeptide comprising an Fc variant in which part of the amino acid sequence of a human antibody Fc domain is substituted with another amino acid sequence, or an antibody comprising the same.
  • the present invention provides a method for preparing the polypeptide or antibody.
  • the Fc variant of the present invention can reduce or silence effector functions and maximize half-life in the body through optimization of some amino acid sequences.
  • Figure 1 shows the SDS-PAGE results of Rituximab (IgG1) Fc double mutant.
  • FIG. 2 shows the results of SEC-HPLC analysis of Rituximab (IgG1) Fc double mutant.
  • FIG. 3 shows the SDS-PAGE results of Nivolumab (IgG4) Fc double mutant.
  • Figure 4 shows the results of SEC-HPLC analysis of Nivolumab (IgG4) Fc double mutant.
  • Figure 5 shows the conditions for measuring the binding force of Rituximab (IgG1) Fc double mutant with Fc ⁇ RIIIa.
  • 6a, 6b, 6c, 6d, and 6e show binding patterns of Rituximab (IgG1) Fc double mutants to Fc ⁇ RIIIa.
  • Figures 7a and 7b show the results of measuring the binding force between human FcRn and Fc double variants at pH 6.0
  • Fig. 7a results of measuring the binding force of four variants (YTE, LS, PFc29, PFc41) compared to Rituximab
  • Fig. 7b effector function As a result of introducing additional TL mutatants that decrease).
  • Figure 8 shows the conditions for measuring the binding force of Nivolumab (IgG4) Fc double mutant with Fc ⁇ RIIIa.
  • FIGS. 9a, 9b, and 9c show the results of analysis of binding patterns of Nivolumab (IgG4) Fc Double Variants to Fc ⁇ RIIIa (FIGS. 9a and 9b: KD values for variants in which SPTL and SPLETL were additionally introduced; FIG. 9c: WT, LS, KD values of PFc29, PFc41).
  • Figures 10a and 10b show ADCC assay results between Rituximab (IgG1) Fc double mutants (Figure 10a: comparison between original form and LALAPG mutant form; Figure 10b: comparison between original form and TL mutant form).
  • FIG. 11a and 11b show CDC assay results between Rituximab (IgG1) Fc double mutants (FIG. 11a: comparison between original form and LALAPG mutant form; FIG. 11b: comparison between original form and TL mutant form).
  • a total of 25 variants were produced by introducing heavy chain and light chain constructs using the pcDNA3.4 vector (Thermo Fisher Scientific, 14697) (Table 1).
  • Fc variants including Rituximab wild type, were transiently expressed in animal cells using the Expi293F expression system (Thermo Fisher Scientific, A14527).
  • Expi293F cells were subcultured to 2x10 6 cells/mL, and the next day, when the cell density reached 2.9x10 6 cells/mL, the cells were prepared in an amount of 270 mL.
  • 15 mL of Opti-MEM1 medium (Thermo Fisher Scientific, 31985070)
  • Opti-MEM1 medium mix a total of 300 ug of plasmid DNA with a heavy chain to light chain transfection ratio of 1:1
  • ExpiFectamine293 transfection reagent (Thermo Fisher Scientific, A14524) 0.81 mL each was mixed and left for 5 minutes, then the DNA-mixed Opti-MEM1 medium mixed with the ExpiFectamine293 transfection reagent mixed Opti-MEM1 medium was mixed well and left at room temperature for 20 minutes.
  • the expression supernatant was subjected to affinity chromatography using an AKTA prime plus (GE healthcare, 11001313) equipped with a HiTrap MabSelect SuRe column (GE healthcare, #11003495), and 300 mL of the supernatant was flowed at a rate of 3 mL per minute.
  • Elution buffer 100 mM citrate buffer, 5% sucrose, pH3.0
  • the protein was finally purified by concentrating while changing the buffer to a pH 7.4 1xPBS buffer (ThermoFisher, 10010031).
  • the produced protein was subjected to SDS-PAGE and SEC-HPLC analysis to secure an antibody with a purity of 92% or more (FIGS. 1 and 2).
  • Nivolumab Fc variants in which SPLE (S228P, L235E), SPLETL (S228P, L235E, T299L), and SPTL (S228P, T299L) mutations were introduced into Nivolumab wild type, YTE, LS, pFc29, and pFc41 variants, respectively, were used as pcDNA3.4 vectors.
  • a total of 20 variants were produced by introducing heavy chain and light chain constructs (Table 2).
  • Fc variants including Nivolumab Wild type, were transiently expressed in animal cells using the ExpiCHO expression system (Thermo Fisher Scientific, A29127).
  • ExpiCHO cells were subcultured to 3x10 6 cells/mL, and the next day, when the cell density reached 6x10 6 cells/mL, cells were prepared in an amount of 200 mL.
  • 8 mL of OptiPRO SFM medium (Thermo Fisher Scientific, 12309050)
  • OptiPRO SFM medium OptiPRO SFM medium
  • ExpiFectamineCHO transfection reagent ThermoFisher, A29129
  • the two media were mixed well and added to 200 ml of ExpiCHO prepared in advance, and cultured in a shaking CO 2 incubator at 37°C, 125 rpm, and 8% CO 2 conditions.
  • the expression supernatant was subjected to affinity chromatography using an AKTA prime plus equipped with a HiTrap MabSelect SuRe column (GE healthcare, 11003495). 300 mL of the supernatant was flowed at a rate of 3 mL per minute. Elution buffer (100 mM citrate buffer, 50 mM NaCl, 5% sucrose, pH3.0) was eluted with 6 fractions of 5 mL each at a rate of 3 mL per minute, and 2-4 fractions were collected.
  • Elution buffer 100 mM citrate buffer, 50 mM NaCl, 5% sucrose, pH3.0
  • the protein was finally purified by concentrating while changing the buffer to a pH 7.4 1xPBS buffer (ThermoFisher, 10010031).
  • the produced protein was subjected to SDS-PAGE and SEC-HPLC analysis to secure an antibody of 95% or more purity (FIGS. 3 and 4).
  • binding force was measured by capturing Fc ⁇ RIIIa as a ligand using Biacore T200 equipment (GE healthcare, 28975001) (FIG. 5).
  • the binding force was measured by capturing the Rituximab-Fc variant as a ligand on a CM5 chip (GE Healthcare, BR100012) using Biacore T200 equipment (GE Healthcare, 28975001).
  • CM5 chip GE Healthcare, BR100012
  • Biacore T200 equipment GE Healthcare, 28975001.
  • the binding force of 4 variants YTE, LS, PFc29, PFc41
  • the binding force was maintained compared to before introduction (FIG. 7).
  • binding force was measured by capturing Fc ⁇ RIIIa as a ligand using Biacore T200 equipment (FIG. 8).
  • Example 3 Confirmation of difference in antibody-dependent cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) reactivity between rituximab (IgG1) Fc double variants
  • ADCC Antibody-Dependent Cellular Mediated Cytotoxicity
  • CD20 is stably expressed Raji cell (ATCC, CCL-86) was selected as the target cell and the experiment was conducted did
  • Raji cells are seeded by counting the cell number in the assay buffer so that the cell number of 1.25x10 4 enters each well in a 96 well white plate, and the test substance (original, LALAPG, TL) is assayed
  • the cells were treated in a 96 well white plate seeded so that the buffer had the highest concentration of 1 ug/mL, 1/3 dilution, and 10 points.
  • Take the effector cell out of the liquid nitrogen container melt it quickly, process it on a 96 well white plate so that the ratio of effector cell to target cell is 25:1, and incubate in a CO 2 incubator (37°C, 5% CO 2 conditions) for 6 hours.
  • Raji cells On the day of the CDC assay, Raji cells (ATCC, CCL-86) were placed in a 96-well V-bottom plate (Corning, 3894) by counting the cell numbers in assay buffer so that 1.2x10 4 cells entered each well and counting the cells. After seeding, the test substance (original, LALAPG, TL) is treated in a 96-well V-bottom plate (Corning, 3894) seeded with cells so that the test substance (original, LALAPG, TL) has a maximum concentration of 1 ug/mL, 1/3 dilution, and 10 points in assay buffer and reacted at room temperature for 15 minutes.
  • Nivolumab-Fc double variants were captured on a CM5 chip at about 160 RU level as a ligand to measure binding force.
  • the binding force measurement was performed in the range of 31.25 to 1000 nM as a total of 6 points by diluting hFcRn with an analyte at a concentration of 1/2 at a concentration of 1000 nM.
  • an Fc duplex introduced with a TL mutatant that reduces the therapeutic effector function that can show an excessive immune response to three variants (LS, PFc29, PFc41) compared to Nivolumab.
  • the binding ability of the variant was greatly improved compared to the control group (FIG. 12).
  • Example 5 Confirmation of effector silencing effect in antibody-dependent cytotoxicity (ADCC) between Nivolumab (IgG4) Fc double variants
  • ADCC Antibody-Dependent Cellular Mediated Cytotoxicity
  • TL mutations were added to Nivolumab (IgG4) Fc variants
  • Raji cells overexpressing hPD-1 Raji cells overexpressing hPD-1 (Raji-hPD-1 cell, Invivogen, raji- hpd1) was selected as the target cell and the experiment was conducted.
  • Example 6 hFcRn Tg mouse pharmacokinetics of Nivolumab (IgG4) TL variants with increased half-life and ablated mechanisms of action
  • mice Human FcRn Tg mice (Jackson Lab., JAX#004919) were used for IgG4-based Nivolumab prepared by introducing half-life increasing mutations LS, PFc29, PFc41 and TL (T299L) mutations reported to inhibit the immune mechanism. The blood half-life improvement effect was compared and verified.

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Abstract

La présente invention concerne un polypeptide comprenant un variant Fc, une partie de la séquence d'acides aminés d'un domaine Fc d'anticorps étant substituée par une autre séquence d'acides aminés, ou un anticorps la comprenant. Le variant Fc selon la présente invention peut avoir des utilisations dans une large gamme d'anticorps et de produits de fusion Fc. Selon un aspect, l'anticorps ou la fusion Fc selon la présente invention peut être utilisé en tant que réactif thérapeutique, diagnostique ou de recherche, et de préférence en tant que médicament thérapeutique. Le variant Fc selon la présente invention peut présenter des fonctions effectrices réduites ou silencieuse tout en réduisant au maximum la demi-vie in vivo par optimisation de certaines séquences d'acides aminés.
PCT/KR2022/008336 2021-06-14 2022-06-14 Variants fc à mécanisme immunitaire contrôlé et demi-vie dans le sang accrue Ceased WO2022265331A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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WO2025002113A1 (fr) * 2023-06-26 2025-01-02 上海济煜医药科技有限公司 Protéine de fusion d'agoniste du récepteur de la thrombopoïétine humaine

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US8969526B2 (en) * 2011-03-29 2015-03-03 Roche Glycart Ag Antibody Fc variants
KR20160104009A (ko) * 2014-01-15 2016-09-02 에프. 호프만-라 로슈 아게 변형된 FcRn-결합 성질을 갖는 Fc-영역 변이체
KR20170066421A (ko) * 2014-09-12 2017-06-14 제넨테크, 인크. 항-cll-1 항체 및 면역접합체
KR20170078677A (ko) * 2014-11-06 2017-07-07 에프. 호프만-라 로슈 아게 Fcrn-결합이 개질된 fc-영역 변이체 및 이의 사용 방법
US20190016828A1 (en) * 2014-01-15 2019-01-17 Hoffmann-La Roche Inc. Fc-region variants with modified fcrn- and maintained protein a-binding properties

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8969526B2 (en) * 2011-03-29 2015-03-03 Roche Glycart Ag Antibody Fc variants
KR20160104009A (ko) * 2014-01-15 2016-09-02 에프. 호프만-라 로슈 아게 변형된 FcRn-결합 성질을 갖는 Fc-영역 변이체
US20190016828A1 (en) * 2014-01-15 2019-01-17 Hoffmann-La Roche Inc. Fc-region variants with modified fcrn- and maintained protein a-binding properties
KR20170066421A (ko) * 2014-09-12 2017-06-14 제넨테크, 인크. 항-cll-1 항체 및 면역접합체
KR20170078677A (ko) * 2014-11-06 2017-07-07 에프. 호프만-라 로슈 아게 Fcrn-결합이 개질된 fc-영역 변이체 및 이의 사용 방법

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025002113A1 (fr) * 2023-06-26 2025-01-02 上海济煜医药科技有限公司 Protéine de fusion d'agoniste du récepteur de la thrombopoïétine humaine

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