WO2023043124A1 - GLYCATED FC VARIANTS WITH IMPROVED BINDING AFFINITY FOR FCγRIIIA - Google Patents

GLYCATED FC VARIANTS WITH IMPROVED BINDING AFFINITY FOR FCγRIIIA Download PDF

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WO2023043124A1
WO2023043124A1 PCT/KR2022/013481 KR2022013481W WO2023043124A1 WO 2023043124 A1 WO2023043124 A1 WO 2023043124A1 KR 2022013481 W KR2022013481 W KR 2022013481W WO 2023043124 A1 WO2023043124 A1 WO 2023043124A1
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cancer
antibody
domain
domain variant
amino acid
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PCT/KR2022/013481
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French (fr)
Korean (ko)
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정상택
조미경
김수연
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고려대학교 산학협력단
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Priority claimed from KR1020210124556A external-priority patent/KR20230041219A/en
Priority claimed from KR1020210124555A external-priority patent/KR20230041218A/en
Application filed by 고려대학교 산학협력단 filed Critical 고려대학교 산학협력단
Publication of WO2023043124A1 publication Critical patent/WO2023043124A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], 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 glycosylated Fc variants with improved selective binding ability to Fc ⁇ RIIIa.
  • Antibodies provide a link between the humoral and cellular immune systems and, while the Fab region of an antibody recognizes an antigen, the Fc domain portion is responsible for directing antibodies (immunoglobulins) on cells that are differentially expressed by all immunocompetent cells. Binds to a receptor (Fc receptor or FcR). The Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell, thereby binding to the cell through the Fc region.
  • Fc receptor Fc receptor
  • Antibodies for treatment are considered one of the most effective cancer treatment methods because they show very high target specificity compared to conventional small molecule drugs, have low biotoxicity and fewer side effects, and have an excellent blood half-life of about 3 weeks.
  • large pharmaceutical companies and research institutes around the world are accelerating research and development of therapeutic antibodies that specifically bind to and effectively remove cancer cells, including cancer-causing factors.
  • Pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS are the main companies developing therapeutic antibody drugs. ) are representative products, and these three therapeutic antibodies are not only generating huge profits, such as achieving sales of about 19.5 billion dollars in the global market in 2012, but also leading the global antibody drug market.
  • an antibody as a pharmaceutical is a direct method of inhibiting the action of an antigen by binding to an antigen, and an effector cell (natural killer cell, There is an indirect antigen removal method by phagocytes, etc.).
  • an effector cell naturally killer cell, There is an indirect antigen removal method by phagocytes, etc.
  • the effectiveness of drugs is enhanced by the ADCC mechanism in which effector cells recognize, attack and remove the Fc-gamma region of the antibody while blocking the action of the antigen due to binding to the antigen.
  • FCGR2A protein Fc ⁇ RIIIa
  • NK cells Natural killer cells
  • NK cells are the immune cells that most strongly induce the cancer cell killing effect of therapeutic IgG antibodies through ADCC, and NK cells differ from other immune cells (eg, monocytes, macrophages, dendritic cells, etc.) Otherwise, Fc ⁇ RIIIa is expressed on the surface, and Fc ⁇ RI, Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIIb are not expressed. Therefore, in order to maximize the mechanism of action of cancer cell death, the Fc region of the IgG antibody is optimized to improve affinity with Fc ⁇ RIIIa expressed on the surface of NK cells. it is essential also.
  • the ratio of the ability (A) of the Fc domain of an antibody to bind to activating Fc ⁇ R and the ability to bind to inhibitory Fc ⁇ RIlb (I) (A/I ratio ) Since the higher the ADCC induction ability, it is very urgent to selectively increase the binding force of the activating receptor compared to the binding force of Fc ⁇ RIIb, an inhibitory receptor (Boruchov et al, J Clin Invest, 115 (10): 2914-23, 2005).
  • Fc ⁇ RIIb has 96% homology with activated Fc ⁇ R
  • efforts to increase the A/I ratio by introducing genetic mutations into glycosylated antibodies have not been successful.
  • an object of the present invention is to provide a nucleic acid molecule encoding the Fc domain variant, or the antibody or fragment having immunological activity thereof, a vector containing the same, and a host cell containing the same.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
  • an object of the present invention is to provide a method for preparing human antibody Fc domain variants.
  • an object of the present invention is to provide a use for preventing or treating cancer of an antibody or a fragment having immunological activity thereof.
  • an object of the present invention is to provide a cancer treatment method comprising the step of administering an antibody or immunologically active fragment thereof to a subject suffering from cancer in a pharmaceutically effective amount.
  • the present invention provides novel human antibody Fc domain variants with increased selective binding ability with a specific Fc gamma receptor.
  • the present invention provides a glycosylated antibody comprising the novel human antibody Fc domain variant or a fragment having immunological activity thereof.
  • the present invention provides a nucleic acid molecule encoding the Fc domain variant, or the antibody or fragment having immunological activity thereof, a vector containing the same, and a host cell containing the same.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the Fc domain variant, or the antibody or fragment having immunological activity thereof as an active ingredient.
  • the present invention provides methods for preparing human antibody Fc domain variants.
  • the present invention provides a method for preparing a glycosylated antibody with improved selective binding ability to Fc ⁇ RIIIa.
  • the invention also provides the use of an antibody or immunologically active fragment thereof of the invention for use in the manufacture of an antibody therapeutic.
  • the present invention provides a use of the antibody or immunologically active fragment thereof of the present invention for preventing or treating cancer.
  • the present invention provides a cancer treatment method comprising administering to a subject suffering from cancer a pharmaceutically effective amount of the antibody or immunologically active fragment thereof of the present invention.
  • novel human antibody Fc domain variants of the present invention have reduced binding ability to Fc ⁇ RIIb, an immune inhibitory receptor, and improved binding ability to Fc ⁇ RIIIa, an immune activating receptor, compared to antibodies approved as conventional antibody therapeutics (increased A / I ratio), It has remarkably improved ADCC induction ability and can be usefully used as an antibody drug because it has the effect of maximizing the immune action mechanism of therapeutic protein drugs.
  • FIG. 1 is a diagram showing four types of trastuzumab Fc variants (XMa, XMt, MM and XMM) (A) composed of simple combinations of known mutations and Fc ⁇ RIIIb-GST protein (B) after high-purity purification and then confirmed by SDS-PAGE gel. am.
  • Figure 2 is a diagram showing the results of ELISA analyzing the Fc ⁇ RIIb binding ability of purified trastuzumab Fc variants (trastuzumab-XMa, XMt, MM and XMM).
  • Figure 3 is a diagram showing a schematic diagram of mammalian cell display technology for glycosylated Fc.
  • Figure 5 is a diagram showing the analysis of the binding activity of fluorescently labeled tetramer Fc ⁇ RIIIa-Alexa488, tetramer Fc ⁇ RIIIa-Alexa647, and Protein A-FITC with wild-type Fc or Fc-T299L expressed in CHO cells.
  • FIG. 6 is a schematic diagram of a shuffle library for glycosylation Fc engineering.
  • FIG. 7 is a schematic diagram of glycosylated Fc engineering using CHO cell display and a diagram showing gene logos of mutants of glycosylated Fc variants obtained as a result of screening.
  • FIG. 8 is a diagram showing ELISA results of analyzing Fc ⁇ RIIIa binding ability using Expi293F cell culture medium obtained by cloning glycosylated Fc variants into the heavy chain gene of trastuzumab, a model antibody, and transfecting the expression vector and culturing, and a diagram showing the mutant sequences of the discovered Fc variants. am.
  • Figure 9 is a diagram confirming trastuzumab Fc variants (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 and PS305) containing glycosylated Fc variants showing high Fc ⁇ RIIIa binding ability after purification using SDS-PAGE gel. am.
  • 10 and 11 are ELISA analysis of the binding ability of selected glycosylated trastuzumab Fc variants (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 and PS305) to Fc ⁇ RIIIa-158V, Fc ⁇ RIIIa-158F and Fc ⁇ RIlb it is one degree
  • FIG 12 shows trastuzumab Fc variants (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 and PS305) of the present invention, and previously approved by the US FDA for the treatment of B-cell lymphoma and breast cancer. It is a diagram comparing and analyzing the binding tendencies of trastuzumab Fc variants DE and VLPLL into which the Fc of tafasitamab and margetuximab, respectively, are introduced, to Fc ⁇ RIIIa-158V, Fc ⁇ RIIb, Fc ⁇ RIIIa-158F or Fc ⁇ RIIb.
  • the present invention provides, in a wild type human antibody Fc domain, 222, 239, 243, 247, 252, 292, 300, 305, 330, 332, numbered according to the Kabat numbering system. It relates to a human antibody Fc domain variant in which one or more amino acids selected from the group consisting of amino acids at positions 339, 356, 361, 387, 396, 405, and 415 are substituted with a sequence different from that of the wild-type amino acid.
  • a human antibody Fc domain variant of the invention is a group consisting of K222N, S239D, F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G, N361D, P387Q, P396L, F405L and S415G It may include any one or more amino acid substitutions selected from.
  • the Fc domain variant of the present invention may include any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22 to 35, and any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22 to 31 It is more preferable to include.
  • the human antibody Fc domain variant may be PS301 comprising amino acid substitutions of K222N, F243L, P247I, R292P, Y300L, V305I, A330L and I332E.
  • the human antibody Fc domain variant may be PS303 comprising amino acid substitutions of S239D, P247I, Y300L, V305I, A330L, I332E, A339Q, N361D and P387Q.
  • the human antibody Fc domain variant may be PS305 containing amino acid substitutions of S239D, R292P, Y300L, A330L, A339Q, P387Q and F405L.
  • the human antibody Fc domain variant may be PS205 containing amino acid substitutions of F243L, P247I, R292P, Y300L, A330L, I332E and P396L.
  • the human antibody Fc domain variant may be PS207 including amino acid substitutions of F243L, P247I, R292P, A330L, I332E, A339Q, P396L, S415G.
  • the human antibody Fc domain variant may be PS101 comprising amino acid substitutions of F243L, P247I, R292P, Y300L, V305I, A330L, I332E, A339Q and P387Q.
  • the human antibody Fc domain variant may be PS102 comprising amino acid substitutions of H224R, F243L, P247I, R292P, Y300L, V303I, V305I, A330L, I332E, A339Q and P387Q.
  • the human antibody Fc domain variant may be PS105 containing amino acid substitutions of R292P, Y300L, V305I, A330L, I332E, A339Q and P387Q.
  • the human antibody Fc domain variant may be PS106 containing amino acid substitutions of F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G and P387Q.
  • the human antibody Fc domain variant may be PS107 comprising amino acid substitutions of F243L, P247I, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G and P387Q.
  • the Fc domain variant of the present invention may have improved binding ability to Fc ⁇ RIIIa and reduced binding ability to Fc ⁇ RIlb.
  • the Fc domain variant of the present invention may have improved ADCC (antibody-dependent cell-mediated cytotoxicity) inducing ability.
  • the human antibody may be IgA, IgM, IgE, IgD or IgG, or variants thereof, may be IgG1, IgG2, IgG3 or IgG4, preferably an anti-HER2 antibody; More preferably, it is trastuzumab.
  • Papain digestion of antibodies forms two Fab domains and one Fc domain, 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 wild-type human antibody Fc domain may include the amino acid sequence of SEQ ID NO: 21 and may be encoded by the nucleic acid molecule of SEQ ID NO: 36.
  • variants comprising amino acid mutations in the human antibody Fc region of the present invention are defined according to amino acid modifications constituting the parent antibody Fc region, and conventional antibody numbering follows the EU index by Kabat (Kabat et al ., Sequence of proteins of immunological interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, 1991).
  • Fc domain variant may be used interchangeably with “Fc variant”.
  • wild-type polypeptide refers to an unmodified polypeptide that is subsequently modified to produce a derivative.
  • a wild-type polypeptide can be a naturally occurring polypeptide or a derivative or engineered of a naturally occurring polypeptide.
  • a wild-type polypeptide may refer to the polypeptide itself, a composition comprising the wild-type polypeptide, or an amino acid sequence encoding the same.
  • wild-type antibody refers to an unmodified antibody polypeptide in which amino acid residues are modified to generate a derivative.
  • parent antibody may be used to refer to an unmodified antibody polypeptide into which amino acid modifications have been introduced to give rise to a derivative.
  • amino acid modification/variation refers to substitution, insertion and/or deletion, preferably substitution, of amino acids in a polypeptide sequence.
  • amino acid substitution or “substitution” means that an amino acid at a specific position in a polypeptide sequence of a wild-type human antibody Fc domain is replaced with another amino acid.
  • an Fc variant including T299A substitution means that threonine, which is the 299th amino acid residue in the amino acid sequence of the Fc domain of a wild type antibody, is replaced with alanine.
  • Fc variant is meant to contain a modification of one or more amino acid residues compared to a wild-type antibody Fc domain.
  • the Fc variants of the present invention contain one or more amino acid modifications compared to wild-type antibody Fc domains (regions or fragments) and therefore differ in amino acid sequence.
  • the amino acid sequence of the Fc variant according to the present invention is substantially identical to the amino acid sequence of the wild-type antibody Fc domain.
  • the amino acid sequence of an Fc variant according to the present invention will have about 80% or more, preferably about 90% or more, most preferably about 95% or more homology compared to the amino acid sequence of a wild-type antibody Fc domain.
  • Amino acid modifications may be performed genetically using molecular biological methods, or may be performed using enzymatic or chemical methods.
  • Fc variants of the present invention can be prepared by any method known in the art.
  • an Fc variant of a human antibody according to the present invention encodes a polypeptide sequence comprising specific amino acid modifications and then, if desired, is used to form a nucleic acid that is cloned into a host cell, expressed and assayed.
  • Various methods for this are described in the literature (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).
  • a nucleic acid encoding an Fc variant according to the present invention may be inserted into an expression vector for protein expression.
  • An expression vector usually contains a protein operably linked, i.e., in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements.
  • the Fc variant according to the present invention can be produced by a method of inducing protein expression by culturing a host cell transformed with a nucleic acid, preferably, an expression vector containing a nucleic acid encoding the Fc variant according to the present invention. there is.
  • suitable host cells may be used including, but not limited to, mammalian cells, bacteria, insect cells, and yeast.
  • the Fc variant according to the present invention is produced using E. coli, which has high industrial value due to low production cost, as a host cell.
  • the scope of the present invention includes culturing a host cell into which a nucleic acid encoding an Fc variant has been introduced under conditions suitable for protein expression; and a method for producing an Fc variant comprising purifying or isolating the Fc variant expressed from the host cell.
  • the present invention relates to an antibody comprising an Fc domain variant of the present invention or an immunologically active fragment thereof.
  • the antibody may be a glycosylation antibody.
  • the antibody of the present invention consists of a heavy chain constant region domain 2 ( CH 2) comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 13 and amino acid sequences of SEQ ID NOs: 14 to 20 It may include a heavy chain constant region domain 3 (C H 3) comprising any one selected from the group, and a heavy chain constant region domain 2 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 9 ( C H 2) and a heavy chain constant region domain 3 ( CH 3) comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 20 is more preferred.
  • the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer It may be any one selected from the group consisting of a body, a complementarity determining region fragment, a humanized antibody, a chimeric antibody, and a diabody.
  • an antibody comprising an Fc domain variant of the present invention or a fragment having immunological activity thereof can increase effector action, and compared to a wild-type Fc domain, binding to Fc ⁇ RIIIa is improved and binding to Fc ⁇ RIIb is reduced, thereby Since it has high Fc ⁇ RIIIa binding selectivity and has a high A/I ratio, antibody-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) can be increased.
  • ADCC antibody dependent cellular cytotoxicity
  • the A / I ratio is the ratio (A / I ratio) of the ability of the Fc domain of the antibody to bind to the activating Fc ⁇ R (A) and the ability to bind to the inhibitory Fc ⁇ RIIb (I), the higher the A / I ratio Since it shows excellent ADCC induction ability, it is important to selectively increase the binding force of the activating receptor compared to the binding force of Fc ⁇ RIIb, which is an inhibitory receptor.
  • glycosylated antibodies that are expressed in mammals and are glycosylated have a protein structure stabilized by a sugar chain modified at the Fc region so that the antibody can bind to an Fc receptor, but has binding ability to all Fc ⁇ Rs.
  • aglycosylated antibodies produced in bacteria do not have a hydrocarbon chain bound to the Fc region, they cannot bind to Fc receptors and thus cannot exhibit ADCC function.
  • the antibody of the present invention is an 'aglycosylated' antibody or a fragment having immunological activity thereof, it has the effect of controlling the immune response by selectively enhancing the binding force to Fc ⁇ R.
  • Antibodies can be isolated or purified by a variety of methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunoprecipitation, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, a variety of natural proteins bind antibodies, such as, for example, bacterial proteins A, G, and L, and these proteins can be used for purification. Often, purification by specific fusion partners may be possible.
  • the antibodies include whole antibody forms as well as functional fragments of antibody molecules.
  • a full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond.
  • a functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and examples of antibody fragments include (i) a light chain variable region (VL) and a heavy chain variable region (VH) and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) an isolated CDR region; (vi) a bivalent fragment comprising two
  • F(ab')2 fragments (vii) single-chain Fv molecules (scFv) joined by a peptide linker that links the VH and VL domains to form an antigen-binding site; (viii) bispecific single-chain Fv dimers. (PCT/US92/09965) and (ix) a multivalent or multispecific fragment produced by gene fusion (diabody WO94/13804).
  • the antibody or immunologically active fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof.
  • the antibody may be produced recombinantly or synthetically.
  • the antibody or immunologically active fragment thereof may be isolated from a living body (not present in a living body) or non-naturally occurring, for example, synthetically or recombinantly produced.
  • antibody refers to a substance produced by stimulation of an antigen in the immune system, and the type is not particularly limited, and may be obtained naturally or non-naturally (e.g., synthetically or recombinantly).
  • Antibodies are advantageous for mass expression and production because they are very stable in vitro as well as in vivo and have a long half-life.
  • avidity is very high.
  • a complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond.
  • the antibody constant region is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ) and epsilon ( ⁇ ) types, subclasses It has gamma 1 ( ⁇ 1), gamma 2 ( ⁇ 2), gamma 3 ( ⁇ 3), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1) and alpha 2 ( ⁇ 2).
  • the constant region of the light chain is of the kappa ( ⁇ ) and lambda ( ⁇ ) type.
  • the term “heavy chain” refers to a variable region domain V H comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains C H 1 , C H 2 and It is interpreted as meaning including both full-length heavy chains and fragments thereof including C H 3 and a hinge.
  • the term “light chain” refers to both a full-length light chain comprising a variable region domain V L and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen and fragments thereof. be interpreted in a sense that includes
  • the term "Fc domain”, “Fc fragment” or “Fc region” constitutes an antibody together with a Fab domain/fragment
  • the Fab domain/fragment comprises a light chain variable region (V L ) and a heavy chain variable region (V H ), light chain constant region ( CL ) and heavy chain first constant region (C H 1)
  • the Fc domain / fragment is the heavy chain of the second constant region (CH 2) and the third constant region (C H 2) H 3).
  • the present invention relates to a nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof.
  • the nucleic acid molecule encoding the Fc variant according to the present invention may include any one selected from the group consisting of nucleotide sequences of 37 to 50.
  • the present invention relates to a vector containing the nucleic acid molecule and 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.
  • the isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or an antibody containing the same, or a fragment having immunological activity thereof, has codons preferred in organisms intended to express the same due to codon degeneracy.
  • various modifications may be made to the coding region within the range of not changing the amino acid sequence of the Fc domain variant expressed from the coding region, or an antibody containing the same or a fragment having immunological activity thereof, and a portion other than the coding region. It will be well understood by those skilled in the art that various modifications or modifications may be made within a range that does not affect gene expression, and that such modified genes are also included in the scope of the present invention.
  • nucleic acid molecule of the present invention encodes a protein having an activity equivalent thereto
  • one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention.
  • the sequence of such a nucleic acid molecule may be single- or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.
  • the isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof according to the present invention may be inserted into an expression vector for protein expression.
  • An expression vector usually contains a protein operably linked, i.e., in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements.
  • a host cell transformed with a nucleic acid preferably, an expression vector containing an isolated nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same or an immunologically active fragment thereof is cultured to produce a protein.
  • An Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof may be produced by a method of inducing expression.
  • a variety of suitable host cells may be used including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, it is possible to produce E. coli, which has high industrial value due to low production cost, as a host cell.
  • Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose.
  • the vector's promoter may be constitutive or inducible.
  • the signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an ⁇ -amylase signal sequence and a subtilisin signal when the host is Bacillus sp.
  • Sequences such as MF ⁇ signal sequence, SUC2 signal sequence, etc. can be used when the host is yeast, and insulin signal sequence, ⁇ -interferon signal sequence, antibody molecule signal sequence, etc. can be used when the host is an animal cell. Not limited to this.
  • the vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.
  • vector refers to a delivery vehicle into which a nucleic acid sequence can be inserted for introduction 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, bacteriophages).
  • viruses eg, bacteriophages.
  • 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, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).
  • a promoter, a terminator, a promoter, a terminator, an expression control sequence such as an enhancer, a sequence for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose.
  • expression vector refers to a vector comprising 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 control 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.
  • the host cell may be a bacterial or animal cell
  • the animal cell line may be a CHO cell, a HEK cell or a NSO cell
  • the bacteria may be Escherichia coli.
  • the present invention relates to an antibody therapeutic comprising an Fc domain variant of the present invention.
  • cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcriptional regulators, blood used for the purpose of treating or preventing human diseases are added to the Fc domain variant or protein conjugate comprising the same according to the present invention.
  • Factors, vaccines, structural proteins, ligand proteins or various physiologically active polypeptides such as receptors, cell surface antigens, and receptor antagonists, derivatives and analogs thereof may be used in combination.
  • an antibody drug may be conjugated to the Fc domain variant or a protein conjugate including the same according to the present invention, and the antibody drug for cancer treatment is Trastzumab, cetuximab, or bevacizumab. (bevacizumab), rituximab, basiliximab, infliximab, ipilimumab, pembrolizumab, nivolumab, atezolizumab (Atezolizumab) or Avelumab.
  • the mechanism of recruiting and delivering immune cells to the target antigen is one of the most important mechanisms, and the Fc domain of an antibody plays a crucial role in the recruitment of immune cells and ADCC (antibody-dependent cell-mediated cytotoxicity), so the present invention
  • An Fc variant having increased selective binding ability to the Fc gamma receptor is advantageous for use as a therapeutic antibody.
  • the ADCC function of antibodies depends on interactions with Fc gamma receptors (Fc ⁇ Rs) present on the surface of many cells, and the type of immune cells recruited depending on which Fc receptors the antibody binds to among the five human Fc receptors. Since is determined, attempts to modify antibodies to recruit specific cells are very important in the field of therapy.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the Fc domain variant of the present invention, or an antibody containing the same or a fragment having immunological activity thereof as an active ingredient.
  • the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, Small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and It may be any one selected from the group consisting
  • the antibody comprising the Fc domain variant of the present invention or a fragment having immunological activity thereof has a high Fc ⁇ RIIIa binding selectivity and a high A/I ratio, so effector action through natural killer cells (NK cells)
  • NK cells natural killer cells
  • ADCC antibody dependent cellular cytotoxicity
  • Unlike other immune cells e.g., monocytes, macrophages, dendritic cells
  • natural killer cells NK cells
  • the antibody comprising the Fc domain variant having Fc ⁇ RIIIa binding selectivity or a fragment having immunological activity thereof of the present invention can maximize the cancer cell killing mechanism through NK cells.
  • the composition of the present invention may further include an immunogenic apoptosis inducer, and the immunogenic apoptosis inducer is an anthracycline-based anticancer agent, a taxane-based anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib ( Bortezomib), cardiac glycoside, cyclophosmid anticancer drug, GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone or oxaliplatin It may be any one or more selected, and anthracycline-based anticancer agents include daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, and sabarubicin. ) or valrubicin, and the taxane-based anticancer agent may be paclitaxel or docetaxel.
  • the pharmaceutical composition for preventing or treating cancer of the present invention can increase the cancer treatment effect of conventional anticancer drugs through the killing effect of cancer cells by administering together with chemical anticancer drugs (anticancer drugs). Concomitant administration may be performed simultaneously or sequentially with the anticancer agent.
  • the anticancer agent examples include DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide ( ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin and carboplatin ; dactinomycin (actinomycin D), plicamycin and mitomycin C as anti-cancer antibiotics; and plant alkaloids such as vincristine, vinblastine, etoposide, teniposide, topotecan and iridotecan. , but is not limited thereto.
  • DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide ( ifosfamide, carmustine (BCNU), lomustine (CCNU
  • prevention refers to all activities that inhibit or delay the occurrence, spread, and recurrence of cancer by administration of the pharmaceutical composition according to the present invention.
  • treatment refers to any activity that ameliorates or beneficially alters the death of cancer cells or symptoms of cancer by administration of the composition of the present invention.
  • Those of ordinary skill in the art to which the present invention pertains will be able to determine the degree of improvement, enhancement and treatment by knowing the exact criteria of the disease for which the composition of the present application is effective by referring to the data presented by the Korean Medical Association, etc. will be.
  • terapéuticaally effective amount used in combination with an active ingredient in the present invention refers to an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease, and a therapeutically effective amount of the composition of the present invention It may vary depending on various factors, such as the method of administration, the target site, and the condition of the patient. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining an effective amount can be found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects
  • the effective dose level is the patient's Health condition, cancer type, severity, drug activity, drug sensitivity, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to
  • the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Stearic Acid Calcium, white sugar, dextrose, sorbitol, and talc may be used.
  • the pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but is not limited thereto.
  • composition of the present invention may also include a carrier, diluent, excipient or a combination of two or more commonly used in biological preparations.
  • the pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added.
  • diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • formulations for injection such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
  • it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
  • composition of the present invention may be parenterally administered (for example, intravenously, subcutaneously, intraperitoneally, or topically applied as an injection formulation) or orally, depending on the desired method, and the dosage may be determined by the patient's weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease.
  • the daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide the administration once or several times a day.
  • Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents etc. may be included.
  • Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like.
  • the present invention includes a method for preparing a long-acting drug formulation by covalently linking the Fc domain variant to a physiologically active polypeptide through a non-peptide polymer.
  • the manufacturing method according to the present invention comprises the steps of covalently linking a physiologically active polypeptide and an Fc domain variant through a non-peptide polymer having a reactive group at the terminal; and isolating a conjugate in which the physiologically active polypeptide, the non-peptide polymer, and the Fc domain variant are covalently linked.
  • the present invention comprises the steps of a) culturing a host cell containing a vector containing a nucleic acid molecule encoding an Fc domain variant of the present invention; And b) it relates to a method for preparing a human antibody Fc domain variant with improved selective binding ability to Fc ⁇ RIIIa, comprising the step of recovering the polypeptide expressed by the host cell.
  • the present invention comprises the steps of a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof; and b) purifying the antibody expressed from the host cell.
  • purification of the antibody may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably using Protein A. affinity chromatography can be used.
  • the invention relates to the use of an antibody or immunologically active fragment thereof of the invention for use in the manufacture of an antibody therapeutic.
  • the present invention relates to the use of the antibody or immunologically active fragment thereof of the present invention for preventing or treating cancer.
  • the present invention relates to a method for treating cancer comprising administering to a subject suffering from cancer a pharmaceutically effective amount of an antibody or immunologically active fragment thereof of the present invention.
  • DEL variants S239D/I332E/A330L: A/I ratio improved 9 times compared to wild-type Fc
  • LPLIL variants F243L/R292P/ Fc variants (XMa: S239D/F243L/R292P/XMa: S239D/F243L/R292P/ Y300L/V305I/A330L/I332E/P396L; XMt: S239D/P247I/A330L/I332E/A339Q; MM: F243L/P247I/R292P/Y300L/V305I/A339Q/P396L; V305I/A330L/I332E/A339Q/P396L) (Table 1).
  • the Fc variants were cloned into the heavy chain gene of trastuzumab, a model antibody, and transient expression was induced by co-transfection with PEI (polyethylenimine) (Polyscience, 23966) into Expi293F cells together with the light chain gene. After culturing the co-transfected Expi293F cells at 37°C, 125 rpm, and 8% CO 2 conditions for 7 days, the culture medium was recovered, equilibrated with PBS, and trastuzumab Fc variants were detected through Protein A affinity chromatography. Purified (FIG. 1A).
  • Fc ⁇ RIIb-GST was produced in order to confirm the binding ability of simple combination trastuzumab Fc variants to Fc ⁇ RIIb, an Fc receptor that functions to attenuate ADCC activity by down-regulating the immune response.
  • Expi293F cells were transfected with a plasmid in which a gene in which GST was fused to the C-terminus of Fc ⁇ RIIb was cloned into an animal cell expression vector using PEI, and then incubated at 37°C, 125 rpm, and 8% CO 2 conditions for 7 days. Temporary expression was performed daily. Then, after recovering the cell culture medium, equilibrating with PBS, high purity Fc ⁇ RIIb-GST was secured through anti-GST affinity chromatography (FIG. 1B).
  • ELISA analysis was performed to confirm the binding ability of the trastuzumab Fc variants (XMa, XMt, MM and XMM) purified and prepared in Example 1-1 to Fc ⁇ RIIb.
  • 50 ⁇ l of Fc ⁇ RIIb-GST diluted to 4 ⁇ g/ml in 0.05 M Na 2 CO 3 pH 9.6 was immobilized in a flat bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4° C. for 16 hours, then Blocking was performed at room temperature for 2 hours with 100 ⁇ l of 4% skim milk (GenomicBase, SKI400).
  • trastuzumab Fc variants composed of simple combinations of known mutations are ADCC-enhancing glycosylation Fc variants (B-cell lymphoma treatment (Monjuvi, Tafasitamab) approved by the US FDA for the treatment of B-cell lymphoma and Her2-positive breast cancer, respectively, 2020 DE (S239E/I332E) approved by the US FDA on July 31, 2020 and VLPLL (L235V/F243L/R292P/Y300L/P396L) approved by the US FDA on December 16, 2020 as Her2-positive breast cancer treatment (Margenza, Margetuximab)) It was found to have a significantly high Fc ⁇ RIIb binding affinity (FIG. 2), and it was confirmed that it could have a negative effect on ADCC activity induction.
  • Fc ⁇ RIIb binding affinity Fc ⁇ RIIb binding affinity
  • An Fc library was constructed to find the optimal combination of Fc mutations capable of maximizing the ADCC induction ability and discover new glycosylated Fc variants.
  • the FLP-FRT gene recombination system used for producing stable cell lines was utilized.
  • a platelet-derived growth factor receptor (PDGFR) transmembrane domain was fused to the C-terminus.
  • the Fc-PDGFR gene was prepared by cloning into a pcDNA5/FRT plasmid (Invitrogen, V601020) into which an FRT site was inserted.
  • This plasmid is co-transfected with a pOG44 plasmid (Invitrogen, V600520) expressing FLP, an enzyme that causes genetic recombination, into CHO cells (Invitrogen, R75807) into which an FRT site has been inserted to induce genetic recombination, thereby inducing chromosomes of CHO cells.
  • Fc-PDGFR DNA was integrated into DNA (chromosomal DNA) to construct a CHO cell line stably expressing glycosylated Fc (FIG. 3).
  • hygromycin-B resistance gene was integrated together, and by treatment with 500 ⁇ g/ml hygromycin-B (Invitrogen, 10687010)
  • a mammalian cell display system for screening glycosylated Fc variants was constructed by selecting CHO cell lines stably expressing glycosylated Fc.
  • Fc ⁇ RIIIa and Fc ⁇ RIIb were constructed and produced to analyze the Fc ⁇ Rs binding ability of the Fc variants to be identified.
  • Fc ⁇ Rs Fc ⁇ RIIIa and Fc ⁇ RIIb are known to be low affinity receptors with low binding affinity to Fc, so streptavidin is fused to the C-terminus of each receptor to improve the visible binding affinity with Fc, resulting in efficient FACS screening-capable tetrameric Fc ⁇ RIIIa-158V-streptavidin-His, tetrameric Fc ⁇ RIIIa-158F-streptavidin-His, and tetrameric Fc ⁇ RIIb-streptavidin-His were prepared.
  • Fc ⁇ RIIIa-158V-GST and Fc ⁇ RIIIa-158F-GST were prepared. Each protein was prepared by cloning into an animal cell expression vector, transfected into Expi293F cells using PEI, and then cultured for 7 days at 37°C, 125 rpm and 8% CO 2 conditions. After culturing, the supernatant was collected, equilibrated with PBS, and purified by Ni-NTA (Anti-His) or anti-GST affinity chromatography.
  • the tetrameric Fc ⁇ RIIIa-streptavidin prepared for FACS screening was non-fluorescent by labeling the fluorescent dyes of Alexa488 (Invitrogen, A10235) and Alexa647 (Invitrogen, A20173). It was prepared to enable sorting of Fc variants through competitive binding with tetrameric Fc ⁇ RIIb-streptavidin, and Protein A (Amicogen, 1070020) was prepared by conjugation of FITC (Invitrogen, F6434). Conjugation of fluorescent dyes (alexa488, Alexa647 and FITC) was performed according to the manual provided by each manufacturer.
  • Fluorescence-labeled tetrameric Fc ⁇ RIIIa-streptavidin-Alexa488, tetrameric Fc ⁇ RIIIa-streptavidin-Alexa647, and Protein A-FITC were respectively applied to wild-type Fc and Fc variants (Fc-T299L) with no Fc ⁇ Rs-binding affinity displayed in CHO cells. After binding was induced, activity was confirmed.
  • Protein A-FITC has normal activity through the fluorescence signal by Protein A-FITC binding, and wild-type Fc and Fc-T299L displayed in CHO cells are stably expressed, showing similar expression levels to each other. It was confirmed that shown (FIG. 5).
  • tetrameric Fc ⁇ RIIIa-streptavidin-Alexa488 and tetrameric Fc ⁇ RIIIa-streptavidin-Alexa647 also appeared to normally bind to wild-type Fc, but not to Fc-T299L, an Fc variant in which Fc ⁇ Rs binding ability was removed (FIG. 5) , Alexa488/647 fluorescently labeled Fc ⁇ RIIIa also had excellent activity, and it was successfully verified that Fc displayed in CHO cells were normally expressed and performed their respective functions.
  • Glycosylated Fc was engineered through the established CHO cell Fc display system, and a library was constructed to select glycosylated Fc variants with improved Fc ⁇ RIIIa binding ability. Mutations of DEL variants, LPLIL variants, and IQ variants were shuffled using the library to search for optimal mutation combinations capable of maximizing Fc ⁇ RIIIa binding (FIG. 6).
  • the library gene was co-transfected with the FLP expression plasmid in the same way as the Fc stable expression mammalian cell line production method established in Example 2, and then the glycosylated Fc variant library was stably expressed through a selection process using hygromycin-B medium. A CHO cell line was constructed.
  • an expression vector was prepared by cloning into the model antibody trastuzumab heavy chain gene. Specifically, in 3 ml of Freestyle293 expression culture medium (Gibco, 12338-018), the heavy chain genes and light chain genes of the variants were first mixed at a ratio of 1:1, and PEI (polyethylenimine) (Polyscience, 23966) and the variant genes were mixed at a ratio of 4:1.
  • Freestyle293 expression culture medium Gibco, 12338-018
  • PEI polyethylenimine
  • ELISA analysis was performed to confirm the binding ability of the glycosylated trastuzumab Fc variants expressed and purified in Example 7 to Fc ⁇ RIIIa and Fc ⁇ RIIb.
  • 50 ⁇ l each of Fc ⁇ Rs-GST (Fc ⁇ RIIIa-158V-GST, Fc ⁇ RIIIa-158F-GST, and Fc ⁇ RIIb-GST) diluted to 4 ⁇ g/ml in 0.05 M Na 2 CO 3 (pH 9.6) was added to flat bottom polystyrene high After dispensing to a bind 96-well plate (Costar, 3590), immobilizing at 4° C.
  • the glycated trastuzumab Fc variants of the present invention are Fc variants composed of simple combinations of glycated Fc variants (DE and VLPLL) approved by the US FDA for the treatment of B-cell lymphoma and Her2-positive breast cancer, respectively, and previously known mutations. It was found to have significantly higher Fc ⁇ RIIIa / Fc ⁇ RIIb selective binding ability than those (XMa, XMt, MM and XMM) (Figs. 10 and 11).
  • Fc variants containing optimized mutations by selectively adding and deleting some mutations by searching for a large library of glycosylated antibody Fc variants are very effective in inducing Fc ⁇ RIIIa/Fc ⁇ RIIb selective binding ability. It was found (FIG. 12), and according to their binding characteristics, they were classified into 3 groups as shown in Table 2 below.

Abstract

The present invention relates to a glycated Fc variant having improved selective binding affinity for FcγRIIIa. With the effects of exhibiting decreased binding affinity for the immunity-inhibitory receptor FcγRIIb and increased binding affinity for the immunity-activating receptor FcγRIIIa (A/I ratio increased), compared to conventional antibodies approved as antibody therapeutic agents, having a remarkably improved ADCC-inducing potential, and maximizing immune action mechanisms of therapeutic protein medications, the novel human antibody Fc domain variants of the present invention can be advantageously used as antibody medications.

Description

FCγRⅢA 결합력이 향상된 당화 FC 변이체들Glycosylated FC variants with improved FCγRIIIA binding ability
본 발명은 FcγRⅢa에 대한 선택적 결합력이 향상된 당화 Fc 변이체에 관한 것이다.The present invention relates to glycosylated Fc variants with improved selective binding ability to FcγRIIIa.
전 세계적으로 유전자 재조합, 세포 배양 등 생명공학기술의 발달에 따라 단백질의 구조와 기능에 대한 연구가 활발히 진행되어왔으며, 이는 생명현상에 대한 이해를 높일 뿐만 아니라, 각종 질병들의 발병 기작을 규명하는데 결정적 역할을 함으로써 효과적인 질병 진단과 치료의 길을 마련해 삶의 질 향상에 크게 기여 하고 있다. 특히, 1975년에 B 세포(B Cell)와 골수암 세포(Myeoloma cell)를 융합하여 단일클론항체를 생산하는 하이브리도마 기술(Hybridoma technology)이 개발(Kohler and Milstein, Nature , 256:495-497, 1975)되면서 암, 자가면역질환, 염증, 심혈관 질환, 감염 등의 임상 분야에서 치료용 항체를 이용한 면역 치료(Immunotherapy)에 대한 연구 개발이 활발히 이루어지고 있다Worldwide, with the development of biotechnology such as genetic recombination and cell culture, research on the structure and function of proteins has been actively conducted. By doing so, it provides a path for effective disease diagnosis and treatment, contributing greatly to the improvement of quality of life. In particular, hybridoma technology was developed in 1975 to produce monoclonal antibodies by fusing B cells and myeloma cells (Kohler and Milstein, Nature , 256:495-497, 1975), research and development on immunotherapy using therapeutic antibodies is being actively conducted in the clinical fields of cancer, autoimmune diseases, inflammation, cardiovascular diseases, infections, etc.
항체는 체액성 및 세포성 면역계 사이의 연결고리를 제공하며, 항체의 Fab 영역이 항원을 인식하는 반면, Fc 도메인 부분은 모든 면역 적격 세포에 의해 차별적으로 발현되는 세포 상의 항체(면역글로불린)에 대한 수용체 (Fc 수용체 또는 FcR)에 결합한다. 항체 Fc 영역 상의 Fc 수용체 결합 부위가 세포 상의 Fc 수용체 (FcR)에 결합함으로써 Fc 영역을 통해 세포에 결합하며, 항체가 세포 표면 상의 Fc 수용체에 결합하면 항체-코팅 입자의 포식 및 파괴, 면역 복합체의 제거, 살세포에 의한 항체-코팅 표적 세포의 용해 (항체-의존적 세포-매개 세포독성, antibody-dependent cell-mediated cytotoxicity 또는 ADCC), 염증 매개체의 방출, 태반 이동 및 면역글로불린 생성의 제어를 포함하여 중요하고도 다양한 여러 생물학적 반응을 촉발한다 (Deo, Y.M. et al., Immunol. Today 18(3):127-135 (1997)). 이와 같이, Fc 도메인은 면역세포의 모집과 ADCC 및 ADCP(antibody dependent cellular phagocytosis)에 결정적인 역할을 하며, 특히, 항체의 작용기 기능(effector function)인 ADCC 및 ADCP 기능은 많은 세포의 표면에 존재하는 Fc 수용체와의 상호작용에 의존한다. 사람의 Fc 수용체는 5가지로 분류되며, 항체가 어떠한 Fc 수용체에 결합되는지에 따라 모집되는 면역세포의 종류가 결정된다. 따라서, 특정한 세포를 모집할 수 있도록 항체를 변형하는 시도는 치료 분야에 있어서 매우 중요하다고 할 수 있다. Antibodies provide a link between the humoral and cellular immune systems and, while the Fab region of an antibody recognizes an antigen, the Fc domain portion is responsible for directing antibodies (immunoglobulins) on cells that are differentially expressed by all immunocompetent cells. Binds to a receptor (Fc receptor or FcR). The Fc receptor binding site on the antibody Fc region binds to the Fc receptor (FcR) on the cell, thereby binding to the cell through the Fc region. including clearance, lysis of antibody-coated target cells by killer cells (antibody-dependent cell-mediated cytotoxicity or ADCC), release of inflammatory mediators, control of placental migration and immunoglobulin production It triggers a number of important and diverse biological responses (Deo, Y.M. et al., Immunol. Today 18(3):127-135 (1997)). As such, the Fc domain plays a critical role in the recruitment of immune cells and ADCC and ADCP (antibody dependent cellular phagocytosis). Depends on interaction with receptors. Human Fc receptors are classified into five types, and the type of immune cells recruited depends on which Fc receptor an antibody binds to. Therefore, attempts to modify antibodies to recruit specific cells can be said to be very important in the field of therapy.
치료용 항체는 기존의 저분자 약물에 비해 타깃에 매우 높은 특이성을 보이며, 생체 독성이 낮고 부작용이 적을 뿐만 아니라, 약 3주의 우수한 혈중 반감기를 가지기 때문에 가장 효과적인 암 치료방법 중의 하나로 여겨지고 있다. 실제로 전 세계의 거대 제약회사들과 연구소들에서 암 발병 원인인자를 비롯한 암세포에 특이적으로 결합하여 효과적으로 제거하는 치료용 항체의 연구 개발에 박차를 가하고 있다. 치료용 항체 의약품 개발 기업으로는 로슈, 암젠, 존슨앤존슨, 애보트, 비엠에스 등의 제약 기업이 주를 이루고 있으며, 특히 로슈는 항암 치료 목적의 허셉틴(Herceptin), 아바스틴(Avastin), 리툭산(Rituxan) 등이 대표적 상품으로 이 세 가지 치료용 항체로 2012년 세계시장에서 약 195억 달러의 매출을 달성하는 등 큰 이윤을 창출하고 있을 뿐 아니라, 세계의 항체 의약품 시장을 이끌고 있다. 레미케이드(Remicade)를 개발한 존슨앤존슨 역시 매출의 증가로 세계 항체 시장에서 빠르게 성장해나가고 있으며, 애보트와 비엠에스 등의 제약 기업 역시 개발 막바지 단계의 치료용 항체를 다수 보유하고 있는 것으로 알려져 있다. 이에 따른 결과로 저분자 의약품이 주도권을 가지고 있던 세계 제약 시장에서 질병 타깃에 특이적이고 부작용이 낮은 치료용 항체를 포함한 바이오 의약품이 빠르게 그 자리를 대체해 나가고 있다.Antibodies for treatment are considered one of the most effective cancer treatment methods because they show very high target specificity compared to conventional small molecule drugs, have low biotoxicity and fewer side effects, and have an excellent blood half-life of about 3 weeks. In fact, large pharmaceutical companies and research institutes around the world are accelerating research and development of therapeutic antibodies that specifically bind to and effectively remove cancer cells, including cancer-causing factors. Pharmaceutical companies such as Roche, Amgen, Johnson & Johnson, Abbott, and BMS are the main companies developing therapeutic antibody drugs. ) are representative products, and these three therapeutic antibodies are not only generating huge profits, such as achieving sales of about 19.5 billion dollars in the global market in 2012, but also leading the global antibody drug market. Johnson & Johnson, which developed Remicade, is also growing rapidly in the global antibody market with increased sales, and pharmaceutical companies such as Abbott and BMS are also known to have many therapeutic antibodies in the final stages of development. As a result, biopharmaceuticals, including therapeutic antibodies that are specific to disease targets and have low side effects, are rapidly replacing the position in the global pharmaceutical market, where small-molecule drugs had been dominant.
현재 의약품으로서 항체의 작용은 항원과 결합하여 항원의 작용을 저해하는 직접적인 방식과, 항원과 결합한 항체의 Fc 감마 부위와 Fc 감마 수용체(Fc gamma receptor, FCGR)를 가지는 효과세포(자연살해세포, 대식세포 등)에 의한 간접적인 항원제거 방식이 있다. 최근 개발 중인 항체 치료제의 경우, 항원과의 결합으로 인한 항원의 작용을 막음과 동시에 항체의 Fc 감마 부위를 효과세포가 인식하여 공격 및 제거하는 ADCC 기전으로 약제의 효과를 높이고 있다. 최근 ADCC와 관련된 연구로, Fc 감마 부위 수용체의 유전자형에 따라 Fc 감마와 Fc 감마 수용체의 결합력에 영향을 주는 것으로 밝혀졌으며, 항체 기반 치료제의 효율이 환자의 Fc 감마 수용체인 FCGR2A 단백질(FcγRⅢa)의 131번째 아미노산과 FCGR3A 단백질의 158번째 아미노산의 아미노산 서열의 다양성에 따라 달라진다는 다수의 연구가 발표되었다. 일 예로 유방암 치료제인 허셉틴(trastuzumab)의 경우 전임상 연구에서 FCGR2A 131 H/H 혹은 FCGR3A 158 V/V 유전자형에서 항체 의존성 세포독성이 유의적인 차이를 보이며 증가하였다는 연구가 2008년에 Musolino등에 의하여 보고되었다. 그러나, 포유동물의 항체의 Fc 부위 변형에 의해 특정 Fc 수용체에 대한 결합력이 증가되지만, 다른 Fc 수용체에 대한 결합력도 유지를 하기 때문에 바람직하지 않은 면역반응을 여전히 유지하는 문제가 있다. 인간의 5 종류의 주요 FcγR 중 4가지는 면역 활성화 또는 염증 반응을 유도하고, FcγRⅡb는 면역 저해 또는 항염증 반응을 유도하는데, 대부분의 자연적으로 생산된 항체 또는 재조합된 당화 항체는 활성화 및 저해 Fc 수용체에 모두 결합을 한다. ADCC를 통해 치료용 IgG 항체의 암세포 사멸 효능을 가장 강력하게 유도하는 면역세포는 자연살해세포(NK 세포)로 NK 세포는 다른 면역세포들(예를 들어,　monocytes, macrophages, dendritic cells 등)과는 달리 표면에 FcγRⅢa를 발현하고, FcγRI과 FcγRⅡa, FcγRⅡb 및 FcγRⅢb는 발현하지 않기 때문에 암세포 사멸 작용기작을 극대화하기 위해서는 IgG 항체의 Fc 부위의 최적화를 통해 NK 세포 표면에 발현되는 FcγRⅢa와의 친화도를 향상시키는 것이 필수적이다. 또한. NK 세포 뿐만 아니라 혈액에 존재하는 다양한 면역 세포들의 복합적인 면역 반응을 고려하면 항체의 Fc 도메인이 활성화 FcγR에 결합하는 능력 (A)과 저해 FcγRⅡb에 결합하는 능력 (I)의 비율(A/I ratio)이 높을수록 우수한 ADCC 유도능을 보이기 때문에 저해 수용체인 FcγRⅡb 결합력 대비 활성화 수용체 결합력을 선택적으로 높이는 것이 매우 절실한 실정이다 (Boruchov et al, J Clin Invest, 115(10):2914-23, 2005). 하지만, FcγRⅡb는 활성화 FcγR와 96%의 상동성을 갖는 문제로 인하여, 당화항체에 유전적 돌연변이를 도입하여 A/I ratio를 증가시키기 위한 노력은 큰 결실을 거두지 못하고 있다.Currently, the action of an antibody as a pharmaceutical is a direct method of inhibiting the action of an antigen by binding to an antigen, and an effector cell (natural killer cell, There is an indirect antigen removal method by phagocytes, etc.). In the case of antibody therapeutics currently under development, the effectiveness of drugs is enhanced by the ADCC mechanism in which effector cells recognize, attack and remove the Fc-gamma region of the antibody while blocking the action of the antigen due to binding to the antigen. A recent study related to ADCC revealed that the binding force of Fc gamma and Fc gamma receptors was affected depending on the genotype of the Fc gamma site receptor, and the efficiency of antibody-based therapeutics was found to affect the patient's Fc gamma receptor, FCGR2A protein (FcγRIIIa) 131 A number of studies have been published that the amino acid sequence of the 158th amino acid and the 158th amino acid of the FCGR3A protein differs depending on the diversity. For example, in the case of Herceptin (trastuzumab), a breast cancer treatment, a study in which antibody-dependent cytotoxicity increased with a significant difference in FCGR2A 131 H/H or FCGR3A 158 V/V genotypes in a preclinical study was reported by Musolino et al. in 2008 . However, although the binding ability to a specific Fc receptor is increased by modification of the Fc region of a mammalian antibody, there is still a problem of maintaining an undesirable immune response because the binding force to other Fc receptors is also maintained. Four of the five major FcγRs in humans induce immune activation or inflammatory responses, and FcγRIIb induces immune suppression or anti-inflammatory responses. Most naturally produced or recombinant glycosylated antibodies act on activating and inhibitory Fc receptors. all combine Natural killer cells (NK cells) are the immune cells that most strongly induce the cancer cell killing effect of therapeutic IgG antibodies through ADCC, and NK cells differ from other immune cells (eg, monocytes, macrophages, dendritic cells, etc.) Otherwise, FcγRIIIa is expressed on the surface, and FcγRI, FcγRIIa, FcγRIIb and FcγRIIIb are not expressed. Therefore, in order to maximize the mechanism of action of cancer cell death, the Fc region of the IgG antibody is optimized to improve affinity with FcγRIIIa expressed on the surface of NK cells. it is essential also. Considering the complex immune response of not only NK cells but also various immune cells present in the blood, the ratio of the ability (A) of the Fc domain of an antibody to bind to activating FcγR and the ability to bind to inhibitory FcγRIlb (I) (A/I ratio ) Since the higher the ADCC induction ability, it is very urgent to selectively increase the binding force of the activating receptor compared to the binding force of FcγRIIb, an inhibitory receptor (Boruchov et al, J Clin Invest, 115 (10): 2914-23, 2005). However, due to the problem that FcγRIIb has 96% homology with activated FcγR, efforts to increase the A/I ratio by introducing genetic mutations into glycosylated antibodies have not been successful.
본 발명의 목적은 신규한 인간 항체 Fc 도메인 변이체들을 제공하는 것이다.It is an object of the present invention to provide novel human antibody Fc domain variants.
또한, 본 발명의 목적은 특정 Fc 감마 수용체에 특이적인 당화 항체 또는 이의 면역학적 활성을 가진 단편을 제공하는 것이다.It is also an object of the present invention to provide a glycosylated antibody specific for a specific Fc gamma receptor or a fragment having immunological activity thereof.
또한, 본 발명의 목적은 상기 Fc 도메인 변이체, 또는 상기 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산분자, 이를 포함하는 벡터, 및 이를 포함하는 숙주세포를 제공하는 것이다.In addition, an object of the present invention is to provide a nucleic acid molecule encoding the Fc domain variant, or the antibody or fragment having immunological activity thereof, a vector containing the same, and a host cell containing the same.
또한, 본 발명의 목적은 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer.
또한, 본 발명의 목적은 인간 항체 Fc 도메인 변이체의 제조방법을 제공하는 것이다.In addition, an object of the present invention is to provide a method for preparing human antibody Fc domain variants.
또한, 본 발명의 목적은 FcγRⅢa에 대한 선택적 결합력이 향상된 당화 항체의 제조방법을 제공하는 것이다.It is also an object of the present invention to provide a method for preparing a glycosylated antibody with improved selective binding ability to FcγRIIIa.
또한, 본 발명의 목적은 항체 치료제의 제조에 사용하기 위한 항체 또는 이의 면역학적 활성을 가진 단편의 용도를 제공하는 것이다.It is also an object of the present invention to provide the use of an antibody or an immunologically active fragment thereof for use in the manufacture of an antibody therapeutic.
또한, 본 발명의 목적은 항체 또는 이의 면역학적 활성을 가진 단편의 암의 예방 또는 치료 용도를 제공하는 것이다.In addition, an object of the present invention is to provide a use for preventing or treating cancer of an antibody or a fragment having immunological activity thereof.
아울러, 본 발명의 목적은 항체 또는 이의 면역학적 활성을 가진 단편을 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법을 제공하는 것이다.In addition, an object of the present invention is to provide a cancer treatment method comprising the step of administering an antibody or immunologically active fragment thereof to a subject suffering from cancer in a pharmaceutically effective amount.
상기 과제를 해결하기 위하여, 본 발명은 특정 Fc 감마 수용체와의 선택적 결합력이 증대된 신규한 인간 항체 Fc 도메인 변이체들을 제공In order to solve the above problems, the present invention provides novel human antibody Fc domain variants with increased selective binding ability with a specific Fc gamma receptor.
또한, 본 발명은 상기 신규한 인간 항체 Fc 도메인 변이체를 포함하는 당화 항체 또는 이의 면역학적 활성을 가진 단편을 제공In addition, the present invention provides a glycosylated antibody comprising the novel human antibody Fc domain variant or a fragment having immunological activity thereof.
또한, 본 발명은 상기 Fc 도메인 변이체, 또는 상기 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산분자, 이를 포함하는 벡터, 및 이를 포함하는 숙주세포를 제공한다.In addition, the present invention provides a nucleic acid molecule encoding the Fc domain variant, or the antibody or fragment having immunological activity thereof, a vector containing the same, and a host cell containing the same.
또한, 본 발명은 상기 Fc 도메인 변이체, 또는 상기 항체 또는 이의 면역학적 활성을 가진 단편을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising the Fc domain variant, or the antibody or fragment having immunological activity thereof as an active ingredient.
또한, 본 발명은 인간 항체 Fc 도메인 변이체의 제조방법을 제공한다.In addition, the present invention provides methods for preparing human antibody Fc domain variants.
또한, 본 발명은 FcγRⅢa에 대한 선택적 결합력이 향상된 당화 항체의 제조방법을 제공한다.In addition, the present invention provides a method for preparing a glycosylated antibody with improved selective binding ability to FcγRIIIa.
또한, 본 발명은 항체 치료제의 제조에 사용하기 위한 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편의 용도를 제공한다.The invention also provides the use of an antibody or immunologically active fragment thereof of the invention for use in the manufacture of an antibody therapeutic.
또한, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편의 암의 예방 또는 치료 용도를 제공한다.In addition, the present invention provides a use of the antibody or immunologically active fragment thereof of the present invention for preventing or treating cancer.
아울러, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법을 제공한다.In addition, the present invention provides a cancer treatment method comprising administering to a subject suffering from cancer a pharmaceutically effective amount of the antibody or immunologically active fragment thereof of the present invention.
본 발명의 신규한 인간 항체 Fc 도메인 변이체들은 종래의 항체 치료제로서 승인받은 항체들보다 면역 저해 수용체인 FcγRⅡb와의 결합력이 감소되고, 면역 활성화 수용체인 FcγRⅢa와의 결합력이 향상되어 (A/I 비율 증가), 현저히 향상된 ADCC 유도능을 가지며, 치료용 단백질 의약품의 면역 작용 기작을 극대화할 수 있는 효과가 있으므로 항체 의약품으로 유용하게 이용될 수 있다.The novel human antibody Fc domain variants of the present invention have reduced binding ability to FcγRIIb, an immune inhibitory receptor, and improved binding ability to FcγRIIIa, an immune activating receptor, compared to antibodies approved as conventional antibody therapeutics (increased A / I ratio), It has remarkably improved ADCC induction ability and can be usefully used as an antibody drug because it has the effect of maximizing the immune action mechanism of therapeutic protein drugs.
도 1은 공지된 돌연변이들의 단순조합으로 구성된 4종의 트라스트주맙 Fc 변이체들 (XMa, XMt, MM 및 XMM) (A) 및 FcγRⅡb-GST 단백질 (B)을 고순도 정제한 후 SDS-PAGE 젤로 확인한 도이다.1 is a diagram showing four types of trastuzumab Fc variants (XMa, XMt, MM and XMM) (A) composed of simple combinations of known mutations and FcγRIIIb-GST protein (B) after high-purity purification and then confirmed by SDS-PAGE gel. am.
도 2는 정제된 트라스트주맙 Fc 변이체들 (트라스트주맙-XMa, XMt, MM 및 XMM)의 FcγRⅡb 결합력을 분석한 ELISA 결과를 나타낸 도이다.Figure 2 is a diagram showing the results of ELISA analyzing the FcγRIIb binding ability of purified trastuzumab Fc variants (trastuzumab-XMa, XMt, MM and XMM).
도 3은 당화 Fc의 포유류 세포 디스플레이 기술에 대한 개략도를 나타낸 도이다.Figure 3 is a diagram showing a schematic diagram of mammalian cell display technology for glycosylated Fc.
도 4는 고순도로 정제된 사합체 FcγRⅢa-158V-스트렙타비딘, 사합체 FcγRⅢa-158F-스트렙타비딘, 사합체 FcγRⅡb-스트렙타비딘, FcγRⅢa-158V-GST 및 FcγRⅢa-158F-GST을 SDS-PAGE 젤로 확인한 도이다.4 shows SDS-PAGE of highly purified tetrameric FcγRIIIa-158V-streptavidin, tetrameric FcγRIIIa-158F-streptavidin, tetrameric FcγRIIb-streptavidin, FcγRIIIa-158V-GST and FcγRIIIa-158F-GST It is also confirmed by gel.
도 5는 형광 표지된 사합체 FcγRⅢa-Alexa488, 사합체 FcγRⅢa-Alexa647 및 Protein A-FITC와 CHO 세포 발현 야생형 Fc 또는 Fc-T299L의 결합 활성을 분석한 도이다.Figure 5 is a diagram showing the analysis of the binding activity of fluorescently labeled tetramer FcγRIIIa-Alexa488, tetramer FcγRIIIa-Alexa647, and Protein A-FITC with wild-type Fc or Fc-T299L expressed in CHO cells.
도 6은 당화 Fc 엔지니어링을 위한 셔플(shuffle) 라이브러리의 개략도를 나타낸 도이다.6 is a schematic diagram of a shuffle library for glycosylation Fc engineering.
도 7은 CHO 세포 디스플레이를 이용한 당화 Fc 엔지니어링 모식도 및 스크리닝 결과 확보한 당화 Fc 변이체의 돌연변이들의 유전자 로고(gene logo)를 나타낸 도이다.7 is a schematic diagram of glycosylated Fc engineering using CHO cell display and a diagram showing gene logos of mutants of glycosylated Fc variants obtained as a result of screening.
도 8은 모델 항체인 트라스트주맙 중쇄 유전자에 당화 Fc 변이체들을 클로닝하여 발현 백터를 트랜스펙션하여 배양한 Expi293F 세포 배양액을 이용하여 FcγRⅢa 결합력을 분석한 ELISA 결과 및 발굴한 Fc 변이체들의 돌연변이 서열을 나타낸 도이다.8 is a diagram showing ELISA results of analyzing FcγRIIIa binding ability using Expi293F cell culture medium obtained by cloning glycosylated Fc variants into the heavy chain gene of trastuzumab, a model antibody, and transfecting the expression vector and culturing, and a diagram showing the mutant sequences of the discovered Fc variants. am.
도 9는 높은 FcγRⅢa 결합력을 보이는 당화 Fc 변이체가 포함된 트라스트주맙 Fc 변이체들 (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 및 PS305)을 정제한 후 SDS-PAGE 젤로 확인한 도이다.Figure 9 is a diagram confirming trastuzumab Fc variants (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 and PS305) containing glycosylated Fc variants showing high FcγRIIIa binding ability after purification using SDS-PAGE gel. am.
도 10 및 11은 선별한 당화 트라스트주맙 Fc 변이체들 (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 및 PS305)의 FcγRⅢa-158V, FcγRⅢa-158F 및 FcγRⅡb와의 결합력을 ELISA로 분석한 도이다.10 and 11 are ELISA analysis of the binding ability of selected glycosylated trastuzumab Fc variants (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 and PS305) to FcγRIIIa-158V, FcγRIIIa-158F and FcγRIlb it is one degree
도 12는 본 발명의 트라스트주맙 Fc 변이체들 (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 및 PS305), 및 기존에 B-세포 림프종 및 유방암 치료제로 US FDA 승인을 받은 당화 항체인 tafasitamab 및 margetuximab의 Fc가 각각 도입된 트라스트주맙 Fc 변이체 DE 및 VLPLL의 FcγRⅢa-158V, FcγRⅡb, FcγRⅢa-158F 또는 FcγRⅡb에 대한 결합 경향성을 비교 분석한 도이다.Figure 12 shows trastuzumab Fc variants (PS101, PS102, PS105, PS106, PS107, PS205, PS207, PS301, PS303 and PS305) of the present invention, and previously approved by the US FDA for the treatment of B-cell lymphoma and breast cancer. It is a diagram comparing and analyzing the binding tendencies of trastuzumab Fc variants DE and VLPLL into which the Fc of tafasitamab and margetuximab, respectively, are introduced, to FcγRIIIa-158V, FcγRIIb, FcγRIIIa-158F or FcγRIIb.
이하, 첨부된 도면을 참조하여 본 발명의 구현예로 본 발명을 상세히 설명하기로 한다. 다만, 하기 구현예는 본 발명에 대한 예시로 제시되는 것으로, 당업자에게 주지 저명한 기술 또는 구성에 대한 구체적인 설명이 본 발명의 요지를 불필요하게 흐릴 수 있다고 판단되는 경우에는 그 상세한 설명을 생략할 수 있고, 이에 의해 본 발명이 제한되지는 않는다. 본 발명은 후술하는 특허청구범위의 기재 및 그로부터 해석되는 균등 범주 내에서 다양한 변형 및 응용이 가능하다. Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.
또한, 본 명세서에서 사용되는 용어(terminology)들은 본 발명의 바람직한 실시예를 적절히 표현하기 위해 사용된 용어들로서, 이는 사용자, 운용자의 의도 또는 본 발명이 속하는 분야의 관례 등에 따라 달라질 수 있다. 따라서, 본 용어들에 대한 정의는 본 명세서 전반에 걸친 내용을 토대로 내려져야 할 것이다. 명세서 전체에서, 어떤 부분이 어떤 구성요소를 "포함"한다고 할 때, 이는 특별히 반대되는 기재가 없는 한 다른 구성요소를 제외하는 것이 아니라 다른 구성 요소를 더 포함할 수 있는 것을 의미한다.In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.
본 발명에서 사용되는 모든 기술용어는, 달리 정의되지 않는 이상, 본 발명의 관련 분야에서 통상의 당업자가 일반적으로 이해하는 바와 같은 의미로 사용된다. 또한 본 명세서에는 바람직한 방법이나 시료가 기재되나, 이와 유사하거나 동등한 것들도 본 발명의 범주에 포함된다. 본 명세서에 참고문헌으로 기재되는 모든 간행물의 내용은 본 발명에 통합된다.All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.
본 명세서 전반을 통하여, 천연적으로 존재하는 아미노산에 대한 통상의 1문자 및 3문자 코드가 사용될 뿐만 아니라 Aib(α-아미노이소부티르산), Sar(N-methylglycine) 등과 같은 다른 아미노산에 대해 일반적으로 허용되는 3문자 코드가 사용된다. 또한 본 발명에서 약어로 언급된 아미노산은 하기와 같이 IUPAC-IUB 명명법에 따라 기재되었다:Throughout this specification, conventional one-letter and three-letter codes for naturally occurring amino acids are used, as well as generally accepted for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-letter code is used. Amino acids also referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:
알라닌: A, 아르기닌: R, 아스파라긴: N, 아스파르트산: D, 시스테인: C, 글루탐산: E, 글루타민: Q, 글리신: G, 히스티딘: H, 이소류신: I, 류신: L, 리신: K, 메티오닌: M, 페닐알라닌: F, 프롤린: P, 세린: S, 트레오닌: T, 트립토판: W, 티로신: Y 및 발린: V. Alanine: A, Arginine: R, Asparagine: N, Aspartic acid: D, Cysteine: C, Glutamic acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y, and valine: V.
일 측면에서, 본 발명은 야생형(Wild type) 인간 항체 Fc 도메인에서, 카밧 넘버링 시스템(Kabat numbering system)에 따라 넘버링된 222, 239, 243, 247, 252, 292, 300, 305, 330, 332, 339, 356, 361, 387, 396, 405 및 415위치의 아미노산으로 이루어진 군으로부터 선택되는 어느 하나 이상의 위치의 아미노산이 야생형의 아미노산과 다른 서열로 치환된, 인간 항체 Fc 도메인 변이체에 관한 것이다.In one aspect, the present invention provides, in a wild type human antibody Fc domain, 222, 239, 243, 247, 252, 292, 300, 305, 330, 332, numbered according to the Kabat numbering system. It relates to a human antibody Fc domain variant in which one or more amino acids selected from the group consisting of amino acids at positions 339, 356, 361, 387, 396, 405, and 415 are substituted with a sequence different from that of the wild-type amino acid.
일 구현예에서, 본 발명의 인간 항체 Fc 도메인 변이체는 K222N, S239D, F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G, N361D, P387Q, P396L, F405L 및 S415G로 이루어진 군으로부터 선택된 어느 하나 이상의 아미노산 치환을 포함할 수 있다.In one embodiment, a human antibody Fc domain variant of the invention is a group consisting of K222N, S239D, F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G, N361D, P387Q, P396L, F405L and S415G It may include any one or more amino acid substitutions selected from.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 서열번호 22 내지 35의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함할 수 있으며, 서열번호 22 내지 31의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 것이 더욱 바람직하다.In one embodiment, the Fc domain variant of the present invention may include any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22 to 35, and any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 22 to 31 It is more preferable to include.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 K222N, F243L, P247I, R292P, Y300L, V305I, A330L 및 I332E의 아미노산 치환을 포함하는 PS301일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS301 comprising amino acid substitutions of K222N, F243L, P247I, R292P, Y300L, V305I, A330L and I332E.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 S239D, P247I, Y300L, V305I, A330L, I332E, A339Q, N361D 및 P387Q의 아미노산 치환을 포함하는 PS303일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS303 comprising amino acid substitutions of S239D, P247I, Y300L, V305I, A330L, I332E, A339Q, N361D and P387Q.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 S239D, R292P, Y300L, A330L, A339Q, P387Q 및 F405L의 아미노산 치환을 포함하는 PS305일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS305 containing amino acid substitutions of S239D, R292P, Y300L, A330L, A339Q, P387Q and F405L.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 F243L, P247I, R292P, Y300L, A330L, I332E 및 P396L의 아미노산 치환을 포함하는 PS205일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS205 containing amino acid substitutions of F243L, P247I, R292P, Y300L, A330L, I332E and P396L.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 F243L, P247I, R292P, A330L, I332E, A339Q, P396L, S415G의 아미노산 치환을 포함하는 PS207일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS207 including amino acid substitutions of F243L, P247I, R292P, A330L, I332E, A339Q, P396L, S415G.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 F243L, P247I, R292P, Y300L, V305I, A330L, I332E, A339Q 및 P387Q의 아미노산 치환을 포함하는 PS101일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS101 comprising amino acid substitutions of F243L, P247I, R292P, Y300L, V305I, A330L, I332E, A339Q and P387Q.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 H224R, F243L, P247I, R292P, Y300L, V303I, V305I, A330L, I332E, A339Q 및 P387Q의 아미노산 치환을 포함하는 PS102일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS102 comprising amino acid substitutions of H224R, F243L, P247I, R292P, Y300L, V303I, V305I, A330L, I332E, A339Q and P387Q.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 R292P, Y300L, V305I, A330L, I332E, A339Q 및 P387Q의 아미노산 치환을 포함하는 PS105일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS105 containing amino acid substitutions of R292P, Y300L, V305I, A330L, I332E, A339Q and P387Q.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G 및 P387Q의 아미노산 치환을 포함하는 PS106일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS106 containing amino acid substitutions of F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G and P387Q.
일 구현예에서, 상기 인간 항체 Fc 도메인 변이체는 F243L, P247I, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G 및 P387Q의 아미노산 치환을 포함하는 PS107일 수 있다.In one embodiment, the human antibody Fc domain variant may be PS107 comprising amino acid substitutions of F243L, P247I, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G and P387Q.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 야생형 Fc 도메인과 비교하여 FcγRⅢa와의 결합력이 향상되고, FcγRⅡb와의 결합력이 감소될 수 있다.In one embodiment, compared to the wild-type Fc domain, the Fc domain variant of the present invention may have improved binding ability to FcγRIIIa and reduced binding ability to FcγRIlb.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 향상된 ADCC(antibody-dependent cell-mediated cytotoxicity) 유도능을 가질 수 있다.In one embodiment, the Fc domain variant of the present invention may have improved ADCC (antibody-dependent cell-mediated cytotoxicity) inducing ability.
일 구현예에서, 인간 항체 (면역글로불린)가 IgA, IgM, IgE, IgD 또는 IgG, 또는 이들의 변형일 수 있으며, IgG1, IgG2, IgG3 또는 IgG4일 수 있고, 항-HER2 항체인 것이 바람직하고, 트라스트주맙인 것이 더욱 바람직하다. 항체의 파파인 분해는 2개의 Fab 도메인과 1개의 Fc 도메인을 형성하며, 인간 IgG 분자에서, Fc 영역은 Cys 226의 N-말단을 파파인 분해함으로써 생성된다 (Deisenhofer, Biochemistry 20: 2361-2370, 1981).In one embodiment, the human antibody (immunoglobulin) may be IgA, IgM, IgE, IgD or IgG, or variants thereof, may be IgG1, IgG2, IgG3 or IgG4, preferably an anti-HER2 antibody; More preferably, it is trastuzumab. Papain digestion of antibodies forms two Fab domains and one Fc domain, 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) .
일 구현예에서, 야생형 인간 항체 Fc 도메인은 서열번호 21의 아미노산 서열을 포함할 수 있으며, 서열번호 36의 핵산분자로 코딩될 수 있다.In one embodiment, the wild-type human antibody Fc domain may include the amino acid sequence of SEQ ID NO: 21 and may be encoded by the nucleic acid molecule of SEQ ID NO: 36.
본 발명에서, 본 발명의 인간 항체 Fc 영역에서 아미노산의 변이를 포함하는 변이체는 모 항체 Fc 영역을 구성하는 아미노산 변형에 따라 정의되고, 통상의 항체 넘버링은 카밧에 의한 EU 인덱스에 따른다 (Kabat et al., Sequence of proteins of immunological interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, 1991). In the present invention, variants comprising amino acid mutations in the human antibody Fc region of the present invention are defined according to amino acid modifications constituting the parent antibody Fc region, and conventional antibody numbering follows the EU index by Kabat (Kabat et al ., Sequence of proteins of immunological interest, 5th Ed., United States Public Health Service, National Institutes of Health, Bethesda, 1991).
본 발명에서 사용된, 용어 "Fc 도메인 변이체"는 "Fc 변이체"와 혼용되어 사용될 수 있다.As used herein, the term "Fc domain variant" may be used interchangeably with "Fc variant".
본 발명에서 사용된, 용어 "야생형 (wild-type) 폴리펩타이드"는 후에 변형되어 유도체를 생산하는 비변형 폴리 펩타이드를 의미한다. 야생형 폴리펩타이드는 자연에서 발견되는 폴리펩타이드 또는 자연에서 발견되는 폴리펩타이드의 유도체 또는 조작된 것일 수 있다. 야생형 폴리펩타이드는 폴리펩타이드 그 자체, 상기 야생형 폴리펩타이드를 포함하는 조성물, 또는 이를 코딩하는 아미노산 서열을 언급하는 것일 수 있다. 따라서, 본 발명에서 사용된 용어 "야생형 항체"는 아미노산 잔기가 변형되어 유도체를 생성하게 되는 비변형된 항체 폴리펩타이드를 의미한다. 상기 용어와 호환적으로, 아미노산 변형이 도입되어 유도체를 생성하게 되는 비변형된 항체 폴리펩타이드를 의미하는 "모(parent) 항체"이 사용될 수 있다.As used herein, the term "wild-type polypeptide" refers to an unmodified polypeptide that is subsequently modified to produce a derivative. A wild-type polypeptide can be a naturally occurring polypeptide or a derivative or engineered of a naturally occurring polypeptide. A wild-type polypeptide may refer to the polypeptide itself, a composition comprising the wild-type polypeptide, or an amino acid sequence encoding the same. Accordingly, the term "wild-type antibody" as used herein refers to an unmodified antibody polypeptide in which amino acid residues are modified to generate a derivative. Consistent with the above term, "parent antibody" may be used to refer to an unmodified antibody polypeptide into which amino acid modifications have been introduced to give rise to a derivative.
본 발명에서 사용된, 용어 "아미노산 변형/변이"는 폴리펩타이드 서열의 아미노산의 치환, 삽입 및/또는 결실, 바람직하게는 치환을 의미한다. 본 발명에서 사용된, 용어 "아미노산 치환" 또는 "치환"은 야생형 인간 항체 Fc 도메인의 폴리펩타이드 서열의 특정 위치에서의 아미노산이 다른 아미노산으로 대체되는 것을 의미한다. 예를 들면, T299A 치환을 포함하는 Fc 변이체는 야생형 항체의 Fc 도메인의 아미노산 서열에서 299번째 아미노산 잔기인 트레오닌이 알라닌으로 대체된 것을 의미한다.As used herein, the term "amino acid modification/variation" refers to substitution, insertion and/or deletion, preferably substitution, of amino acids in a polypeptide sequence. As used herein, the term "amino acid substitution" or "substitution" means that an amino acid at a specific position in a polypeptide sequence of a wild-type human antibody Fc domain is replaced with another amino acid. For example, an Fc variant including T299A substitution means that threonine, which is the 299th amino acid residue in the amino acid sequence of the Fc domain of a wild type antibody, is replaced with alanine.
본 명세서에 사용된 용어 "Fc 변이체"는 야생형 항체 Fc 도메인과 비교하여 하나 이상의 아미노산 잔기의 변형을 포함하는 것을 의미한다. As used herein, the term "Fc variant" is meant to contain a modification of one or more amino acid residues compared to a wild-type antibody Fc domain.
본 발명의 Fc 변이체는 야생형 항체 Fc 도메인 (영역 또는 단편)과 비교하여 하나 이상의 아미노산 변형을 포함하며, 그로 인해 아미노산 서열에 있어 차이를 갖는다. 본 발명에 따른 Fc 변이체의 아미노산 서열은 야생형 항체 Fc 도메인의 아미노산 서열과 실질적으로 상동하다. 예를 들면, 본 발명에 따른 Fc 변이체의 아미노산 서열은 야생형 항체 Fc 도메인의 아미노산 서열과 비교하여 약 80% 이상, 바람직하게는 약 90% 이상, 가장 바람직하게는 약 95% 이상의 상동성을 가질 것이다. 아미노산 변형은 분자생물학적 방법을 사용하여 유전적으로 수행될 수 있거나, 또는 효소적 또는 화학적 방법을 이용하여 수행될 수도 있다.The Fc variants of the present invention contain one or more amino acid modifications compared to wild-type antibody Fc domains (regions or fragments) and therefore differ in amino acid sequence. The amino acid sequence of the Fc variant according to the present invention is substantially identical to the amino acid sequence of the wild-type antibody Fc domain. For example, the amino acid sequence of an Fc variant according to the present invention will have about 80% or more, preferably about 90% or more, most preferably about 95% or more homology compared to the amino acid sequence of a wild-type antibody Fc domain. . Amino acid modifications may be performed genetically using molecular biological methods, or may be performed using enzymatic or chemical methods.
본 발명의 Fc 변이체는 당해 기술분야에 공지된 임의의 방법으로 제조될 수 있다. 일 실시예에서, 본 발명에 따른 인간 항체의 Fc 변이체는 특정 아미노산 변형을 포함하는 폴리펩타이드 서열을 코딩한 후, 원하는 경우, 숙주세포 내로 클로닝되고, 발현 및 검정되는 핵산 형성에 이용된다. 이를 위한 다양한 방법이 문헌 (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons)에 기재되어 있다.Fc variants of the present invention can be prepared by any method known in the art. In one embodiment, an Fc variant of a human antibody according to the present invention encodes a polypeptide sequence comprising specific amino acid modifications and then, if desired, is used to form a nucleic acid that is cloned into a host cell, expressed and assayed. Various methods for this are described in the literature (Molecular Cloning - A Laboratory Manual, 3rd Ed., Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001; Current Protocols in Molecular Biology, John Wiley & Sons).
본 발명에 따른 Fc 변이체를 코딩하는 핵산은 단백질 발현을 위해 발현벡터에 삽입될 수 있다. 발현벡터는, 통상 조절 또는 제어(regulatory) 서열, 선별마커, 임의의 융합 파트너, 및/또는 추가적 요소와 작동가능하게 연결된, 즉, 기능적 관계에 놓인 단백질을 포함한다. 적절한 상태에서, 핵산으로 형질전환된 숙주세포, 바람직하게는, 본 발명에 따른 Fc 변이체를 코딩하는 핵산 함유 발현벡터를 배양하여 단백질 발현을 유도하는 방법에 의해 본 발명에 따른 Fc 변이체가 생산될 수 있다. 포유류 세포, 박테리아, 곤충 세포, 및 효모를 포함하는 다양한 적절한 숙주세포가 사용될 수 있으나, 이에 제한하는 것은 아니다. 외인성 핵산을 숙주세포에 도입하는 방법은 당해 기술분야에 공지되어 있으며, 사용되는 숙주세포에 따라 달라질 것이다. 바람직하게는, 생산비가 저렴하여 산업적 이용가치가 높은 대장균을 숙주세포로 하여 본 발명에 따른 Fc 변이체를 생산한다.A nucleic acid encoding an Fc variant according to the present invention may be inserted into an expression vector for protein expression. An expression vector usually contains a protein operably linked, i.e., in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements. Under appropriate conditions, the Fc variant according to the present invention can be produced by a method of inducing protein expression by culturing a host cell transformed with a nucleic acid, preferably, an expression vector containing a nucleic acid encoding the Fc variant according to the present invention. there is. A variety of suitable host cells may be used including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, the Fc variant according to the present invention is produced using E. coli, which has high industrial value due to low production cost, as a host cell.
따라서, 본 발명의 범위에는 Fc 변이체를 코딩하는 핵산이 도입된 숙주세포를 단백질 발현에 적합한 조건 하에서 배양하는 단계; 및 숙주세포로부터 발현된 Fc 변이체를 정제 또는 분리하는 단계를 포함하는 Fc 변이체의 제조방법이 포함된다.Therefore, the scope of the present invention includes culturing a host cell into which a nucleic acid encoding an Fc variant has been introduced under conditions suitable for protein expression; and a method for producing an Fc variant comprising purifying or isolating the Fc variant expressed from the host cell.
일 측면에서, 본 발명은 본 발명의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편에 관한 것이다.In one aspect, the present invention relates to an antibody comprising an Fc domain variant of the present invention or an immunologically active fragment thereof.
일 구현예에서, 상기 항체는 당화 항체일 수 있다.In one embodiment, the antibody may be a glycosylation antibody.
일 구현예에서, 본 발명의 항체는 서열번호 1 내지 13의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 2(CH2) 및 서열번호 14 내지 20의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 3(CH3)를 포함할 수 있으며, 서열번호 1 내지 9의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 2(CH2) 및 서열번호 14 내지 20의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 3(CH3)을 포함하는 것이 더욱 바람직하다.In one embodiment, the antibody of the present invention consists of a heavy chain constant region domain 2 ( CH 2) comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 1 to 13 and amino acid sequences of SEQ ID NOs: 14 to 20 It may include a heavy chain constant region domain 3 (C H 3) comprising any one selected from the group, and a heavy chain constant region domain 2 comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 9 ( C H 2) and a heavy chain constant region domain 3 ( CH 3) comprising any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 14 to 20 is more preferred.
일 구현예에서, 면역학적 활성을 가진 단편은 Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv(single chain fragment variable), Fv, 단일쇄 항체, Fv 이량체, 상보성 결정 영역 단편, 인간화 항체, 키메라 항체 및 디아바디(diabody)로 이루어진 군으로부터 선택되는 어느 하나일 수 있다.In one embodiment, the fragment having immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single chain antibody, Fv dimer It may be any one selected from the group consisting of a body, a complementarity determining region fragment, a humanized antibody, a chimeric antibody, and a diabody.
일 구현예에서, 본 발명의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편은 효과기 작용을 증가시킬 수 있으며, 야생형 Fc 도메인과 비교하여 FcγRⅢa와의 결합력이 향상되고 FcγRⅡb와의 결합력이 감소됨으로써 높은 FcγRⅢa 결합 선택성을 가져 A/I 비율이 높은 특성을 가지므로, 항체-매개성 세포독성작용(antibody dependent cellular cytotoxicity, ADCC)이 증가될 수 있다.In one embodiment, an antibody comprising an Fc domain variant of the present invention or a fragment having immunological activity thereof can increase effector action, and compared to a wild-type Fc domain, binding to FcγRIIIa is improved and binding to FcγRIIb is reduced, thereby Since it has high FcγRIIIa binding selectivity and has a high A/I ratio, antibody-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) can be increased.
본 발명에서, A/I 비율은 항체의 Fc 도메인이 활성화 FcγR에 결합하는 능력(A)과 저해 FcγRⅡb에 결합하는 능력(I)의 비율(A/I ratio)로서, A/I 비율이 높을수록 우수한 ADCC 유도능을 보이기 때문에 저해 수용체인 FcγRⅡb 결합력 대비 활성화 수용체 결합력을 선택적으로 높이는 것이 중요하다.In the present invention, the A / I ratio is the ratio (A / I ratio) of the ability of the Fc domain of the antibody to bind to the activating FcγR (A) and the ability to bind to the inhibitory FcγRIIb (I), the higher the A / I ratio Since it shows excellent ADCC induction ability, it is important to selectively increase the binding force of the activating receptor compared to the binding force of FcγRIIb, which is an inhibitory receptor.
일반적으로, 포유동물이 발현하여 당화(glycosylated)되어 있는 당화 항체는 Fc 부위에 수식된 당 사슬에 의해 단백질의 구조가 안정화되어 항체가 Fc 수용체에 결합할 수 있지만, 모든 FcγR에 대한 결합력을 가지기 때문에 면역 반응 활성화와 동시에 저해가 나타나는 문제가 있어왔으며, 이와 반대로, 박테리아에서 생산되는 무당화(aglycosylated) 항체는 Fc 부위에 결합된 탄화수소 사슬이 없기 때문에 Fc 수용체에 결합을 하지 못하여 ADCC 기능을 나타낼 수 없는 문제가 있으나, 본 발명의 항체는 '무당화' 항체 또는 이의 면역학적 활성을 가진 단편이기 때문에 선택적으로 FcγR에 대한 결합력을 향상시켜 면역 반응을 조절할 수 있는 효과가 있다. In general, glycosylated antibodies that are expressed in mammals and are glycosylated have a protein structure stabilized by a sugar chain modified at the Fc region so that the antibody can bind to an Fc receptor, but has binding ability to all FcγRs. There has been a problem of inhibition at the same time as activation of the immune response. On the contrary, since aglycosylated antibodies produced in bacteria do not have a hydrocarbon chain bound to the Fc region, they cannot bind to Fc receptors and thus cannot exhibit ADCC function. However, since the antibody of the present invention is an 'aglycosylated' antibody or a fragment having immunological activity thereof, it has the effect of controlling the immune response by selectively enhancing the binding force to FcγR.
항체는 당해 기술분야에서 공지된 다양한 방법으로 분리 또는 정제될 수 있다. 표준 정제방법은 크로마토그래피 기술, 전기영동, 면역, 침강, 투석, 여과, 농축, 및 크로마토포커싱 (chromatofocusing) 기술을 포함한다. 당해 기술분야에 공지된 바와 같이, 예를 들어 박테리아 단백질 A, G, 및 L과 같은 다양한 천연 단백질이 항체와 결합하며, 상기 단백질은 정제에 이용될 수 있다. 종종, 특정 융합 파트너에 의한 정제가 가능할 수 있다. Antibodies can be isolated or purified by a variety of methods known in the art. Standard purification methods include chromatographic techniques, electrophoresis, immunoprecipitation, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques. As is known in the art, a variety of natural proteins bind antibodies, such as, for example, bacterial proteins A, G, and L, and these proteins can be used for purification. Often, purification by specific fusion partners may be possible.
상기 항체는 전체(whole) 항체 형태일 뿐 아니라 항체 분자의 기능적인 단편을 포함한다. 전체 항체는 2개의 전체 길이의 경쇄(light chain) 및 2개의 전체 길이의 중쇄(heavy chain)를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합(disulfide bond)으로 연결되어 있다. 항체 분자의 기능적인 단편이란 항원 결합 기능을 보유하고 있는 단편을 뜻하며, 항체 단편의 예는 (i) 경쇄의 가변영역(VL) 및 중쇄의 가변영역(VH)과 경쇄의 불변영역(CL) 및 중쇄의 첫번째 불변 영역(CH1)으로 이루어진 Fab 단편; (ii) VH 및 CH1 도메인으로 이루어진 Fd 단편; (iii) 단일 항체의 VL 및 VH 도메인으로 이루어진 Fv 단편; (iv) VH 도메인으로 이루어진 dAb 단편(Ward ES et al., Nature 341:544-546 (1989)]; (v) 분리된 CDR 영역; (vi) 2개의 연결된 Fab 단편을 포함하는 2가 단편인 F(ab')2 단편; (vii) VH 도메인 및 VL 도메인이 항원 결합 부위를 형성하도록 결합시키는 펩타이드 링커에 의해 결합된 단일쇄 Fv 분자(scFv); (viii) 이특이적인 단일쇄 Fv 이량체(PCT/US92/09965) 및 (ix) 유전자 융합에 의해 제작된 다가 또는 다특이적인 단편인 디아바디(diabody) WO94/13804) 등을 포함한다. The antibodies include whole antibody forms as well as functional fragments of antibody molecules. A full antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. A functional fragment of an antibody molecule refers to a fragment having an antigen-binding function, and examples of antibody fragments include (i) a light chain variable region (VL) and a heavy chain variable region (VH) and a light chain constant region (CL) and a Fab fragment consisting of the first constant region of the heavy chain (CH1); (ii) a Fd fragment consisting of the VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)]; (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments. F(ab')2 fragments; (vii) single-chain Fv molecules (scFv) joined by a peptide linker that links the VH and VL domains to form an antigen-binding site; (viii) bispecific single-chain Fv dimers. (PCT/US92/09965) and (ix) a multivalent or multispecific fragment produced by gene fusion (diabody WO94/13804).
본 발명의 항체 또는 이의 면역학적 활성을 가진 단편은 동물 유래 항체, 키메릭 항체, 인간화 항체, 인간 항체, 및 이들의 면역학적 활성을 가진 단편으로 이루어진 군에서 선택된 것일 수 있다. 상기 항체는 재조합적 또는 합성적으로 생산된 것일 수 있다.The antibody or immunologically active fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The antibody may be produced recombinantly or synthetically.
상기 항체 또는 이의 면역학적 활성을 가진 단편은 생체에서 분리된 (생체에 존재하지 않는) 것 또는 비자연적으로 생산(non-naturally occurring)된 것일 수 있으며, 예컨대, 합성적 또는 재조합적으로 생산된 것일 수 있다.The antibody or immunologically active fragment thereof may be isolated from a living body (not present in a living body) or non-naturally occurring, for example, synthetically or recombinantly produced. can
본 발명에서 "항체"라 함은, 면역계 내에서 항원의 자극에 의하여 만들어지는 물질을 의미하는 것으로서, 그 종류는 특별히 제한되지 않으며, 자연적 또는 비자연적(예컨대, 합성적 또는 재조합적)으로 얻어질 수 있다. 항체는 생체 외뿐 아니라 생체 내에서도 매우 안정하고 반감기가 길기 때문에 대량 발현 및 생산에 유리하다. 또한, 항체는 본질적으로 다이머(dimer) 구조를 가지므로 접착능(avidity)이 매우 높다. 완전한 항체는 2개의 전장(full length) 경쇄 및 2개의 전장 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 이황화 결합으로 연결되어 있다. 항체의 불변 영역은 중쇄 불변 영역과 경쇄 불변 영역으로 나뉘어지며, 중쇄 불변 영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고, 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변 영역은 카파(κ) 및 람다(λ) 타입을 가진다.In the present invention, "antibody" refers to a substance produced by stimulation of an antigen in the immune system, and the type is not particularly limited, and may be obtained naturally or non-naturally (e.g., synthetically or recombinantly). can Antibodies are advantageous for mass expression and production because they are very stable in vitro as well as in vivo and have a long half-life. In addition, since antibodies essentially have a dimer structure, avidity is very high. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond. The antibody constant region is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types, subclasses It has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain is of the kappa (κ) and lambda (λ) type.
본 발명에서 용어, "중쇄(heavy chain)"는 항원에 특이성을 부여하기 위해 충분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VH 및 3개의 불변 영역 도메인 CH1 , CH2 및 CH3과 힌지(hinge)를 포함하는 전장 중쇄 및 이의 단편을 모두 포함하는 의미로 해석된다. 또한, 용어 "경쇄(light chain)"는 항원에 특이성을 부여하기 위한 충분한 가변 영역 서열을 갖는 아미노산 서열을 포함하는 가변 영역 도메인 VL 및 불변 영역 도메인 CL을 포함하는 전장 경쇄 및 이의 단편을 모두 포함하는 의미로 해석된다.As used herein, the term "heavy chain" refers to a variable region domain V H comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen and three constant region domains C H 1 , C H 2 and It is interpreted as meaning including both full-length heavy chains and fragments thereof including C H 3 and a hinge. In addition, the term "light chain" refers to both a full-length light chain comprising a variable region domain V L and a constant region domain CL comprising an amino acid sequence having sufficient variable region sequence to impart specificity to an antigen and fragments thereof. be interpreted in a sense that includes
본 발명에서 용어, "Fc 도메인", "Fc 단편" 또는 "Fc 영역"은 Fab 도메인/단편과 함께 항체를 이루며, Fab 도메인/단편은 경쇄의 가변영역(VL) 및 중쇄의 가변영역(VH)과, 경쇄의 불변영역(CL) 및 중쇄의 첫번째 불변 영역(CH1)으로 이루어지고, Fc 도메인/단편은 중쇄의 두 번째 불변 영역(CH2) 및 세 번째 불변 영역(CH3)로 이루어진다.In the present invention, the term "Fc domain", "Fc fragment" or "Fc region" constitutes an antibody together with a Fab domain/fragment, and the Fab domain/fragment comprises a light chain variable region (V L ) and a heavy chain variable region (V H ), light chain constant region ( CL ) and heavy chain first constant region (C H 1), the Fc domain / fragment is the heavy chain of the second constant region (CH 2) and the third constant region (C H 2) H 3).
일 측면에서, 본 발명은 본 발명의 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산분자에 관한 것이다.In one aspect, the present invention relates to a nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof.
일 구현예에서, 본 발명에 따른 Fc 변이체를 코딩하는 핵산분자는 37 내지 50의 염기서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함할 수 있다.In one embodiment, the nucleic acid molecule encoding the Fc variant according to the present invention may include any one selected from the group consisting of nucleotide sequences of 37 to 50.
일 측면에서, 본 발명은 상기 핵산분자를 포함하는 벡터, 상기 벡터를 포함하는 숙주세포에 관한 것이다.In one aspect, the present invention relates to a vector containing the nucleic acid molecule and a host cell containing the vector.
본 발명의 핵산분자는 단리된 것이거나 재조합된 것일 수 있으며, 단일쇄 및 이중쇄 형태의 DNA 및 RNA뿐만 아니라 대응하는 상보성 서열이 포함된다. 단리된 핵산은 천연 생성 원천에서 단리된 핵산의 경우, 핵산이 단리된 개체의 게놈에 존재하는 주변 유전 서열로부터 분리된 핵산이다. 주형으로부터 효소적으로 또는 화학적으로 합성된 핵산, 예컨대 PCR 산물, cDNA 분자, 또는 올리고뉴클레오타이드의 경우, 이러한 절차로부터 생성된 핵산이 단리된 핵산분자로 이해될 수 있다. 단리된 핵산분자는 별도 단편의 형태 또는 더 큰 핵산 구축물의 성분으로서의 핵산 분자를 나타낸다. 핵산은 다른 핵산 서열과 기능적 관계로 배치될 때 작동가능하게 연결된다. 예를 들면, 전서열 또는 분비 리더(leader)의 DNA는 폴리펩타이드가 분비되기 전의 형태인 전단백질(preprotein)로서 발현되는 경우 폴리펩타이드의 DNA에 작동가능하게 연결되고, 프로모터 또는 인핸서는 폴리펩타이드 서열의 전사에 영향을 주는 경우 코딩 서열에 작동가능하게 연결되며, 또는 리보솜 결합 부위는 번역을 촉진하도록 배치될 때 코딩 서열에 작동가능하게 연결된다. 일반적으로 작동가능하게 연결된은 연결될 DNA 서열들이 인접하여 위치함을 의미하며, 분비 리더의 경우 인접하여 동일한 리딩 프레임 내에 존재하는 것을 의미한다. 그러나 인핸서는 인접하여 위치할 필요는 없다. 연결은 편리한 제한 효소 부위에서 라이게이션에 의해 달성된다. 이러한 부위가 존재하지 않는 경우, 합성 올리고뉴클레오타이드 어댑터 또는 링커를 통상적인 방법에 따라 사용한다. 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. In the case of a nucleic acid synthesized enzymatically or chemically from a template, such as a PCR product, cDNA molecule, or oligonucleotide, the nucleic acid resulting from such a procedure can be understood as an isolated nucleic acid molecule. An isolated 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. For example, 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. In general, 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. However, 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.
본 발명의 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자는 코돈의 축퇴성(degeneracy)으로 인하여 또는 상기 이를 발현시키고자 하는 생물에서 선호되는 코돈을 고려하여, 코딩영역으로부터 발현되는 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편의 아미노산 서열을 변화시키지 않는 범위 내에서 코딩영역에 다양한 변형이 이루어질 수 있고, 코딩영역을 제외한 부분에서도 유전자의 발현에 영향을 미치지 않는 범위 내에서 다양한 변형 또는 수식이 이루어질 수 있으며, 그러한 변형 유전자 역시 본 발명의 범위에 포함됨을 당업자는 잘 이해할 수 있을 것이다. 즉, 본 발명의 핵산 분자는 이와 동등한 활성을 갖는 단백질을 코딩하는 한, 하나 이상의 핵산 염기가 치환, 결실, 삽입 또는 이들의 조합에 의해 변이될 수 있으며, 이들 또한 본 발명의 범위에 포함된다. 이러한 핵산 분자의 서열은 단쇄 또는 이중쇄일 수 있으며, DNA 분자 또는 RNA(mRNA)분자일 수 있다.The isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or an antibody containing the same, or a fragment having immunological activity thereof, has codons preferred in organisms intended to express the same due to codon degeneracy. In consideration of this, various modifications may be made to the coding region within the range of not changing the amino acid sequence of the Fc domain variant expressed from the coding region, or an antibody containing the same or a fragment having immunological activity thereof, and a portion other than the coding region. It will be well understood by those skilled in the art that various modifications or modifications may be made within a range that does not affect gene expression, and that such modified genes are also included in the scope of the present invention. That is, as long as the nucleic acid molecule of the present invention encodes a protein having an activity equivalent thereto, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included in the scope of the present invention. The sequence of such a nucleic acid molecule may be single- or double-stranded, and may be a DNA molecule or an RNA (mRNA) molecule.
본 발명에 따른 본 발명의 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자는 단백질 발현을 위해 발현벡터에 삽입될 수 있다. 발현벡터는, 통상 조절 또는 제어 (regulatory) 서열, 선별마커, 임의의 융합 파트너, 및/또는 추가적 요소와 작동가능하게 연결된, 즉, 기능적 관계에 놓인 단백질을 포함한다. 적절한 상태에서, 핵산으로 형질전환된 숙주세포, 바람직하게는, 본 발명의 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 단리된 핵산 분자 함유 발현벡터를 배양하여 단백질 발현을 유도하는 방법에 의해 본 발명의 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편이 생산될 수 있다. 포유류 세포, 박테리아, 곤충 세포, 및 효모를 포함하는 다양한 적절한 숙주세포가 사용될 수 있으나, 이에 제한하는 것은 아니다. 외인성 핵산을 숙주세포에 도입하는 방법은 당해 기술분야에 공지되어 있으며, 사용되는 숙주세포에 따라 달라질 것이다. 바람직하게는, 생산비가 저렴하여 산업적 이용가치가 높은 대장균을 숙주세포로 생산할 수 있다.The isolated nucleic acid molecule encoding the Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof according to the present invention may be inserted into an expression vector for protein expression. An expression vector usually contains a protein operably linked, i.e., in a functional relationship, with regulatory or regulatory sequences, selectable markers, optional fusion partners, and/or additional elements. Under appropriate conditions, a host cell transformed with a nucleic acid, preferably, an expression vector containing an isolated nucleic acid molecule encoding an Fc domain variant of the present invention, or an antibody comprising the same or an immunologically active fragment thereof is cultured to produce a protein. An Fc domain variant of the present invention, or an antibody comprising the same, or a fragment having immunological activity thereof may be produced by a method of inducing expression. A variety of suitable host cells may be used including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, it is possible to produce E. coli, which has high industrial value due to low production cost, as a host cell.
본 발명의 벡터는 플라스미드 벡터, 코즈미드 벡터, 박테리오 파아지 벡터 및 바이러스 벡터 등을 포함하나 이에 제한되지 않는다. 적합한 벡터는 프로모터, 오퍼레이터, 개시코돈, 종결코돈, 폴리아데닐화 시그널 및 인핸서 같은 발현 조절 엘리먼트 외에도 막 표적화 또는 분비를 위한 시그널 서열 또는 리더 서열을 포함하며 목적에 따라 다양하게 제조될 수 있다. 벡터의 프로모터는 구성적 또는 유도성일 수 있다. 상기 시그널 서열에는 숙주가 에쉐리키아속(Escherichia sp.)균인 경우에는 PhoA 시그널 서열, OmpA 시그널 서열 등이, 숙주가 바실러스속(Bacillus sp.)균인 경우에는 α-아밀라아제 시그널 서열, 서브틸리신 시그널 서열 등이, 숙주가 효모(yeast)인 경우에는 MFα 시그널 서열, SUC2 시그널 서열 등이, 숙주가 동물세포인 경우에는 인슐린 시그널 서열, α-인터페론 시그널 서열, 항체 분자 시그널 서열 등을 이용할 수 있으나, 이에 제한되지 않는다. 또한 벡터는 벡터를 함유하는 숙주 세포를 선택하기 위한 선택 마커를 포함할 수 있고, 복제 가능한 발현벡터인 경우 복제 기원을 포함한다.Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors and viral vectors. Suitable vectors include expression control elements such as promoters, operators, initiation codons, stop codons, polyadenylation signals and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and may be prepared in various ways depending on the purpose. The vector's promoter may be constitutive or inducible. The signal sequence includes a PhoA signal sequence and an OmpA signal sequence when the host is Escherichia sp., and an α-amylase signal sequence and a subtilisin signal when the host is Bacillus sp. Sequences such as MFα signal sequence, SUC2 signal sequence, etc. can be used when the host is yeast, and insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used when the host is an animal cell. Not limited to this. In addition, the vector may include a selectable marker for selecting a host cell containing the vector, and in the case of a replicable expression vector, an origin of replication.
본 발명에서 용어, "벡터"는 핵산 서열을 복제할 수 있는 세포로의 도입을 위해서 핵산 서열을 삽입할 수 있는 전달체를 의미한다. 핵산 서열은 외생 (exogenous) 또는 이종 (heterologous)일 수 있다. 벡터로서는 플라스미드, 코스미드 및 바이러스(예를 들면 박테리오파지)를 들 수 있으나, 이에 제한되지 않는다. 당업자는 표준적인 재조합 기술에 의해 벡터를 구축할 수 있다(Maniatis, et al., Molecular Cloning , A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; 및 Ausubel et al., In: Current Protocols in Molecular Biology , John, Wiley & Sons, Inc, NY, 1994 등).As used herein, the term "vector" refers to a delivery vehicle into which a nucleic acid sequence can be inserted for introduction 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, bacteriophages). 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, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).
일 구현예에서, 상기 벡터의 제작 시, 상기 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을생산하고자 하는 숙주세포의 종류에 따라 프로모터(promoter), 종결자(terminator), 인핸서(enhancer) 등과 같은 발현조절 서열, 막 표적화 또는 분비를 위한 서열 등을 적절히 선택하고 목적에 따라 다양하게 조합할 수 있다.In one embodiment, when constructing the vector, a promoter, a terminator, a promoter, a terminator, An expression control sequence such as an enhancer, a sequence for membrane targeting or secretion, etc. may be appropriately selected and combined in various ways according to the purpose.
본 발명에서, 용어 "발현 벡터"는 전사되는 유전자 산물 중 적어도 일부분을 코딩하는 핵산 서열을 포함한 벡터를 의미한다. 일부의 경우에는 그 후 RNA 분자가 단백질, 폴리펩타이드, 또는 펩타이드로 번역된다. 발현 벡터에는 다양한 조절서열을 포함할 수 있다. 전사 및 번역을 조절하는 조절서열과 함께 벡터 및 발현 벡터에는 또 다른 기능도 제공하는 핵산 서열도 포함될 수 있다.In the present invention, the term "expression vector" refers to a vector comprising 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 control sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.
본 발명에서, 용어 "숙주세포"는 진핵생물 및 원핵생물을 포함하며, 상기 벡터를 복제할 수 있거나 벡터에 의해 코딩되는 유전자를 발현할 수 있는 임의의 형질 전환 가능한 생물을 의미한다. 숙주세포는 상기 벡터에 의해 형질감염(transfected) 또는 형질전환(transformed) 될 수 있으며, 이는 외생의 핵산분자가 숙주세포 내에 전달되거나 도입되는 과정을 의미한다.In the present invention, 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.
일 구현예에서, 상기 숙주 세포는 박테리아 또는 동물세포일 수 있으며, 동물 세포주는 CHO 세포, HEK 세포 또는 NSO 세포일 수 있고, 박테리아는 대장균일 수 있다.In one embodiment, the host cell may be a bacterial or animal cell, the animal cell line may be a CHO cell, a HEK cell or a NSO cell, and the bacteria may be Escherichia coli.
일 측면에서, 본 발명은 본 발명의 Fc 도메인 변이체를 포함하는 항체 치료제에 관한 것이다.In one aspect, the present invention relates to an antibody therapeutic comprising an Fc domain variant of the present invention.
일 구현예에서, 본 발명에 따른 Fc 도메인 변이체 또는 이를 포함하는 단백질 결합체에 인간의 질병을 치료 또는 예방할 목적으로 사용되는 싸이토카인, 인터루킨, 인터루킨 결합 단백질, 효소, 항체, 성장인자, 전사조절인자, 혈액인자, 백신, 구조단백질, 리간드 단백질 또는 수용체, 세포표면항원, 수용체 길항물질과 같은 다양한 생리활성 폴리펩타이드, 이들의 유도체 및 유사체가 결합되어 사용될 수 있다.In one embodiment, cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcriptional regulators, blood used for the purpose of treating or preventing human diseases are added to the Fc domain variant or protein conjugate comprising the same according to the present invention. Factors, vaccines, structural proteins, ligand proteins or various physiologically active polypeptides such as receptors, cell surface antigens, and receptor antagonists, derivatives and analogs thereof may be used in combination.
일 구현예에서, 본 발명에 따른 Fc 도메인 변이체 또는 이를 포함하는 단백질 결합체에 항체 약물이 결합될 수 있으며, 암 치료용 항체 약물은 트라스투주맙(Trastzumab), 세툭시맙(cetuximab), 베바시주맙(bevacizumab), 리툭시맙(rituximab), 바실릭시맙(basiliximab), 인플릭시맙(infliximab), 이필리무맙(Ipilimumab), 펨브롤리주맙(Pembrolizumab), 니볼루맙(Nivolumab), 아테졸리주맙(Atezolizumab) 또는 아벨루맙(Avelumab)일 수 있다.In one embodiment, an antibody drug may be conjugated to the Fc domain variant or a protein conjugate including the same according to the present invention, and the antibody drug for cancer treatment is Trastzumab, cetuximab, or bevacizumab. (bevacizumab), rituximab, basiliximab, infliximab, ipilimumab, pembrolizumab, nivolumab, atezolizumab (Atezolizumab) or Avelumab.
항체 치료제에서 타깃 항원으로 면역세포들을 모집하여 전달하는 기작은 가장 중요한 기작 중 하나이며, 항체의 Fc 도메인이 면역세포의 모집과 ADCC(antibody-dependent cell-mediated cytotoxicity)에 결정적인 역할을 하므로, 본 발명의 Fc 감마 수용체에 선택적 결합력이 증가된 Fc 변이체는 치료용 항체로 이용되기에 유리하다. 특히, 항체의 ADCC 기능은 많은 세포의 표면에 존재하는 Fc감마 수용체(FcγR)와의 상호작용에 의존하며, 사람의 5가지 Fc 수용체 중 항체가 어떠한 Fc 수용체에 결합되는지에 따라 모집되는 면역세포의 종류가 결정되기 때문에 특정한 세포를 모집할 수 있도록 항체를 변형하는 시도는 치료 분야에 있어서 매우 중요하다.In antibody therapeutics, the mechanism of recruiting and delivering immune cells to the target antigen is one of the most important mechanisms, and the Fc domain of an antibody plays a crucial role in the recruitment of immune cells and ADCC (antibody-dependent cell-mediated cytotoxicity), so the present invention An Fc variant having increased selective binding ability to the Fc gamma receptor is advantageous for use as a therapeutic antibody. In particular, the ADCC function of antibodies depends on interactions with Fc gamma receptors (FcγRs) present on the surface of many cells, and the type of immune cells recruited depending on which Fc receptors the antibody binds to among the five human Fc receptors. Since is determined, attempts to modify antibodies to recruit specific cells are very important in the field of therapy.
일 측면에서, 본 발명은 본 발명의 Fc 도메인 변이체, 또는 이를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물에 관한 것이다.In one aspect, the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising the Fc domain variant of the present invention, or an antibody containing the same or a fragment having immunological activity thereof as an active ingredient.
일 구현예에서, 암은 뇌종양, 흑색종, 골수종, 비소세포성폐암, 구강암, 간암, 위암, 결장암, 유방암, 폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 나팔관암종, 항문부근암, 자궁내막암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 중추신경계 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나일 수 있다. In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colorectal cancer, Small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma, and It may be any one selected from the group consisting of pituitary adenoma.
일 구현예에서, 본 발명의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편은 높은 FcγRⅢa 결합 선택성을 가져 높은 A/I 비율을 가지므로, 자연살해세포(NK 세포)를 통해 효과기 작용을 증가시켜 항체-매개성 세포독성작용 (antibody dependent cellular cytotoxicity, ADCC)을 증가시킬 수 있다. 가장 강력한 암세포 사멸 효능을 가지고 있는 자연살해세포(NK 세포)는 다른 면역세포들 (예:　monocytes, macrophages, dendritic cells)과는 달리 표면에 FcγRⅢa를 발현하고, FcγRI과 FcγRⅡa, FcγRⅡb 및 FcγRⅢb는 발현하지 않으므로, 본 발명의 FcγRⅢa 결합 선택성을 가지는 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편이 NK 세포를 통해 암세포 사멸 작용 기작을 극대화할 수 있다.In one embodiment, the antibody comprising the Fc domain variant of the present invention or a fragment having immunological activity thereof has a high FcγRIIIa binding selectivity and a high A/I ratio, so effector action through natural killer cells (NK cells) By increasing the antibody-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) can be increased. Unlike other immune cells (e.g., monocytes, macrophages, dendritic cells), natural killer cells (NK cells), which have the strongest cancer cell killing effect, express FcγRIIIa on their surface and do not express FcγRI, FcγRIIa, FcγRIIb, or FcγRIIIb. Therefore, the antibody comprising the Fc domain variant having FcγRIIIa binding selectivity or a fragment having immunological activity thereof of the present invention can maximize the cancer cell killing mechanism through NK cells.
일 구현예에서, 본 발명의 조성물은 면역원성 세포사멸 유도제를 추가로 포함할 수 있으며, 면역원성 세포사멸 유도제는 안트라사이클린계열 항암제, 탁산 계열 항암제, 항-EGFR 항체, BK 채널 작용제, 보르테조밉(Bortezomib), 강심성 배당체(cardiac glycoside), 사이클로포스마이드 계열 항암제, GADD34/PP1 저해제, LV-tSMAC, Measles 바이러스, 블레오마이신(bleomycin), 미토잔트론(mitoxantrone) 또는 옥살리플라틴(oxaliplatin)으로 이루어진 군으로부터 선택되는 어느 하나 이상일 수 있으며, 안트라사이클린 계열 항암제는 다우노루비신(daunorubicin), 독소루비신(doxorubicin), 에피루비신(epirubicin), 이다루비신(idarubicin), 픽산트론(pixantrone), 사바루비신(sabarubicin) 또는 발루비신(valrubicin)일 수 있고, 탁산계열 항암제는 파클리탁셀(paclitaxel) 또는 도세탁셀(docetaxel)일 수 있다.In one embodiment, the composition of the present invention may further include an immunogenic apoptosis inducer, and the immunogenic apoptosis inducer is an anthracycline-based anticancer agent, a taxane-based anticancer agent, an anti-EGFR antibody, a BK channel agonist, bortezomib ( Bortezomib), cardiac glycoside, cyclophosmid anticancer drug, GADD34/PP1 inhibitor, LV-tSMAC, Measles virus, bleomycin, mitoxantrone or oxaliplatin It may be any one or more selected, and anthracycline-based anticancer agents include daunorubicin, doxorubicin, epirubicin, idarubicin, pixantrone, and sabarubicin. ) or valrubicin, and the taxane-based anticancer agent may be paclitaxel or docetaxel.
본 발명의 암의 예방 또는 치료용 약학적 조성물은 화학적 항암 약물(항암제) 등과 함께 투여함으로써, 암세포의 사멸 효과를 통해 종래의 항암제의 암치료 효과를 증가시킬 수 있다. 병용 투여는 상기 항암제와 동시에 또는 순차적으로 이루어질 수 있다. 상기 항암제의 예시에는 DNA 알킬화제(DNA alkylating agents)로 메클로에타민(mechloethamine), 클로람부칠(chlorambucil), 페닐알라닌(phenylalanine), 무스타드(mustard), 사이클로포스파미드(cyclophosphamide), 이포스파미드(ifosfamide), 카르무스틴(carmustine: BCNU), 로무스틴(lomustine: CCNU), 스트렙토조토신(streptozotocin), 부술판(busulfan), 티오테파(thiotepa), 시스플라틴(cisplatin) 및 카보플라틴(carboplatin); 항암 항생제(anti-cancer antibiotics)로 닥티노마이신(dactinomycin: actinomycin D), 플리카마이신(plicamycin) 및 마이토마이신 C(mitomycin C); 및 식물 알카로이드(plant alkaloids)로 빈크리스틴(vincristine), 빈블라스틴(vinblastine), 에토포시드(etoposide), 테니포시드(teniposide), 토포테칸(topotecan) 및 이리도테칸(iridotecan) 등이 포함되지만, 이에 한정되는 것은 아니다.The pharmaceutical composition for preventing or treating cancer of the present invention can increase the cancer treatment effect of conventional anticancer drugs through the killing effect of cancer cells by administering together with chemical anticancer drugs (anticancer drugs). Concomitant administration may be performed simultaneously or sequentially with the anticancer agent. Examples of the anticancer agent are DNA alkylating agents such as mechloethamine, chlorambucil, phenylalanine, mustard, cyclophosphamide, ifosfamide ( ifosfamide, carmustine (BCNU), lomustine (CCNU), streptozotocin, busulfan, thiotepa, cisplatin and carboplatin ; dactinomycin (actinomycin D), plicamycin and mitomycin C as anti-cancer antibiotics; and plant alkaloids such as vincristine, vinblastine, etoposide, teniposide, topotecan and iridotecan. , but is not limited thereto.
본 발명에서, 용어 "예방"이란 본 발명에 따른 약학적 조성물의 투여에 의해 암의 발생, 확산 및 재발을 억제 또는 지연시키는 모든 행위를 의미한다.In the present invention, the term "prevention" refers to all activities that inhibit or delay the occurrence, spread, and recurrence of cancer by administration of the pharmaceutical composition according to the present invention.
본 발명에서 사용되는 용어 "치료"란 본 발명의 조성물의 투여로 암세포의 사멸 또는 암의 증세를 호전시키거나 이롭게 변경하는 모든 행위를 의미한다. 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자라면, 대한의학협회 등에서 제시된 자료를 참조하여 본원의 조성물이 효과가 있는 질환의 정확한 기준을 알고, 개선, 향상 및 치료된 정도를 판단할 수 있을 것이다.The term "treatment" used in the present invention refers to any activity that ameliorates or beneficially alters the death of cancer cells or symptoms of cancer by administration of the composition of the present invention. Those of ordinary skill in the art to which the present invention pertains will be able to determine the degree of improvement, enhancement and treatment by knowing the exact criteria of the disease for which the composition of the present application is effective by referring to the data presented by the Korean Medical Association, etc. will be.
본 발명에서 유효성분과 결합하여 사용된 "치료학적으로 유효한 양"이란 용어는 대상 질환을 예방 또는 치료하는데 유효한 조성물의 약학적으로 허용가능한 염의 양을 의미하며, 본 발명의 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. 유효한 양의 결정시 고려할 이러한 사항은, 예를 들면 Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed.(2001), Pergamon Press; 및 E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed.(1990), Mack Publishing Co.에 기술되어있다.The term "therapeutically effective amount" used in combination with an active ingredient in the present invention refers to an amount of a pharmaceutically acceptable salt of a composition effective for preventing or treating a target disease, and a therapeutically effective amount of the composition of the present invention It may vary depending on various factors, such as the method of administration, the target site, and the condition of the patient. Therefore, when used in the human body, the dosage should be determined in an appropriate amount considering both safety and efficiency. It is also possible to estimate the amount to be used in humans from the effective amount determined through animal experiments. These considerations in determining an effective amount can be found, for example, in Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.
본 발명의 약학 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에서 사용되는 용어, "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분하며 부작용을 일으키지 않을 정도의 양을 의미하며, 유효용량 수준은 환자의 건강상태, 암의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 방법, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 배합 또는 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고, 종래의 치료제와 순차적으로 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여, 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is the patient's Health condition, cancer type, severity, drug activity, drug sensitivity, method of administration, time of administration, route of administration and excretion rate, duration of treatment, factors including drugs used in combination or concurrently, and other factors well known in the medical field can be determined according to The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Considering all of the above factors, it is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
본 발명의 약학 조성물은 약학적으로 허용 가능한 첨가제를 더 포함할 수 있으며, 이때 약학적으로 허용 가능한 첨가제로는 전분, 젤라틴화 전분, 미결정셀룰로오스, 유당, 포비돈, 콜로이달실리콘디옥사이드, 인산수소칼슘, 락토스, 만니톨, 엿, 아라비아고무, 전호화전분, 옥수수전분, 분말셀룰로오스, 히드록시프로필셀룰로오스, 오파드라이, 전분글리콜산나트륨, 카르나우바 납, 합성규산알루미늄, 스테아린산, 스테아린산마그네슘, 스테아린산알루미늄, 스테아린산칼슘, 백당, 덱스트로스, 소르비톨 및 탈크 등이 사용될 수 있다. 본 발명에 따른 약학적으로 허용 가능한 첨가제는 상기 조성물에 대해 0.1 중량부 내지 90 중량부 포함되는 것이 바람직하나, 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, Lactose, Mannitol, Taffy, Gum Arabic, Pregelatinized Starch, Corn Starch, Powdered Cellulose, Hydroxypropyl Cellulose, Opadry, Sodium Starch Glycolate, Carnauba Lead, Synthetic Aluminum Silicate, Stearic Acid, Magnesium Stearate, Aluminum Stearate, Stearic Acid Calcium, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 part by weight to 90 parts by weight based on the composition, but is not limited thereto.
본 발명의 조성물은 또한 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약학적으로 허용 가능한 담체는 조성물을 생체 내 전달에 적합한 것이면 특별히 제한되지 않으며, 예를 들면, Merck Index, 13th ed., Merck & Co. Inc. 에 기재된 화합물, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition of the present invention may also include a carrier, diluent, excipient or a combination of two or more commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. , saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used. Customary additives may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate formulations for injection, such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by using an appropriate method in the art or by using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 조성물은 목적하는 방법에 따라 비 경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 주사 제형으로 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설률 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 조성물의 일일 투여량은 0.0001 ~ 10 ㎎/㎖이며, 바람직하게는 0.0001 ~ 5 ㎎/㎖이며, 하루 일 회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The composition of the present invention may be parenterally administered (for example, intravenously, subcutaneously, intraperitoneally, or topically applied as an injection formulation) or orally, depending on the desired method, and the dosage may be determined by the patient's weight, age, sex, The range varies according to health status, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to divide the administration once or several times a day.
본 발명의 조성물의 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 통상적으로 사용되는 단순 희석제인 물, 액체 파라핀 이외에 다양한 부형제, 예컨대 습윤제, 감미제, 방향제, 보존제 등이 함께 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성 용제, 현탁제, 유제, 동결건조 제제, 좌제 등이 포함된다.Liquid formulations for oral administration of the composition of the present invention include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents etc. may be included. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, suppositories, and the like.
본 발명은 상기 Fc 도메인 변이체를 비펩타이드성 중합체를 통해 생리활성 폴리펩타이드에 공유결합으로 연결하여 지속성 약물 제제를 제조하는 방법을 포함한다.The present invention includes a method for preparing a long-acting drug formulation by covalently linking the Fc domain variant to a physiologically active polypeptide through a non-peptide polymer.
본 발명에 따른 제조방법은 말단에 반응기를 갖는 비펩타이드성 중합체를 통해 생리활성 폴리펩타이드 및 Fc 도메인 변이체를 공유결합으로 연결하는 단계; 및 생리활성 폴리펩타이드, 비펩타이드성 중합체 및 Fc 도메인 변이체가 공유결합으로 연결된 결합체를 분리하는 단계를 포함할 수 있다.The manufacturing method according to the present invention comprises the steps of covalently linking a physiologically active polypeptide and an Fc domain variant through a non-peptide polymer having a reactive group at the terminal; and isolating a conjugate in which the physiologically active polypeptide, the non-peptide polymer, and the Fc domain variant are covalently linked.
일 측면에서, 본 발명은 a) 본 발명의 Fc 도메인 변이체를 코딩하는 핵산분자를 포함하는 벡터를 포함하는 숙주세포를 배양하는 단계; 및 b) 숙주세포에 의해 발현된 폴리펩타이드를 회수하는 단계를 포함하는, FcγRⅢa에 대한 선택적 결합력이 향상된, 인간 항체 Fc 도메인 변이체의 제조방법에 관한 것이다.In one aspect, the present invention comprises the steps of a) culturing a host cell containing a vector containing a nucleic acid molecule encoding an Fc domain variant of the present invention; And b) it relates to a method for preparing a human antibody Fc domain variant with improved selective binding ability to FcγRIIIa, comprising the step of recovering the polypeptide expressed by the host cell.
일 측면에서, 본 발명은 a) 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산분자를 포함하는 벡터를 포함하는 숙주세포를 배양하는 단계; 및 b) 숙주세포로부터 발현된 항체를 정제하는 단계를 포함하는 FcγRⅢa에 대한 선택적 결합력이 향상된 당화 항체의 제조방법에 관한 것이다.In one aspect, the present invention comprises the steps of a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the antibody of the present invention or a fragment having immunological activity thereof; and b) purifying the antibody expressed from the host cell.
일 구현예에서, 항체의 정제는 여과, HPLC, 음이온 교환 또는 양이온 교환, 고속 액체 크로마토그래피(HPLC), 친화도 크로마토그래피, 또는 이들의 조합을 하는 것이 포함될 수 있으며, 바람직하게는 Protein A를 사용하는 친화 크로마토그래피를 이용할 수 있다.In one embodiment, purification of the antibody may include filtration, HPLC, anion exchange or cation exchange, high performance liquid chromatography (HPLC), affinity chromatography, or a combination thereof, preferably using Protein A. affinity chromatography can be used.
일 측면에서, 본 발명은 항체 치료제의 제조에 사용하기 위한 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편의 용도에 관한 것이다.In one aspect, the invention relates to the use of an antibody or immunologically active fragment thereof of the invention for use in the manufacture of an antibody therapeutic.
일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편의 암의 예방 또는 치료 용도에 관한 것이다.In one aspect, the present invention relates to the use of the antibody or immunologically active fragment thereof of the present invention for preventing or treating cancer.
일 측면에서, 본 발명은 본 발명의 항체 또는 이의 면역학적 활성을 가진 단편을 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법에 관한 것이다.In one aspect, the present invention relates to a method for treating cancer comprising administering to a subject suffering from cancer a pharmaceutically effective amount of an antibody or immunologically active fragment thereof of the present invention.
하기의 실시예를 통하여 본 발명을 보다 상세하게 설명한다. 그러나 하기 실시예는 본 발명의 내용을 구체화하기 위한 것일 뿐 이에 의해 본 발명이 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. However, the following examples are only for specifying the content of the present invention, and the present invention is not limited thereto.
실시예 1. Fc 돌연변이들의 단순 조합으로 구성된 트라스트주맙 Fc 변이체들 제작 및 분석Example 1. Construction and analysis of trastuzumab Fc variants composed of simple combinations of Fc mutations
1-1. 단순 조합 트라스트주맙 Fc 변이체들 제작1-1. Construction of Simple Combination Trastuzumab Fc Variants
기존에 공지된 트라스트주맙(trastuzumab) Fc 변이체들의 단순 조합에 의한 효과를 확인하기 위해 DEL 변이체 (S239D/I332E/A330L: 야생형 Fc에 비해 A/I ratio 9배 향상), LPLIL 변이체 (F243L/R292P/Y300L/V305I/P396L: FcγRⅢa-158V에 대한 결합력 10배 향상) 및 IQ 변이체 (P247I/A339Q: ADCC 유도능 6배 향상)들의 단순 조합으로 구성된 Fc 변이체들을 제작하였다 (XMa: S239D/F243L/R292P/Y300L/V305I/A330L/I332E/P396L, XMt: S239D/P247I/A330L/I332E/A339Q, MM: F243L/P247I/R292P/Y300L/V305I/A339Q/P396L 및 XMM: S239D/F243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/P396L) (표 1). 상기 Fc 변이체들을 모델 항체인 트라스트주맙의 중쇄 유전자에 클로닝하고, 경쇄 유전자와 함께 Expi293F 세포에 PEI(polyethylenimine) (Polyscience, 23966)로 co-트랜스펙션(transfection)함으로써 임시 발현을 유도하였다. co-트랜스펙션한 Expi293F 세포를 37℃, 125 rpm 및 8%　CO2 조건에서 7일간의 배양 후, 배양액을 회수하여 PBS로 평형을 맞춘 다음 Protein A 친화도 크로마토그래피를 통해 트라스트주맙 Fc 변이체들을 정제하였다 (도 1A). In order to confirm the effect of a simple combination of previously known trastuzumab Fc variants, DEL variants (S239D/I332E/A330L: A/I ratio improved 9 times compared to wild-type Fc), LPLIL variants (F243L/R292P/ Fc variants (XMa: S239D/F243L/R292P/XMa: S239D/F243L/R292P/ Y300L/V305I/A330L/I332E/P396L; XMt: S239D/P247I/A330L/I332E/A339Q; MM: F243L/P247I/R292P/Y300L/V305I/A339Q/P396L; V305I/A330L/I332E/A339Q/P396L) (Table 1). The Fc variants were cloned into the heavy chain gene of trastuzumab, a model antibody, and transient expression was induced by co-transfection with PEI (polyethylenimine) (Polyscience, 23966) into Expi293F cells together with the light chain gene. After culturing the co-transfected Expi293F cells at 37°C, 125 rpm, and 8% CO 2 conditions for 7 days, the culture medium was recovered, equilibrated with PBS, and trastuzumab Fc variants were detected through Protein A affinity chromatography. Purified (FIG. 1A).
1-2. FcγRⅡb-GST 생산1-2. Production of FcγRIIb-GST
면역 반응을 하향 조절(down-regulation)하여 ADCC 활성을 약화시키는 역할을 하는 Fc 수용체인 FcγRⅡb에 대한 단순 조합 트라스트주맙 Fc 변이체들의 결합력을 확인하기 위해 FcγRⅡb-GST를 생산하였다. 구체적으로, GST가 FcγRⅡb의 C-말단에 융합된 유전자가 동물세포 발현용 벡터에 클로닝되어 있는 플라스미드를 PEI를 이용해 Expi293F 세포에 트랜스펙션한 뒤 37℃, 125 rpm 및 8%　CO2 조건에서 7일간 임시발현을 진행하였다. 그런 다음, 세포 배양액을 회수한 뒤, PBS로 평형을 맞추고, anti-GST 친화도 크로마토그래피를 통해 고순도의 FcγRⅡb-GST을 확보하였다 (도 1B).FcγRIIb-GST was produced in order to confirm the binding ability of simple combination trastuzumab Fc variants to FcγRIIb, an Fc receptor that functions to attenuate ADCC activity by down-regulating the immune response. Specifically, Expi293F cells were transfected with a plasmid in which a gene in which GST was fused to the C-terminus of FcγRIIb was cloned into an animal cell expression vector using PEI, and then incubated at 37°C, 125 rpm, and 8% CO 2 conditions for 7 days. Temporary expression was performed daily. Then, after recovering the cell culture medium, equilibrating with PBS, high purity FcγRIIb-GST was secured through anti-GST affinity chromatography (FIG. 1B).
1-3. 단순 조합 트라스트주맙 Fc 변이체들의 FcγRⅡb 결합력 분석1-3. Analysis of FcγRIIb binding ability of simple combinatorial trastuzumab Fc variants
상기 실시예 1-1에서 정제하여 준비한 트라스트주맙 Fc 변이체들 (XMa, XMt, MM 및 XMM)의 FcγRⅡb에 대한 결합력을 확인하기 위해 ELISA 분석을 진행하였다. 구체적으로, 0.05 M Na2CO3 pH 9.6에 4 μg/ml로 희석한 FcγRⅡb-GST를 50 μl씩 flat bottom polystyrene high bind 96웰 마이크로플레이트 (Costar, 3590)에 4℃에서 16시간 동안 고정화한 후 100 μl의 4% 스킴밀크 (GenomicBase, SKI400)로 상온에서 2시간 동안 블로킹(blocking)하였다. 그 후, 0.05% PBST 180 μl로 4회씩 세척하고 1% 스킴밀크로 희석하여 준비한 상기 실시예 1-1의 당화 트라스트주맙 Fc 변이체들을 50 μl씩 각 웰에 분주하여 상온에서 1 시간 동안 반응시켰다. 이를 세척한 후 HRP-Protein L (GenScript, M00098) 50 μl씩 첨가하여 상온에서 1시간 동안 항체 반응을 진행하고 세척하였다. 그 후, 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028)을 50 μl씩 첨가하여 발색한 뒤 2 M H2SO4을 50 μl씩 처리하여 반응을 종료시키고 Epoch microplate spectrophotometer (BioTek)을 이용해 흡광도를 분석하였다. 그 결과, 공지된 돌연변이들의 단순 조합으로 구성된 트라스트주맙 Fc 변이체들이 각각 B-세포 림프종 및 Her2 양성 유방암 치료제로 US FDA 승인을 받은 ADCC 향상 당화 Fc 변이체들 (B 세포 림프종 치료제(Monjuvi, Tafasitamab)로 2020년 7월 31일 US FDA 승인받은 DE (S239E/I332E) 및 Her2 양성 유방암 치료제(Margenza, Margetuximab)로 2020년 12월 16일 US FDA 승인받은 VLPLL (L235V/F243L/R292P/Y300L/P396L))보다 현저히 높은 FcγRⅡb 결합력을 가지는 것으로 나타나 (도 2), ADCC 활성 유도에 부정적 영향을 끼칠 수 있음을 확인하였다.ELISA analysis was performed to confirm the binding ability of the trastuzumab Fc variants (XMa, XMt, MM and XMM) purified and prepared in Example 1-1 to FcγRIIb. Specifically, 50 μl of FcγRIIb-GST diluted to 4 μg/ml in 0.05 M Na 2 CO 3 pH 9.6 was immobilized in a flat bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4° C. for 16 hours, then Blocking was performed at room temperature for 2 hours with 100 μl of 4% skim milk (GenomicBase, SKI400). Thereafter, 50 μl of the glycated trastuzumab Fc variants of Example 1-1 prepared by washing with 180 μl of 0.05% PBST and diluting with 1% skim milk were dispensed into each well and reacted at room temperature for 1 hour. After washing, 50 μl of HRP-Protein L (GenScript, M00098) was added each time, followed by antibody reaction at room temperature for 1 hour, followed by washing. After that, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to develop color, and 2 MH 2 SO 4 was treated with 50 μl of 50 μl each to terminate the reaction. Epoch microplate spectrophotometer (BioTek) Absorbance was analyzed using As a result, trastuzumab Fc variants composed of simple combinations of known mutations are ADCC-enhancing glycosylation Fc variants (B-cell lymphoma treatment (Monjuvi, Tafasitamab) approved by the US FDA for the treatment of B-cell lymphoma and Her2-positive breast cancer, respectively, 2020 DE (S239E/I332E) approved by the US FDA on July 31, 2020 and VLPLL (L235V/F243L/R292P/Y300L/P396L) approved by the US FDA on December 16, 2020 as Her2-positive breast cancer treatment (Margenza, Margetuximab)) It was found to have a significantly high FcγRIIb binding affinity (FIG. 2), and it was confirmed that it could have a negative effect on ADCC activity induction.
실시예 2.Example 2. 당화 Fc 변이체 스크리닝을 위한 포유류 세포 디스플레이 시스템 구축Establishment of a mammalian cell display system for screening glycosylated Fc variants
ADCC 유도능을 극대화할 수 있는 Fc 돌연변이들의 최적의 조합을 찾고 새로운 당화 Fc 변이체를 발굴하기 위해 Fc 라이브러리를 제작하였다. 구체적으로, 당화 Fc를 포유류 세포 표면에 디스플레이하고 안정적으로 스크리닝하기 위해 스테이블 세포주(stable cell line) 제작에 활용되는 FLP-FRT 유전자 재조합(gene recombination) 시스템을 활용하였으며, 세포막에 디스플레이하기 위해 Fc의 C-말단에 PDGFR(Platelet-derived growth factor receptor) 트랜스멤브레인(transmembrane) 도메인을 융합하였다. Fc-PDGFR 유전자는 FRT 사이트(site)가 삽입되어 있는 pcDNA5/FRT 플라스미드(Invitrogen, V601020)에 클로닝하여 준비하였다. 해당 플라스미드를 유전자 재조합을 일으키는 효소인 FLP를 발현하는 pOG44플라스미드 (Invitrogen, V600520)와 함께 FRT 사이트가 삽입된 CHO 세포 (Invitrogen, R75807)에 co-트랜스펙션하여 유전자 재조합을 유도함으로써 CHO 세포의 염색체 DNA(chromosomal DNA)에 Fc-PDGFR DNA가 통합(Integration)되어 당화 Fc 안정적 발현 CHO 세포주가 제작되었다 (도 3). 이 때 트랜스펙션 후 유전자 통합이 일어난 CHO 세포들만 선별하기 위해 하이그로마이신(hygromycin)-B 저항성 유전자를 함께 통합되도록 하였으며, 500 μg/ml의 하이그로마이신-B (Invitrogen, 10687010)를 처리함으로써 당화 Fc 안정적 발현 CHO 세포주를 선별하여 당화 Fc 변이체 스크리닝을 위한 포유류 세포 디스플레이 시스템을 구축하였다.An Fc library was constructed to find the optimal combination of Fc mutations capable of maximizing the ADCC induction ability and discover new glycosylated Fc variants. Specifically, in order to display and stably screen glycosylated Fc on the surface of mammalian cells, the FLP-FRT gene recombination system used for producing stable cell lines was utilized. A platelet-derived growth factor receptor (PDGFR) transmembrane domain was fused to the C-terminus. The Fc-PDGFR gene was prepared by cloning into a pcDNA5/FRT plasmid (Invitrogen, V601020) into which an FRT site was inserted. This plasmid is co-transfected with a pOG44 plasmid (Invitrogen, V600520) expressing FLP, an enzyme that causes genetic recombination, into CHO cells (Invitrogen, R75807) into which an FRT site has been inserted to induce genetic recombination, thereby inducing chromosomes of CHO cells. Fc-PDGFR DNA was integrated into DNA (chromosomal DNA) to construct a CHO cell line stably expressing glycosylated Fc (FIG. 3). At this time, in order to select only CHO cells in which gene integration occurred after transfection, the hygromycin-B resistance gene was integrated together, and by treatment with 500 μg/ml hygromycin-B (Invitrogen, 10687010) A mammalian cell display system for screening glycosylated Fc variants was constructed by selecting CHO cell lines stably expressing glycosylated Fc.
실시예 3. 결합력 분석을 위한 FcγRs 제작Example 3. Fabrication of FcγRs for avidity analysis
3-1. 발현 및 정제3-1. Expression and purification
확립된 당화 Fc의 CHO 세포 디스플레이 시스템 및 Fc 라이브러리 안정적 발현 세포주를 FACS 스크리닝하고, 발굴할 Fc 변이체들의 FcγRs 결합력을 분석하기 위해 FcγRⅢa 및 FcγRⅡb를 제작 및 생산하였다. FcγRs 중 FcγRⅢa 및 FcγRⅡb는 Fc와의 결합력이 낮은 저친화성 수용체(low affinity receptor)로 알려져 있기 때문에 각 수용체의 C-말단에 스트렙타비딘(streptavidin)을 융합하여, Fc와의 가시적 결합 친화도를 향상시켜 효율적인 FACS 스크리닝이 가능한 사합체(tetrameric) FcγRⅢa-158V-스트렙타비딘-His, 사합체 FcγRⅢa-158F-스트렙타비딘-His 및 사합체 FcγRⅡb-스트렙타비딘-His를 제조하였다. 또한, 발굴할 당화 Fc 변이체들의 FcγRs 결합력을 ELISA로 분석하기 위해, FcγRⅢa-158V-GST 및 FcγRⅢa-158F-GST를 제작하였다. 각각의 단백질들은 동물세포 발현용 벡터에 클로닝하여 준비한 다음, PEI를 이용해 Expi293F 세포에 트랜스펙션한 뒤, 37℃, 125 rpm 및 8%　CO2 조건에서 7일간 배양하여 얻었다. 배양 후, 상등액을 회수하여 PBS로 평형을 맞추고 Ni-NTA(Anti-His) 또는 anti-GST 친화도 크로마토그래피로 정제하였다. 그 결과, 고순도의 사합체 FcγRⅢa-158V-스트렙타비딘, 사합체 FcγRⅢa-158F-스트렙타비딘, 사합체 FcγRⅡb-스트렙타비딘, FcγRⅢa-158V-GST 및 FcγRⅢa-158F-GST가 정제되었다 (도 4).Established glycosylated Fc CHO cell display system and Fc library stably expressing cell lines were subjected to FACS screening, and FcγRIIIa and FcγRIIb were constructed and produced to analyze the FcγRs binding ability of the Fc variants to be identified. Among the FcγRs, FcγRIIIa and FcγRIIb are known to be low affinity receptors with low binding affinity to Fc, so streptavidin is fused to the C-terminus of each receptor to improve the visible binding affinity with Fc, resulting in efficient FACS screening-capable tetrameric FcγRIIIa-158V-streptavidin-His, tetrameric FcγRIIIa-158F-streptavidin-His, and tetrameric FcγRIIb-streptavidin-His were prepared. In addition, in order to analyze the FcγRs binding ability of glycosylated Fc variants to be identified by ELISA, FcγRIIIa-158V-GST and FcγRIIIa-158F-GST were prepared. Each protein was prepared by cloning into an animal cell expression vector, transfected into Expi293F cells using PEI, and then cultured for 7 days at 37°C, 125 rpm and 8% CO 2 conditions. After culturing, the supernatant was collected, equilibrated with PBS, and purified by Ni-NTA (Anti-His) or anti-GST affinity chromatography. As a result, high-purity tetrameric FcγRIIIa-158V-streptavidin, tetrameric FcγRIIIa-158F-streptavidin, tetrameric FcγRIIb-streptavidin, FcγRIIIa-158V-GST, and FcγRIIIa-158F-GST were purified (FIG. 4 ).
3-2. 기능 검증3-2. functional verification
상기 실시예 3-1에서 제작 및 생산한 단백질들 중 FACS 스크리닝을 위해 준비한 사합체 FcγRⅢa-스트렙타비딘은 Alexa488(Invitrogen, A10235) 및 Alexa647(Invitrogen, A20173)의 형광 염료(dye)를 표지하여 무형광 사합체 FcγRⅡb-스트렙타비딘와의 경쟁적 결합을 통한 Fc 변이체들의 선별(sorting)이 가능하도록 준비하였으며, 디스플레이된 Fc 변이체들의 발현 여부 및 레벨을 확인하기 위해 FcγRs와 결합부위가 겹치지 않는 Protein A (Amicogen, 1070020)에 FITC (Invitrogen, F6434)를 컨쥬게이션(conjugation)하여 준비하였다. 형광 염료들 (alexa488, Alexa647 및 FITC)의 컨쥬게이션은 각각의 제조사에서 제공한 메뉴얼에 따라 수행하였다. 형광 표지된 사합체 FcγRⅢa-스트렙타비딘-Alexa488, 사합체 FcγRⅢa-스트렙타비딘-Alexa647 및 Protein A-FITC를 각각 CHO 세포에 디스플레이 된 야생형 Fc 및 FcγRs 결합력이 제거된 Fc 변이체 (Fc-T299L)에 결합 유도한 뒤 활성을 확인하였다. Among the proteins prepared and produced in Example 3-1, the tetrameric FcγRIIIa-streptavidin prepared for FACS screening was non-fluorescent by labeling the fluorescent dyes of Alexa488 (Invitrogen, A10235) and Alexa647 (Invitrogen, A20173). It was prepared to enable sorting of Fc variants through competitive binding with tetrameric FcγRIIb-streptavidin, and Protein A (Amicogen, 1070020) was prepared by conjugation of FITC (Invitrogen, F6434). Conjugation of fluorescent dyes (alexa488, Alexa647 and FITC) was performed according to the manual provided by each manufacturer. Fluorescence-labeled tetrameric FcγRIIIa-streptavidin-Alexa488, tetrameric FcγRIIIa-streptavidin-Alexa647, and Protein A-FITC were respectively applied to wild-type Fc and Fc variants (Fc-T299L) with no FcγRs-binding affinity displayed in CHO cells. After binding was induced, activity was confirmed.
그 결과, Protein A-FITC 결합에 의한 형광 신호를 통해 Protein A-FITC가 정상적인 활성을 가지며, CHO 세포에 디스플레이된 야생형(wild-type) Fc 및 Fc-T299L이 안정적으로 발현되어 서로 유사한 발현 수준을 나타내는 것을 확인하였다 (도 5). 또한, 사합체 FcγRⅢa-스트렙타비딘-Alexa488 및 사합체 FcγRⅢa-스트렙타비딘-Alexa647 역시 야생형 Fc에는 정상적으로 결합하고, FcγRs 결합력이 제거된 Fc 변이체인 Fc-T299L에는 결합하지 않는 것으로 나타나 (도 5), Alexa488/647 형광 표지 FcγRⅢa 역시 우수한 활성을 가지며, CHO 세포에 디스플레이 된 Fc들이 정상적으로 발현되고 각각의 기능을 수행하는 것을 성공적으로 검증하였다.As a result, Protein A-FITC has normal activity through the fluorescence signal by Protein A-FITC binding, and wild-type Fc and Fc-T299L displayed in CHO cells are stably expressed, showing similar expression levels to each other. It was confirmed that shown (FIG. 5). In addition, tetrameric FcγRIIIa-streptavidin-Alexa488 and tetrameric FcγRIIIa-streptavidin-Alexa647 also appeared to normally bind to wild-type Fc, but not to Fc-T299L, an Fc variant in which FcγRs binding ability was removed (FIG. 5) , Alexa488/647 fluorescently labeled FcγRIIIa also had excellent activity, and it was successfully verified that Fc displayed in CHO cells were normally expressed and performed their respective functions.
실시예 4. 포유류 세포 디스플레이를 이용한 FcγRⅢa 선택적 결합력 향상 당화 Fc 변이체 라이브러리 구축Example 4. Construction of FcγRIIIa selective binding affinity-enhancing glycosylated Fc variant library using mammalian cell display
확립된 CHO 세포 Fc 디스플레이 시스템을 통해 당화 Fc를 엔지니어링하고, FcγRⅢa 결합력이 향상된 당화 Fc 변이체를 선별하기 위해 라이브러리를 제작하였다. 라이브러리를 이용하여 DEL 변이체, LPLIL 변이체 및 IQ 변이체의 돌연변이들을 셔플링(shuffling)하여 FcγRⅢa 결합을 극대화할 수 있는 최적의 돌연변이 조합을 탐색하였다 (도 6). 라이브러리 유전자는 상기 실시예 2에서 확립한 Fc 안정적 발현 포유류 세포주 제조 방법과 동일한 방법으로 FLP 발현 플라스미드와 co-트랜스펙션한 후 하이그로마이신-B 배지를 이용한 선별 과정을 거쳐 당화 Fc 변이체 라이브러리 안정적 발현 CHO 세포주를 제작하였다.Glycosylated Fc was engineered through the established CHO cell Fc display system, and a library was constructed to select glycosylated Fc variants with improved FcγRIIIa binding ability. Mutations of DEL variants, LPLIL variants, and IQ variants were shuffled using the library to search for optimal mutation combinations capable of maximizing FcγRIIIa binding (FIG. 6). The library gene was co-transfected with the FLP expression plasmid in the same way as the Fc stable expression mammalian cell line production method established in Example 2, and then the glycosylated Fc variant library was stably expressed through a selection process using hygromycin-B medium. A CHO cell line was constructed.
실시예 5. 포유류 세포 디스플레이를 이용한 FcγRⅢa 선택적 결합력 향상 Fc 변이체 선별Example 5. Selection of Fc γRIIIa selective avidity-enhancing Fc variants using mammalian cell display
상기 실시예 2에서 확립한 CHO 세포 Fc 디스플레이 시스템 및 상기 실시예 4에서 확립한 Fc 라이브러리 안정적 발현 CHO 세포주를 이용하여, FcγRⅢa 결합력이 향상된 당화 Fc를 선별하기 위해, 라이브러리 안정적 발현 CHO 세포주에서 FcγRⅢa-Alexa647, Protein A-FITC 또는 FcγRⅢa-Alexa647과 무형광 FcγRⅡb, Protein A-FITC의 결합을 유도한 뒤, Protein A-FITC에 의한 발현 수준과 FcγRⅢa 결합을 동시에 모니터링하여 1R의 FACS 선별을 수행하였다. 이로써 우수한 FcγRⅢa 결합력 및 선택성을 보일 것으로 예상되는 세포 집단(population)을 선별하였고, 선별된 당화 Fc 변이체 발현 CHO 세포들을 유전체 DNA(genomic DNA) 프렙(prep.)을 통해 회수한 뒤, 염기서열을 확인하여 최종적으로 조작된(engineered) 당화 Fc 변이체들을 선별하였다 (도 7).In order to select a glycosylated Fc with improved FcγRIIIa binding ability using the CHO cell Fc display system established in Example 2 and the CHO cell line stably expressing the Fc library established in Example 4, FcγRIIIa-Alexa647 in the CHO cell line stably expressing FcγRIIIa. , After inducing the binding of Protein A-FITC or FcγRIIIa-Alexa647 to non-fluorescent FcγRIIb and Protein A-FITC, the expression level by Protein A-FITC and FcγRIIIa binding were simultaneously monitored to perform FACS screening of 1R. As a result, a cell population expected to show excellent FcγRIIIa binding affinity and selectivity was selected, and the selected glycosylated Fc variant-expressing CHO cells were recovered through genomic DNA prep. and finally engineered glycosylated Fc variants were selected (FIG. 7).
실시예 6. 트라스트주맙 Fc 변이체들의 FcγRⅢa 결합력 분석Example 6. FcγRIIIa binding force analysis of trastuzumab Fc variants
당화 Fc 변이체들을 발현 및 정제하기 위해, 모델 항체인 트라스트주맙 중쇄 유전자에 클로닝하여 발현 백터를 제조하였다. 구체적으로, Freestyle293 발현 배양액 (Gibco, 12338-018) 3 ml에 변이체들의 중쇄유전자와 경쇄유전자를 1:1의 비율로 먼저 섞고, PEI(polyethylenimine) (Polyscience, 23966) 및 변이체 유전자를 4:1의 비율로 섞어 상온에서 20분간 인큐베이션한 뒤, 2x106 cells/ml의 밀도로 30 ml 계대배양한 Expi293F 세포에 섞어 shaking CO2 인큐베이터에서 37℃, 125 rpm 및 8%　CO2의 조건으로 7일간 배양한 후 원심분리하여 상등액만 취하였다. 상등액은 25×PBS로 평형을 맞춘 후 0.2 μm 시린지 필터로 여과하였다. Her2 단백질의 세포외 도메인(extracellular domain)을 4℃에서 하룻밤 동안 코팅해 둔 high binding 96웰 플레이트 (Costar, 3590)에 상기 여과한 트라스트주맙 Fc 변이체 발현 배양액을 50 μl씩 넣어 1시간 동안 고정화하였다. 그 후, 100 μl의 4% 스킴밀크 (GenomicBase, SKI400)로 상온에서 2시간 동안 블로킹하였다. 0.05% PBST 180 μl로 4 회씩 세척한 뒤 1% 스킴밀크에 20 μg/ml 및 0.02 μg/ml의 FcγRⅢa-GST를 각각 50 μl씩 각 웰에 넣어 상온에서 1시간 동안 반응시켰다. 세척 후 고트 항-GST-HRP (GE Healthcare, RPN1236V) 50 μl을 각각 처리하여 상온에서 1시간 동안 항체 반응을 진행하고 세척하였다. 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028)을 50 μl씩 첨가해 발색한 뒤 2 M H2SO4을 50 μl씩 넣어주어 반응을 종료시킨 다음, Epoch microplate spectrophotometer (BioTek)을 이용해 흡광도를 분석하였다. 그 결과, 본 발명에서 개발한 신규한 Fc 변이체들 중 PS301, PS303, PS305, PS205, PS207, PS101, PS102, PS105, PS106 및 PS107 (표 1)이 Her2 양성 유방암 치료제로 승인 받은 기존에 공지된 마르게툭시맙(margetuximab)인 VLPLL 변이체와 유사하거나 현저히 높은 FcγRⅢa 결합력을 가지는 것을 확인하였다 (도 8). To express and purify the glycosylated Fc variants, an expression vector was prepared by cloning into the model antibody trastuzumab heavy chain gene. Specifically, in 3 ml of Freestyle293 expression culture medium (Gibco, 12338-018), the heavy chain genes and light chain genes of the variants were first mixed at a ratio of 1:1, and PEI (polyethylenimine) (Polyscience, 23966) and the variant genes were mixed at a ratio of 4:1. After incubation at room temperature for 20 minutes at room temperature, mixed with Expi293F cells subcultured in 30 ml at a density of 2x10 6 cells/ml and cultured for 7 days in a shaking CO 2 incubator at 37°C, 125 rpm and 8% CO 2 conditions. After centrifugation, only the supernatant was taken. The supernatant was equilibrated with 25 × PBS and filtered through a 0.2 μm syringe filter. 50 μl of the filtered trastuzumab Fc mutant expression culture medium was added to a high binding 96-well plate (Costar, 3590) coated with the extracellular domain of Her2 protein overnight at 4° C., and then fixed for 1 hour. Then, blocking was performed at room temperature for 2 hours with 100 μl of 4% skim milk (GenomicBase, SKI400). After washing four times with 180 μl of 0.05% PBST, 50 μl of 20 μg/ml and 0.02 μg/ml FcγRIIIa-GST in 1% skim milk were added to each well and reacted at room temperature for 1 hour. After washing, 50 μl of goat anti-GST-HRP (GE Healthcare, RPN1236V) was treated, followed by antibody reaction at room temperature for 1 hour, followed by washing. After adding 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) to develop color, add 50 μl of 2 MH 2 SO 4 to terminate the reaction, and then use an Epoch microplate spectrophotometer (BioTek). Absorbance was analyzed. As a result, among the novel Fc variants developed in the present invention, PS301, PS303, PS305, PS205, PS207, PS101, PS102, PS105, PS106 and PS107 (Table 1) are previously known Marges approved as therapeutic agents for Her2-positive breast cancer. It was confirmed that it had a similar or significantly higher FcγRIIIa binding affinity to that of the VLPLL variant of tuximab (margetuximab) (FIG. 8).
## 돌연변이mutation 서열번호 sequence number
PS301PS301 K222N/F243L/P247I/R292P/Y300L/V305I/A330L/I332EK222N/F243L/P247I/R292P/Y300L/V305I/A330L/I332E 22 및 3722 and 37
PS303PS303 S239D/P247I/Y300L/V305I/A330L/I332E/A339Q/N361D/P387QS239D/P247I/Y300L/V305I/A330L/I332E/A339Q/N361D/P387Q 23 및 3823 and 38
PS305PS305 S239D/R292P/Y300L/A330L/A339Q/P387Q/F405LS239D/R292P/Y300L/A330L/A339Q/P387Q/F405L 24 및 3924 and 39
PS205PS205 F243L/P247I/R292P/Y300L/A330L/I332E/P396LF243L/P247I/R292P/Y300L/A330L/I332E/P396L 25 및 4025 and 40
PS207PS207 F243L/P247I/R292P/A330L/I332E/A339Q/P396L/S415GF243L/P247I/R292P/A330L/I332E/A339Q/P396L/S415G 26 및 4126 and 41
PS101PS101 F243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/P387QF243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/P387Q 27 및 4227 and 42
PS102PS102 H224R/F243L/P247I/R292P/Y300L/V303I/V305I/A330L/I332E/A339Q/P387QH224R/F243L/P247I/R292P/Y300L/V303I/V305I/A330L/I332E/A339Q/P387Q 28 및 4328 and 43
PS105PS105 R292P/Y300L/V305I/A330L/I332E/A339Q/P387QR292P/Y300L/V305I/A330L/I332E/A339Q/P387Q 29 및 4429 and 44
PS106PS106 F243L/P247I/M252V/R292P/Y300L/V305I/A330L/I332E/A339Q/D356G/P387QF243L/P247I/M252V/R292P/Y300L/V305I/A330L/I332E/A339Q/D356G/P387Q 30 및 4530 and 45
PS107PS107 F243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/D356G/P387QF243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/D356G/P387Q 31 및 4631 and 46
XMaXMa S239D/F243L/R292P/Y300L/V305I/A330L/I332E/P396LS239D/F243L/R292P/Y300L/V305I/A330L/I332E/P396L 32 및 4732 and 47
XMtXMt S239D/P247I/A330L/I332E/A339QS239D/P247I/A330L/I332E/A339Q 33 및 4833 and 48
MMMM F243L/P247I/R292P/Y300L/V305I/A339Q/P396LF243L/P247I/R292P/Y300L/V305I/A339Q/P396L 34 및 4934 and 49
XMMXMM S239D/F243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/P396LS239D/F243L/P247I/R292P/Y300L/V305I/A330L/I332E/A339Q/P396L 35 및 5035 and 50
실시예 7. FcγRⅢa 결합력 향상 당화 트라스트주맙 Fc 변이체의 정제Example 7. Purification of FcγRIIIa binding affinity-enhancing glycosylated trastuzumab Fc variants
상기 실시예 6에서 배양액 ELISA를 통해 높은 FcγRⅢa 결합력을 가지는 것으로 확인된 트라스트주맙 Fc 변이체들을 정제하기 위해, 변이체들에 Protein A 레진(resin)을 첨가하여 4℃에서 16시간 동안 교반한 후 스핀다운(spin down)하여 레진을 회수한 후 2 ml PBS로 세척하고 300 μl의 100 mM 글라이신 pH 2.7 버퍼로 용출(elution)한 뒤, 100 μl의 1 M Tris-HCl pH 8.0을 이용하여 중화하였다. 버퍼를 바꾸기 위하여 Amicon Ultra-4 centrifugal filter units 30K (Merck Millipore, UFC503096)을 사용하였다. 그 결과, 높은 순도의 당화 항체 트라스트주맙 Fc 변이체들이 정제되었음을 확인하였다 (도 9).In order to purify the trastuzumab Fc variants confirmed to have high FcγRIIIa binding affinity through culture medium ELISA in Example 6, Protein A resin was added to the variants, stirred at 4 ° C. for 16 hours, and then spun down ( The resin was recovered by spin down), washed with 2 ml PBS, eluted with 300 μl of 100 mM glycine pH 2.7 buffer, and neutralized using 100 μl of 1 M Tris-HCl pH 8.0. Amicon Ultra-4 centrifugal filter units 30K (Merck Millipore, UFC503096) were used to change the buffer. As a result, it was confirmed that highly purified glycosylated antibody trastuzumab Fc variants were purified (FIG. 9).
실시예 8. 당화 트라스트주맙 Fc 변이체들의 FcγRⅢa 및 FcγRⅡb 결합력 ELISA 분석Example 8. ELISA analysis of FcγRIIIa and FcγRIIb binding affinity of glycosylated trastuzumab Fc variants
상기 실시예 7에서 발현 및 정제한 당화 트라스트주맙 Fc 변이체들의 FcγRⅢa 및 FcγRⅡb에 대한 결합력을 확인하기 위해 ELISA 분석을 진행하였다. 구체적으로, 0.05 M Na2CO3 (pH 9.6)에 4 μg/ml로 희석한 FcγRs-GST (FcγRⅢa-158V-GST, FcγRⅢa-158F-GST 및 FcγRⅡb-GST)를 각각 50 μl씩 flat bottom polystyrene high bind 96웰 플레이트 (Costar, 3590)에 분주하고 4℃에서 16시간 동안 고정화한 후, 100 μl의 4% 스킴밀크 (GenomicBase, SKI400)로 상온에서 2시간 동안 블로킹하였다. 그 후, 0.05% PBST 180 μl로 4 회씩 세척한 뒤 1% 스킴밀크로 연속 희석된 각각의 당화 트라스트주맙 Fc 변이체들을 50 μl 각 웰에 분주하여 상온에서 1시간 동안 반응시켰다. 이를 세척한 후 HRP-Protein L (GenScript, M00098) 50 μl씩을 처리하여 상온에서 1시간 동안 항체 반응을 진행하고 세척하였다. 그 뒤, 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) 50 μl씩 첨가해 발색한 뒤 2 M H2SO4을 50 μl씩 첨가하여 반응을 종료시킨 다음 Epoch microplate spectrophotometer (BioTek)을 이용해 흡광도를 분석하였다. ELISA analysis was performed to confirm the binding ability of the glycosylated trastuzumab Fc variants expressed and purified in Example 7 to FcγRIIIa and FcγRIIb. Specifically, 50 μl each of FcγRs-GST (FcγRIIIa-158V-GST, FcγRIIIa-158F-GST, and FcγRIIb-GST) diluted to 4 μg/ml in 0.05 M Na 2 CO 3 (pH 9.6) was added to flat bottom polystyrene high After dispensing to a bind 96-well plate (Costar, 3590), immobilizing at 4° C. for 16 hours, blocking with 100 μl of 4% skim milk (GenomicBase, SKI400) at room temperature for 2 hours. Then, after washing four times with 180 μl of 0.05% PBST, 50 μl of each of the glycated Trastuzumab Fc variants serially diluted with 1% skim milk was dispensed into each well and reacted at room temperature for 1 hour. After washing, 50 μl of HRP-Protein L (GenScript, M00098) was treated, followed by antibody reaction at room temperature for 1 hour, followed by washing. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to develop color, and 2 MH 2 SO 4 was added in 50 μl of each to terminate the reaction, followed by Epoch microplate spectrophotometer (BioTek). Absorbance was analyzed using
그 결과, 본 발명의 당화 트라스트주맙 Fc 변이체들이 각각 B-세포 림프종 및 Her2 양성 유방암 치료제로 US FDA 승인을 받은 당화 Fc 변이체들 (DE 및 VLPLL) 및 기존에 공지된 돌연변이들의 단순 조합으로 구성된 Fc 변이체들 (XMa, XMt, MM 및 XMM)보다 현저히 높은 FcγRⅢa/FcγRⅡb 선택적 결합력을 가지는 것으로 나타났다 (도 10 및 11). 특히, 공지된 기능 향상 Fc 변이체 돌연변이들의 단순 조합이 아닌 당화 항체 Fc 변이체 거대 라이브러리 탐색으로 선별적으로 일부 돌연변이들이 추가되고 삭제되어 최적화된 돌연변이들이 포함된 Fc 변이체들이 FcγRⅢa/FcγRⅡb 선택적 결합력 유도에 매우 효과적인 것으로 나타났으며 (도 12), 이들의 결합 특성에 따라 하기 표 2와 같이 3 그룹으로 구분하였다. As a result, the glycated trastuzumab Fc variants of the present invention are Fc variants composed of simple combinations of glycated Fc variants (DE and VLPLL) approved by the US FDA for the treatment of B-cell lymphoma and Her2-positive breast cancer, respectively, and previously known mutations. It was found to have significantly higher FcγRIIIa / FcγRIIb selective binding ability than those (XMa, XMt, MM and XMM) (Figs. 10 and 11). In particular, Fc variants containing optimized mutations by selectively adding and deleting some mutations by searching for a large library of glycosylated antibody Fc variants, rather than simple combinations of known function-enhancing Fc variant mutations, are very effective in inducing FcγRIIIa/FcγRIIb selective binding ability. It was found (FIG. 12), and according to their binding characteristics, they were classified into 3 groups as shown in Table 2 below.
Figure PCTKR2022013481-appb-img-000001
Figure PCTKR2022013481-appb-img-000001

Claims (25)

  1. 야생형(Wild type) 인간 항체 Fc 도메인에서, 카밧 넘버링 시스템(Kabat numbering system)에 따라 넘버링된 222, 239, 243, 247, 252, 292, 300, 305, 330, 332, 339, 356, 361, 387, 396, 405 및 415 위치의 아미노산으로 이루어진 군으로부터 선택되는 어느 하나 이상의 위치의 아미노산이 야생형의 아미노산과 다른 서열로 치환된, 인간 항체 Fc 도메인 변이체.In the wild type human antibody Fc domain, 222, 239, 243, 247, 252, 292, 300, 305, 330, 332, 339, 356, 361, 387 numbered according to the Kabat numbering system. , 396, 405 and 415 position amino acids selected from the group consisting of amino acids at any one or more positions are substituted with a sequence different from the amino acid of the wild type, human antibody Fc domain variant.
  2. 제 1항에 있어서, K222N, S239D, F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G, N361D, P387Q, P396L, F405L 및 S415G로 이루어진 군으로부터 선택된 어느 하나 이상의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The method of claim 1, wherein any one or more amino acid substitutions selected from the group consisting of K222N, S239D, F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G, N361D, P387Q, P396L, F405L and S415G Including, human antibody Fc domain variants.
  3. 제 1항에 있어서, F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G 및 P387Q의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 , comprising the amino acid substitutions of F243L, P247I, M252V, R292P, Y300L, V305I, A330L, I332E, A339Q, D356G and P387Q.
  4. 제 1항에 있어서, F243L, P247I, R292P, Y300L, A330L, I332E 및 P396L의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 , comprising amino acid substitutions of F243L, P247I, R292P, Y300L, A330L, I332E and P396L.
  5. 제 1항에 있어서, K222N, F243L, P247I, R292P, Y300L, V305I, A330L 및 I332E의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 , comprising the amino acid substitutions of K222N, F243L, P247I, R292P, Y300L, V305I, A330L and I332E.
  6. 제 1항에 있어서, S239D, P247I, Y300L, V305I, A330L, I332E, A339Q, N361D 및 P387Q의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 comprising the amino acid substitutions of S239D, P247I, Y300L, V305I, A330L, I332E, A339Q, N361D and P387Q.
  7. 제 1항에 있어서, S239D, R292P, Y300L, A330L, A339Q, P387Q 및 F405L의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 comprising the amino acid substitutions of S239D, R292P, Y300L, A330L, A339Q, P387Q and F405L.
  8. 제 1항에 있어서, R292P, Y300L, V305I, A330L, I332E, A339Q 및 P387Q의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 comprising the amino acid substitutions of R292P, Y300L, V305I, A330L, I332E, A339Q and P387Q.
  9. 제 1항에 있어서, F243L, P247I, R292P, A330L, I332E, A339Q, P396L 및 S415G의 아미노산 치환을 포함하는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant of claim 1 , comprising the amino acid substitutions of F243L, P247I, R292P, A330L, I332E, A339Q, P396L and S415G.
  10. 제 1항에 있어서, 야생형 Fc 도메인과 비교하여 FcγRⅢa와의 결합력이 향상된, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 1, wherein binding ability to FcγRIIIa is improved compared to the wild-type Fc domain.
  11. 제 1항에 있어서, 야생형 Fc 도메인과 비교하여 FcγRⅡb와의 결합력이 감소된, 인간 항체 Fc 도메인 변이체. According to claim 1, compared to the wild-type Fc domain FcγRIIb binding force is reduced, human antibody Fc domain variants.
  12. 제 1항에 있어서, 향상된 ADCC(antibody-dependent cell-mediated cytotoxicity) 유도능을 가지는, 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 1, having improved ADCC (antibody-dependent cell-mediated cytotoxicity) induction ability.
  13. 제 1항의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편.An antibody comprising the Fc domain variant of claim 1 or a fragment having immunological activity thereof.
  14. 제 13항에 있어서, 상기 항체는 당화 항체인, 항체 또는 이의 면역학적 활성을 가진 단편.The antibody or immunologically active fragment thereof according to claim 13, wherein the antibody is a glycosylated antibody.
  15. 제 13항에 있어서, 서열번호 1 내지 9의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 2(CH2) 및 서열번호 14 내지 20의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 3(CH3)를 포함하는, 항체 또는 이의 면역학적 활성을 가진 단편.The method of claim 13, wherein the heavy chain constant region domain 2 ( CH 2) comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 9 and SEQ ID NOs: 14 to 20 selected from the group consisting of amino acid sequences An antibody or immunologically active fragment thereof, comprising a heavy chain constant region domain 3 (C H 3 ) comprising any one.
  16. 제 1항의 Fc 도메인 변이체, 또는 제 13항의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산분자.A nucleic acid molecule encoding the Fc domain variant of claim 1, or the antibody of claim 13 or a fragment having immunological activity thereof.
  17. 제 16항의 핵산분자를 포함하는 벡터.A vector comprising the nucleic acid molecule of claim 16.
  18. 제 17항의 벡터를 포함하는 숙주세포.A host cell comprising the vector of claim 17.
  19. 제 1항의 Fc 도메인 변이체, 또는 제 13항의 항체 또는 이의 면역학적 활성을 가진 단편을 유효성분으로 포함하는 암의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating cancer comprising the Fc domain variant of claim 1, or the antibody of claim 13 or a fragment having immunological activity thereof as an active ingredient.
  20. 제 19항에 있어서, 암은 뇌종양, 흑색종, 골수종, 비소세포성폐암, 구강암, 간암, 위암, 결장암, 유방암, 폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 자궁경부암, 난소암, 대장암, 소장암, 직장암, 나팔관암종, 항문부근암, 자궁내막암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구 림프종, 신장 또는 수뇨관암, 신장세포 암종, 신장골반암종, 중추신경계 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 및 뇌하수체 선종으로 이루어진 군으로부터 선택되는 어느 하나인, 암의 예방 또는 치료용 약학적 조성물.The method of claim 19, wherein the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, colon cancer , small intestine cancer, rectal cancer, fallopian tube carcinoma, perianal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, kidney or ureteric cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma And any one selected from the group consisting of pituitary adenoma, a pharmaceutical composition for the prevention or treatment of cancer.
  21. a) 제 1항의 Fc 도메인 변이체를 코딩하는 핵산분자를 포함하는 벡터를 포함하는 숙주세포를 배양하는 단계; 및a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the Fc domain variant of claim 1; and
    b) 숙주세포에 의해 발현된 폴리펩타이드를 회수하는 단계를 포함하는, FcγRⅢa에 대한 선택적 결합력이 향상된, 인간 항체 Fc 도메인 변이체의 제조방법.b) a method for producing a human antibody Fc domain variant with improved selective binding ability to FcγRIIIa, comprising the step of recovering a polypeptide expressed by a host cell.
  22. a) 제 13항의 항체 또는 이의 면역학적 활성을 가진 단편을 코딩하는 핵산분자를 포함하는 벡터를 포함하는 숙주세포를 배양하는 단계; 및a) culturing a host cell containing a vector containing a nucleic acid molecule encoding the antibody of claim 13 or a fragment having immunological activity thereof; and
    b) 숙주세포로부터 발현된 항체를 정제하는 단계를 포함하는 FcγRⅢa에 대한 선택적 결합력이 향상된 당화 항체의 제조방법.b) a method for preparing a glycosylated antibody with improved selective binding ability to FcγRIIIa, comprising the step of purifying the antibody expressed from a host cell.
  23. 항체 치료제의 제조에 사용하기 위한, 제 1항의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편의 용도.Use of an antibody comprising the Fc domain variant of claim 1 or an immunologically active fragment thereof for use in the manufacture of an antibody therapeutic.
  24. 제 1항의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편의 암의 예방 또는 치료 용도.Use of an antibody comprising the Fc domain variant of claim 1 or a fragment having immunological activity for the prevention or treatment of cancer.
  25. 제 1항의 Fc 도메인 변이체를 포함하는 항체 또는 이의 면역학적 활성을 가진 단편을 약학적으로 유효한 양으로 암에 걸린 개체에 투여하는 단계를 포함하는 암 치료 방법.A cancer treatment method comprising administering an antibody comprising the Fc domain variant of claim 1 or a fragment having immunological activity thereof to a subject suffering from cancer in a pharmaceutically effective amount.
PCT/KR2022/013481 2021-09-17 2022-09-07 GLYCATED FC VARIANTS WITH IMPROVED BINDING AFFINITY FOR FCγRIIIA WO2023043124A1 (en)

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KR20010102976A (en) * 1999-01-15 2001-11-17 제넨테크, 인크. Polypeptide Variants with Altered Effector Function
US7317091B2 (en) * 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US20100322924A1 (en) * 2006-05-26 2010-12-23 Macrogenics, Inc. Humanized Fc gamma RIIB-Specific Antibodies And Methods Of Use Thereof
KR20180113717A (en) * 2017-04-07 2018-10-17 국민대학교산학협력단 Antibody Fc variants for Prolonged Serum Half-Life
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US7317091B2 (en) * 2002-03-01 2008-01-08 Xencor, Inc. Optimized Fc variants
US20100322924A1 (en) * 2006-05-26 2010-12-23 Macrogenics, Inc. Humanized Fc gamma RIIB-Specific Antibodies And Methods Of Use Thereof
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