WO2023043127A1 - Fc variant having increased affinity for binding to fc gamma receptors - Google Patents

Fc variant having increased affinity for binding to fc gamma receptors Download PDF

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WO2023043127A1
WO2023043127A1 PCT/KR2022/013492 KR2022013492W WO2023043127A1 WO 2023043127 A1 WO2023043127 A1 WO 2023043127A1 KR 2022013492 W KR2022013492 W KR 2022013492W WO 2023043127 A1 WO2023043127 A1 WO 2023043127A1
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cancer
antibody
present
amino acid
domain variant
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PCT/KR2022/013492
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French (fr)
Korean (ko)
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WO2023043127A9 (en
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정상택
조미경
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고려대학교 산학협력단
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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

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  • the present invention relates to Fc variants with increased binding ability to Fc gamma receptors, and to aglycosylated Fc variants with improved selective binding ability to Fc ⁇ RIIIa among Fc gamma receptors.
  • 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. Clearance, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, control of placental migration and immunoglobulin production trigger a biological response (Deo, Y.M.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the Fc domain plays a critical role in the recruitment of immune cells, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP). It depends on interactions with Fc receptors present on the surface. 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.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody dependent cellular phagocytosis
  • 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 object of the present invention is to provide an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor (Fc ⁇ R).
  • Another object of the present invention is to provide an aglycosylated antibody specific for a specific Fc gamma receptor or a fragment having immunological activity thereof.
  • 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 an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor.
  • an object of the present invention is to provide a use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity thereof for use in the manufacture of an antibody therapeutic agent.
  • an object of the present invention is to provide a use of an Fc gamma receptor-specific aglycosylated antibody or a fragment having immunological activity for preventing or treating cancer.
  • an object of the present invention is to provide a cancer treatment method comprising the step of administering a pharmaceutically effective amount of an Fc gamma receptor-specific aglycosylated antibody or immunologically active fragment thereof to a subject suffering from cancer.
  • the present invention provides an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor.
  • the present invention provides an aglycosylated antibody specific for a specific Fc gamma receptor, including an Fc domain variant, or a fragment having immunological activity thereof.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an Fc domain variant, or an antibody containing the same or a fragment having immunological activity thereof as an active ingredient.
  • the present invention provides a method for preparing an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor.
  • the present invention provides a method for producing an aglycosylated antibody specific for a specific Fc gamma receptor.
  • the present invention provides the use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity thereof for use in the manufacture of an antibody therapeutic.
  • the present invention provides a use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity for preventing or treating cancer.
  • the present invention provides a cancer treatment method comprising the step of administering a pharmaceutically effective amount of an Fc gamma receptor-specific aglycosylated antibody or immunologically active fragment thereof to a subject suffering from cancer.
  • human antibody Fc domain variants of the present invention have improved selective binding to Fc ⁇ RIIIa among Fc gamma receptors and increased A/I ratio, they can be usefully used as antibody drugs with improved ADCC effect, and since they are aglycosylated variants, they can be used as protein therapeutics. There is no problem of glycan heterogeneity, it is easy to mass-produce cheaply even in bacteria, and there is an effect that enables the manufacture of biological drugs without problems according to cell lines, culture processes and purification processes.
  • Figure 1 shows the results of FACS analysis of the binding ability to Fc ⁇ RIIIa (A) and the selective binding ability to Fc ⁇ RIIIa (B) of aglycosylated Fc variants displayed on the E. coli inner membrane.
  • Figure 2 is based on the aFc-22 variant (P247L / V264E / T299A / A330I) T225I, T366A and K334E mutations on Fc ⁇ RIIIa binding ability (A) and Fc ⁇ RIIIa selective binding ability (B) analyzed by FACS This is the result.
  • FIG. 3 is a diagram illustrating expression and purification of trastuzumab Fc variants including aglycosylated Fc variants aFc44, aFc44-ITKE and aFc44-ATKE in Expi293F cells and then confirmed by SDS-PAGE.
  • Figure 4 shows the results of measuring the binding constants of trastuzumab Fc variants including aglycosylated Fc variants.
  • Figure 5 shows the results of analyzing the binding ability to Fc ⁇ Rs of trastuzumab Fc variants including aglycosylated Fc variants by ELISA.
  • FIG. 6 is a result of analyzing the selective binding tendency for Fc ⁇ RIIIa and Fc ⁇ RIIb of trastuzumab Fc variants including wild-type glycosylated trastuzumab and aglycosylated Fc variants.
  • the present invention provides from the group consisting of amino acids at positions 225, 247, 264, 299, 330, 334 and 366 numbered according to the Kabat numbering system in the wild type human antibody Fc domain. It relates to a human antibody Fc domain variant with increased binding ability to an Fc gamma receptor (Fc ⁇ R) in which amino acids at any one or more selected positions are substituted with a sequence different from that of the wild-type amino acid.
  • Fc ⁇ R Fc gamma receptor
  • the Fc domain variant of the present invention may contain any one or more amino acid substitutions selected from the group consisting of P247L, V264E, T299A and A330I.
  • the Fc domain variant of the present invention may further comprise an amino acid substitution of T225I, K334E or T366A.
  • the Fc domain variant of the present invention may be aFc44 comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I and T366A.
  • the Fc domain variant of the present invention may be aFc44-ITKE comprising amino acid substitutions of P247L, V264E, T299A, A330I, K334E and T366A.
  • the Fc domain variant of the present invention may be Fc44-ATKE comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I and K334E.
  • an Fc domain variant of the present invention comprises aFc22 comprising the amino acid substitutions of P247L, V264E, T299A and A330I; aFc24 comprising amino acid substitutions of P247L, V264E, T299A, A330I and K334E; aFc44-IT comprising amino acid substitutions of P247L, V264E, T299A, A330I and T366A; aFc44-AT comprising amino acid substitutions of T225I, P247L, V264E, T299A and A330I; or aFc44-KE comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I, K334E and T366A.
  • the Fc domain variant of the present invention may be an Fc domain variant with improved selective binding ability to the Fc gamma receptor Fc ⁇ RIIIa.
  • 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 Fc region of the wild-type human antibody may include the amino acid sequence of SEQ ID NO: 6 and may be encoded by the nucleic acid molecule of SEQ ID NO: 16.
  • 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: 7 to 15.
  • 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.
  • 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.
  • the Fc variant in the present invention comprises a modification of one or more amino acid residues selected from the group consisting of T225I, P247L, V264E, T299A, A330I, K334E and T366A (the numbering is according to the EU index as described in Kabat).
  • 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 aglycosylated antibody specific for an Fc gamma receptor, including an Fc variant of the present invention, or a fragment having immunological activity thereof.
  • the antibody of the present invention comprises a 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 3 and the amino acid sequence of SEQ ID NOs: 4 or 5 and a heavy chain constant region domain 3 (C H 3).
  • CH 2 heavy chain constant region domain 2
  • C H 3 heavy chain constant region domain 3
  • 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, have high Fc ⁇ RIIIa binding selectivity and have a high A/I ratio, thus antibody-mediated It may increase antibody dependent cellular cytotoxicity (ADCC).
  • 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 17 to 25.
  • 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 regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.
  • the term "host cell” includes eukaryotes and prokaryotes, and refers to any transformable organism capable of replicating the vector or expressing a gene encoded by the vector.
  • the host cell may be transfected or transformed by the vector, which means a process in which an exogenous nucleic acid molecule is delivered or introduced into the host cell.
  • 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) a method for producing a human antibody Fc domain variant having increased binding ability to an Fc gamma receptor, comprising recovering a polypeptide expressed by a 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 present invention relates to the use of an aglycosylated antibody specific for an Fc gamma receptor of the present invention or an immunologically active fragment thereof for use in the manufacture of an antibody therapeutic.
  • the present invention relates to the use of the Fc gamma receptor-specific aglycosylated antibody of the present invention or a fragment having immunological activity for the prevention or treatment of 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 aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity of the present invention. will be.
  • Fc ⁇ RIIIa selective binding aglycosylated Fc variants were amplified (enrichment), and fluorescence intensity (i.e., Fc ⁇ RIIIa avidity) due to Fc ⁇ RIIIa-Alexa488 binding of individual clones aglycosylated Fc variants displayed on the E. coli inner membrane ( Figure 1A) and fluorescence intensity by Fc ⁇ RIIIa-Alexa488 binding in a state masked by non-fluorescent Fc ⁇ RIIb (ie, Fc ⁇ RIIIa selective binding intensity) (FIG.
  • CO 2 was cultured for 7 days under conditions of 37 °C, 125 rpm and 8% CO 2 in a shaking incubator, and only the supernatant was taken by centrifugation.
  • the supernatant was equilibrated with 25x PBS and filtered through a 0.2 ⁇ m filter (Corning, 430513) using a bottle top filter.
  • Add 500 ⁇ l of Protein A resin to the filtered culture medium stir at 4 ° C for 16 hours, recover the resin through the column, wash with 5 ml PBS, elute with 3 ml of 100 mM glycine buffer at pH 2.7, and Neutralized using Tris-HCl pH 8.0.
  • the biosensor was quenched with 1 M ethanolamine for 5 minutes, followed by baseline, Fc ⁇ RIIIa-158V-His association and dissociation for 60 seconds each to increase binding force. Confirmed.
  • Fc ⁇ RIIIa-158V-His was diluted by 1/2 from 40 ⁇ M and the binding ability was analyzed by concentration, and the regeneration of the biosensor between each measurement was performed in 10 mM glycine (pH 1.5) buffer and manufacturer. A total of 5 cycles were performed by alternately reacting the provided kinetics buffer for 5 seconds each.
  • the binding constant for Fc ⁇ RIIIa-158V-His of each aglycosylated trastuzumab Fc variant was calculated by a steady-state method using the equilibrium response value.
  • aFc44-ITKE and aFc44-ATKE were significantly better against Fc ⁇ RIIIa compared to the wild-type glycosylated trastuzumab antibody (Herceptin used clinically). It was found to have similar or higher binding strength (FIG. 4).
  • ELISA analysis was performed to confirm the binding ability of the three aglycosylated trastuzumab Fc variants (aFc44, aFc44-ITKE and aFc44-ATKE) to Fc gamma receptors (Fc ⁇ Rs).
  • Fc ⁇ Rs-GST Fc ⁇ RIIIa-158V-GST and Fc ⁇ RIIb-GST
  • 50 ⁇ l each of Fc ⁇ Rs-GST diluted to 4 ⁇ g/ml in 0.05 M Na 2 CO 3 pH 9.6 was placed in a flat bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, and then blocked with 100 ⁇ l of 4% skim milk (GenomicBase) (in PBS) for 2 hours at room temperature.
  • aglycosylated trastuzumab Fc variants (aFc44, aFc44-ITKE and aFc44-ATKE) serially diluted with 1% skim milk (in PBS) was dispensed into each well. and reacted at room temperature for 1 hour.
  • the antibody reaction was carried out with 50 ⁇ l of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, respectively, and then washed.
  • aglycosylated trastuzumab-aFc44-ITKE and aglycosylated trastuzumab-aFc44-ATKE discovered in the present invention were found to have higher Fc ⁇ RIIIa binding ability and significantly reduced Fc ⁇ RIlb binding affinity than wild-type trastuzumab, and wild-type aglycosylated Fc Compared to the introduced trastuzumab-aFc, it was found to have significantly improved Fc ⁇ RIIIa binding ability while maintaining the non-binding properties to Fc ⁇ RIIb, confirming that it had very good Fc ⁇ RIIIa selective binding ability (FIG. 6).

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Abstract

The present invention relates to an aglycosylated Fc variant having improved selective binding to Fc gamma receptors, particularly FcγRⅢa. Human antibody Fc domain variants of the present invention have improved selective binding to FcγRⅢa from among Fc gamma receptors so as to have an increased A/I ratio, and thus can be effectively used as antibody drugs having an improved ADCC effect, and the variants are aglycosylated Fc variants, thereby having no problems with respect to glycosylation heterogenity (glycan heterogeneity) of protein therapeutic agents, is easily mass-produced at low cost even in bacteria, and enables biologics to be produced without any problems with respect to cell lines, culturing and purification.

Description

FC 감마 수용체와의 결합력이 증대된 FC 변이체FC variants with increased binding to FC gamma receptors
본 발명은 Fc 감마 수용체와의 결합력이 증대된 Fc 변이체에 관한 것으로, Fc 감마 수용체 중 FcγRⅢa에 대한 선택적 결합력이 향상된 무당화 Fc 변이체에 관한 것이다.The present invention relates to Fc variants with increased binding ability to Fc gamma receptors, and to aglycosylated Fc variants with improved selective binding ability to FcγRIIIa among Fc gamma receptors.
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, 또는 ADCC 라 알려짐), 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(antibody-dependent cell-mediated cytotoxicity) 및 ADCP(antibody dependent cellular phagocytosis)에 결정적인 역할을 하며, 특히, 항체의 effetor function인 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. Clearance, lysis of antibody-coated target cells by killer cells (known as antibody-dependent cell-mediated cytotoxicity, or ADCC), release of inflammatory mediators, control of placental migration and immunoglobulin production trigger a biological response (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, antibody-dependent cell-mediated cytotoxicity (ADCC), and antibody dependent cellular phagocytosis (ADCP). It depends on interactions with Fc receptors present on the surface. 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.
본 발명의 목적은 특정 Fc 감마 수용체(FcγR)와의 선택적 결합력이 증대된 무당화 인간 항체 Fc 도메인 변이체를 제공하는 것이다.An object of the present invention is to provide an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor (FcγR).
또한, 본 발명의 목적은 특정 Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편을 제공하는 것이다.Another object of the present invention is to provide an aglycosylated antibody specific for a specific Fc gamma receptor or a fragment having immunological activity thereof.
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 특정 Fc 감마 수용체와의 선택적 결합력이 증대된 무당화 인간 항체 Fc 도메인 변이체의 제조방법을 제공하는 것이다.In addition, an object of the present invention is to provide a method for preparing an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor.
또한, 본 발명의 목적은 특정 Fc 감마 수용체에 특이적인 무당화 항체의 제조방법을 제공하는 것이다.It is also an object of the present invention to provide a method for preparing an aglycosylated antibody specific for a specific Fc gamma receptor.
또한, 본 발명의 목적은 항체 치료제의 제조에 사용하기 위한, Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편의 용도를 제공하는 것이다.In addition, an object of the present invention is to provide a use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity thereof for use in the manufacture of an antibody therapeutic agent.
또한, 본 발명의 목적은 Fc 감마 수용체에 특이적인 무당화 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 of an Fc gamma receptor-specific aglycosylated antibody or a fragment having immunological activity for preventing or treating cancer.
아울러, 본 발명의 목적은 Fc 감마 수용체에 특이적인 무당화 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 a pharmaceutically effective amount of an Fc gamma receptor-specific aglycosylated antibody or immunologically active fragment thereof to a subject suffering from cancer.
상기 과제를 해결하기 위하여, 본 발명은 특정 Fc 감마 수용체와의 선택적 결합력이 증대된 무당화 인간 항체 Fc 도메인 변이체를 제공한다.In order to solve the above problems, the present invention provides an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor.
또한, 본 발명은 Fc 도메인 변이체를 포함하는 특정 Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편을 제공한다.In addition, the present invention provides an aglycosylated antibody specific for a specific Fc gamma receptor, including an Fc domain variant, or a fragment having immunological activity thereof.
또한, 본 발명은 Fc 도메인 변이체, 또는 이를 포함하는 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 an Fc domain variant, or an antibody containing the same or a fragment having immunological activity thereof as an active ingredient.
또한, 본 발명은 특정 Fc 감마 수용체와의 선택적 결합력이 증대된 무당화 인간 항체 Fc 도메인 변이체의 제조방법을 제공한다.In addition, the present invention provides a method for preparing an aglycosylated human antibody Fc domain variant with increased selective binding ability to a specific Fc gamma receptor.
또한, 본 발명은 특정 Fc 감마 수용체에 특이적인 무당화 항체의 제조방법을 제공한다.In addition, the present invention provides a method for producing an aglycosylated antibody specific for a specific Fc gamma receptor.
또한, 본 발명은 항체 치료제의 제조에 사용하기 위한, Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편의 용도를 제공한다.In addition, the present invention provides the use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity thereof for use in the manufacture of an antibody therapeutic.
또한, 본 발명은 Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편의 암의 예방 또는 치료 용도를 제공한다.In addition, the present invention provides a use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity for preventing or treating cancer.
아울러, 본 발명은 Fc 감마 수용체에 특이적인 무당화 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 the step of administering a pharmaceutically effective amount of an Fc gamma receptor-specific aglycosylated antibody or immunologically active fragment thereof to a subject suffering from cancer.
본 발명의 인간 항체 Fc 도메인 변이체들은 Fc 감마 수용체 중 FcγRⅢa에 대한 선택적인 결합이 향상되어 A/I 비율이 증가되었으므로 ADCC 효과가 향상된 항체 의약품으로 유용하게 이용될 수 있고, 무당화 변이체이므로 단백질 치료제의 당화 비균질성(glycan heterogenity) 문제점이 없으며, 박테리아에서도 저렴하게 대량 생산이 용이하고, 세포주, 배양공정 및 정제공정에 따른 문제가 없는 생물의약품 제조가 가능한 효과가 있다. Since the human antibody Fc domain variants of the present invention have improved selective binding to FcγRIIIa among Fc gamma receptors and increased A/I ratio, they can be usefully used as antibody drugs with improved ADCC effect, and since they are aglycosylated variants, they can be used as protein therapeutics. There is no problem of glycan heterogeneity, it is easy to mass-produce cheaply even in bacteria, and there is an effect that enables the manufacture of biological drugs without problems according to cell lines, culture processes and purification processes.
도 1은 E. coli 내막에 디스플레이 된 무당화 Fc 변이체들의 FcγRⅢa에 대한 결합력 (A) 및 FcγRⅢa에 대한 선택적 결합력 (B)을 FACS로 분석한 결과이다.Figure 1 shows the results of FACS analysis of the binding ability to FcγRIIIa (A) and the selective binding ability to FcγRIIIa (B) of aglycosylated Fc variants displayed on the E. coli inner membrane.
도 2는 aFc-22 변이체 (P247L/V264E/T299A/A330I)를 기반으로 T225I, T366A 및 K334E 돌연변이가 FcγRⅢa에 대한 결합력 (A) 및 FcγRⅢa에 대한 선택적 결합력 (B)에 미치는 영향을 FACS로 분석한 결과이다.Figure 2 is based on the aFc-22 variant (P247L / V264E / T299A / A330I) T225I, T366A and K334E mutations on FcγRIIIa binding ability (A) and FcγRIIIa selective binding ability (B) analyzed by FACS This is the result.
도 3은 무당화 Fc 변이체 aFc44, aFc44-ITKE 및 aFc44-ATKE를 포함하는 트라스트주맙 Fc 변이체를 Expi293F 세포에서 발현 및 정제한 후 SDS-PAGE로 확인한 도이다.FIG. 3 is a diagram illustrating expression and purification of trastuzumab Fc variants including aglycosylated Fc variants aFc44, aFc44-ITKE and aFc44-ATKE in Expi293F cells and then confirmed by SDS-PAGE.
도 4는 무당화 Fc 변이체가 포함된 트라스트주맙 Fc 변이체들의 결합 상수를 측정한 결과이다.Figure 4 shows the results of measuring the binding constants of trastuzumab Fc variants including aglycosylated Fc variants.
도 5는 무당화 Fc 변이체가 포함된 트라스트주맙 Fc 변이체들의 FcγRs에 대한 결합력을 ELISA로 분석한 결과이다.Figure 5 shows the results of analyzing the binding ability to FcγRs of trastuzumab Fc variants including aglycosylated Fc variants by ELISA.
도 6은 야생형 당화 트라스트주맙과 무당화 Fc 변이체가 포함된 트라스트주맙 Fc 변이체들의 FcγRⅢa 및 FcγRⅡb에 대한 선택적 결합 경향성을 분석한 결과이다.6 is a result of analyzing the selective binding tendency for FcγRIIIa and FcγRIIb of trastuzumab Fc variants including wild-type glycosylated trastuzumab and aglycosylated Fc variants.
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 225, 247, 264, 299, 330, 334 및 366 위치의 아미노산으로 이루어진 군으로부터 선택되는 어느 하나 이상의 위치의 아미노산이 야생형의 아미노산과 다른 서열로 치환된, Fc 감마 수용체(FcγR)와의 결합력이 증대된 인간 항체 Fc 도메인 변이체에 관한 것이다.In one aspect, the present invention provides from the group consisting of amino acids at positions 225, 247, 264, 299, 330, 334 and 366 numbered according to the Kabat numbering system in the wild type human antibody Fc domain. It relates to a human antibody Fc domain variant with increased binding ability to an Fc gamma receptor (FcγR) in which amino acids at any one or more selected positions are substituted with a sequence different from that of the wild-type amino acid.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 P247L, V264E, T299A 및 A330I로 이루어진 군으로부터 선택된 어느 하나 이상의 아미노산 치환을 포함할 수 있다.In one embodiment, the Fc domain variant of the present invention may contain any one or more amino acid substitutions selected from the group consisting of P247L, V264E, T299A and A330I.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 T225I, K334E 또는 T366A의 아미노산 치환을 추가로 포함할 수 있다.In one embodiment, the Fc domain variant of the present invention may further comprise an amino acid substitution of T225I, K334E or T366A.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 T225I, P247L, V264E, T299A, A330I 및 T366A의 아미노산 치환을 포함하는 aFc44일 수 있다.In one embodiment, the Fc domain variant of the present invention may be aFc44 comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I and T366A.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 P247L, V264E, T299A, A330I, K334E 및 T366A의 아미노산 치환을 포함하는 aFc44-ITKE일 수 있다.In one embodiment, the Fc domain variant of the present invention may be aFc44-ITKE comprising amino acid substitutions of P247L, V264E, T299A, A330I, K334E and T366A.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 T225I, P247L, V264E, T299A, A330I 및 K334E의 아미노산 치환을 포함하는 Fc44-ATKE일 수 있다.In one embodiment, the Fc domain variant of the present invention may be Fc44-ATKE comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I and K334E.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 P247L, V264E, T299A 및 A330I의 아미노산 치환을 포함하는 aFc22; P247L, V264E, T299A, A330I 및 K334E의 아미노산 치환을 포함하는 aFc24; P247L, V264E, T299A, A330I 및 T366A의 아미노산 치환을 포함하는 aFc44-IT; T225I, P247L, V264E, T299A 및 A330I의 아미노산 치환을 포함하는 aFc44-AT; 또는 T225I, P247L, V264E, T299A, A330I, K334E 및 T366A의 아미노산 치환을 포함하는 aFc44-KE일 수 있다.In one embodiment, an Fc domain variant of the present invention comprises aFc22 comprising the amino acid substitutions of P247L, V264E, T299A and A330I; aFc24 comprising amino acid substitutions of P247L, V264E, T299A, A330I and K334E; aFc44-IT comprising amino acid substitutions of P247L, V264E, T299A, A330I and T366A; aFc44-AT comprising amino acid substitutions of T225I, P247L, V264E, T299A and A330I; or aFc44-KE comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I, K334E and T366A.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 Fc 감마 수용체 FcγRⅢa에 대한 선택적 결합력이 향상된 Fc 도메인 변이체일 수 있다.In one embodiment, the Fc domain variant of the present invention may be an Fc domain variant with improved selective binding ability to the Fc gamma receptor FcγRIIIa.
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 항체의 Fc 영역은 서열번호 6의 아미노산 서열을 포함할 수 있으며, 서열번호 16의 핵산분자로 코딩될 수 있다.In one embodiment, the Fc region of the wild-type human antibody may include the amino acid sequence of SEQ ID NO: 6 and may be encoded by the nucleic acid molecule of SEQ ID NO: 16.
일 구현예에서, 본 발명의 Fc 도메인 변이체는 서열번호 7 내지 15의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함할 수 있다.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: 7 to 15.
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. 바람직하게는, 본 발명에서 Fc 변이체는 T225I, P247L, V264E, T299A, A330I, K334E 및 T366A (상기 넘버링은 카밧에 기재된 EU 인덱스에 따른 것임)로 이루어진 군으로부터 선택되는 하나 이상의 아미노산 잔기의 변형을 포함하여 야생형 항체 Fc 도메인 (영역)에 비해 Fc 감마 수용체에 대한 선택적 결합력이 증가된다. 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. Preferably, the Fc variant in the present invention comprises a modification of one or more amino acid residues selected from the group consisting of T225I, P247L, V264E, T299A, A330I, K334E and T366A (the numbering is according to the EU index as described in Kabat). As a result, the selective binding ability to the Fc gamma receptor is increased compared to the wild-type antibody Fc domain (region).
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 변이체를 포함하는 Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편에 관한 것이다.In one aspect, the present invention relates to an aglycosylated antibody specific for an Fc gamma receptor, including an Fc variant of the present invention, or a fragment having immunological activity thereof.
일 구현예에서, 본 발명의 항체는 서열번호 1 내지 3의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 2(CH2) 및 서열번호 4 또는 5의 아미노산 서열을 포함하는 중쇄 불변 영역 도메인 3(CH3)를 포함할 수 있다.In one embodiment, the antibody of the present invention comprises a 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 3 and the amino acid sequence of SEQ ID NOs: 4 or 5 and a heavy chain constant region domain 3 (C H 3).
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, 높은 FcγRⅢa 결합 선택성을 가져 높은 A/I 비율을 가지므로, 항체-매개성 세포독성작용 (antibody dependent cellular cytotoxicity, ADCC)을 증가시킬 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, have high FcγRIIIa binding selectivity and have a high A/I ratio, thus antibody-mediated It may increase antibody dependent cellular cytotoxicity (ADCC).
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 17 내지 25의 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 17 to 25.
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 단편을 생산하고자 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 regulatory sequences. Along with regulatory sequences that control transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that serve other functions.
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, Fc 감마 수용체와의 결합력이 증대된 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) a method for producing a human antibody Fc domain variant having increased binding ability to an Fc gamma receptor, comprising recovering a polypeptide expressed by a 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 Fc 감마 수용체에 특이적인 무당화 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 Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편의 용도에 관한 것이다.In one aspect, the present invention relates to the use of an aglycosylated antibody specific for an Fc gamma receptor of the present invention or an immunologically active fragment thereof for use in the manufacture of an antibody therapeutic.
일 측면에서, 본 발명은 본 발명의 Fc 감마 수용체에 특이적인 무당화 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 Fc gamma receptor-specific aglycosylated antibody of the present invention or a fragment having immunological activity for the prevention or treatment of cancer.
일 측면에서, 본 발명은 본 발명의 Fc 감마 수용체에 특이적인 무당화 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 aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity of the present invention. will be.
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.Example 1. 선택적 결합력이 향상된 무당화 Fc 변이체 선별Selection of aglycosylated Fc variants with improved selective binding ability
FcγRⅢa에 대한 선택적 결합력이 향상된 무당화 트라스트주맙(trastuzumab) Fc 변이체 선별을 위해, 항체 Fc의 canonical N-당화(glycosylation) 변형(modification)이 일어나는 위치인 299번째 아마노산 Thr을 Leu으로 치환한 후 본 발명자들이 구축한 박테리아 디스플레이된 인간 scFv 항체 라이브러리를 제작하였다. 라이브러리는 Alexa488 접합된(conjugated) 사합체(tetrameric) FcγRⅢa 및 비형광 FcγRⅡb의 경쟁 결합을 유도한 뒤 높은 형광 신호를 나타내는 클론들의 집단(population)을 골라내는 방식으로 총 6 라운드의 선별(sorting) 과정을 거쳐 FcγRⅢa 선택적 결합 무당화 Fc 변이체를 증폭(enrichment) 시켰으며, E. coli 내막에 디스플레이된 개별 클론(individual clones) 무당화 Fc 변이체들의 FcγRⅢa-Alexa488 결합에 의한 형광세기 (즉, FcγRⅢa 결합력) (도 1A) 및 무형광 FcγRⅡb에 의해 마스킹(masking)된 상태에서의 FcγRⅢa-Alexa488 결합에 의한 형광세기 (즉, FcγRⅢa 선택적 결합 세기) (도 1B)를 FACS로 분석하여, 최종적으로 FcγRⅢa/FcγRⅡb에 대한 선택적 결합력이 향상된 무당화 Fc 변이체들로서 aFc22 (P247L/V264E/T299A/A330I), aFc24 (P247L/V264E/T299A/A330I/K334E) 및 aFc44 (T225I/P247L/V264E/T299A/A330I/T366A)을 선별하였다 (표 1). In order to screen for aglycosylated trastuzumab Fc variants with improved selective binding ability to FcγRIIIa, canonical N -glycosylation modification of antibody Fc, the 299th amino acid Thr, which is the position where modification occurs, is substituted with Leu, and then A bacteria-displayed human scFv antibody library constructed by the inventors was constructed. The library undergoes a total of 6 rounds of sorting process by inducing competitive binding of Alexa488 conjugated tetrameric FcγRIIIa and non-fluorescent FcγRIIb and then selecting a population of clones showing high fluorescence signals. Through this, FcγRIIIa selective binding aglycosylated Fc variants were amplified (enrichment), and fluorescence intensity (i.e., FcγRIIIa avidity) due to FcγRIIIa-Alexa488 binding of individual clones aglycosylated Fc variants displayed on the E. coli inner membrane ( Figure 1A) and fluorescence intensity by FcγRIIIa-Alexa488 binding in a state masked by non-fluorescent FcγRIIb (ie, FcγRIIIa selective binding intensity) (FIG. 1B) was analyzed by FACS, finally selective for FcγRIIIa / FcγRIIb aFc22 (P247L/V264E/T299A/A330I), aFc24 (P247L/V264E/T299A/A330I/K334E) and aFc44 (T225I/P247L/V264E/T299A/A330I/T366A) were selected as aglycosylated Fc variants with improved binding ability. Table 1).
## 돌연변이mutation 서열번호 sequence number
aFcaFc T299LT299L 77
aFc22aFc22 P247L, V264E, T299A, A330IP247L, V264E, T299A, A330I 88
aFc24aFc24 P247L, V264E, T299A, A330I, K334EP247L, V264E, T299A, A330I, K334E 99
aFc44aFc44 T225I, P247L, V264E, T299A, A330I, T366AT225I, P247L, V264E, T299A, A330I, T366A 1010
aFc44-ITaFc44-IT P247L, V264E, T299A, A330I, T366AP247L, V264E, T299A, A330I, T366A 1111
aFc44-ATaFc44-AT T225I, P247L, V264E, T299A, A330IT225I, P247L, V264E, T299A, A330I 1212
aFc44-KEaFc44-KE T225I, P247L, V264E, T299A, A330I, K334E, T366AT225I, P247L, V264E, T299A, A330I, K334E, T366A 1313
aFc44-ITKEaFc44-ITKE P247L, V264E, T299A, A330I, K334E, T366AP247L, V264E, T299A, A330I, K334E, T366A 1414
aFc44-ATKEaFc44-ATKE T225I, P247L, V264E, T299A, A330I, K334ET225I, P247L, V264E, T299A, A330I, K334E 1515
실시예 2. Fc 변이체들의 선택적 결합 확인Example 2. Confirmation of selective binding of Fc variants
상기 실시예 1에서 선별한 FcγRⅢa/FcγRⅡb 선택적 결합력이 가장 높은 세 클론 (aFc22, aFc24 및 aFc44)의 서열 분석을 통해 공통적인 아미노산 변이 P247L/V264E/T299A/A330I를 확인하였으며, 중복되지 않는 돌연변이인 T225I, K334E 및 T366A가 FcγRⅢa/FcγRⅡb에 대한 선택적 결합에 미치는 영향을 확인하기 위해 T225I, K334E 및 T366A의 돌연변이가 조합된 무당화 Fc 변이체들을 제작한 뒤 (aFc44-IT, aFc44-AT, aFc44-KE, aFc44-ITKE 및 aFc44-ATKE) (표 1), E. coli 내막에 디스플레이하여 FcγRⅢa 결합력 및 FcγRⅢa/FcγRⅡb 선택적 결합력을 FACS로 분석하였다. 그 결과, aFc44 클론 및 aFc44-ATKE가 가장 우수한 FcγRⅢa 결합 대비 FcγRⅢa/FcγRⅡb 선택적 결합을 가지는 것으로 나타났다 (도 2). Through sequence analysis of the three clones (aFc22, aFc24 and aFc44) with the highest FcγRIIIa/FcγRIIb selective binding ability selected in Example 1, a common amino acid mutation P247L/V264E/T299A/A330I was identified, and the non-overlapping mutation T225I , In order to confirm the effect of K334E and T366A on the selective binding to FcγRIIIa / FcγRIIb, aglycosylated Fc variants in which T225I, K334E and T366A mutations were combined were prepared (aFc44-IT, aFc44-AT, aFc44-KE, aFc44-ITKE and aFc44-ATKE) (Table 1), displayed on E. coli inner membrane, and FcγRIIIa binding ability and FcγRIIIa/FcγRIIb selective binding ability were analyzed by FACS. As a result, it was found that the aFc44 clone and aFc44-ATKE had the most excellent FcγRIIIa binding versus FcγRIIIa/FcγRIIb selective binding (FIG. 2).
실시예 3. 선택적 결합력이 향상된 무당화 Fc 변이체의 발현 및 정제Example 3. Expression and purification of aglycosylated Fc variants with improved selective binding ability
상기 실시예에서 선발한 무당화 Fc 변이체들을 포함하는 트라스트주맙 Fc 변이체를 발현 및 정제하기 위해, Fc 변이체 aFc44, aFc44-ITKE 및 aFc44-ATKE의 중쇄(heavy chain) 및 경쇄(light chain) 발현 벡터를 각각 제작한 뒤, Freestyle 293 expression 배양액 (Gibco, 12338-018) 100 ml에 각 변이체들의 중쇄유전자와 경쇄유전자를 1:1의 비율로 먼저 섞고 PEI:변이체유전자=4:1의 비율로 섞어 상온에서 20 분간 두었다가 전날 2x106 cells/ml의 밀도로 계대배양해 놓은 Expi293F 세포에 트랜스펙션하였다. 그 후 CO2 진탕배양기에서 37 ℃, 125 rpm 및 8 %　CO2의 조건으로 7일간 배양하고 원심 분리하여 상등액만 취하였다. 상등액을 25x PBS를 이용해 평형을 맞춘 뒤 바틀탑 필터를 이용해 0.2 μm 필터 (Corning, 430513)로 여과하였다. 여과된 배양액에 Protein A 레진 500 μl을 넣어 주고 4 ℃에서 16 시간 교반한 뒤 컬럼을 통해 레진을 회수한 후 5 ml PBS로 세척하고 pH 2.7의 100 mM 글리신(glycine) 버퍼 3 ml로 용출하고 1M Tris-HCl pH 8.0을 이용하여 중화하였다. Amicon Ultra-4 centrifugal filter units 3K(Merck Millipore, UFC800324)을 사용하여 버퍼를 교체한 후 높은 순도로 정제된 무당화 항체 트라스트주맙 Fc 변이체들 (aFc44, aFc44-ITKE 및 aFc44-ATKE)의 크기와 순도를 SDS-PAGE를 통해 분석하였다 (도 3).In order to express and purify the trastuzumab Fc variants including the aglycosylated Fc variants selected in the above example, heavy chain and light chain expression vectors of Fc variants aFc44, aFc44-ITKE and aFc44-ATKE were prepared. After making each, first mix the heavy chain gene and light chain gene of each variant in 100 ml of Freestyle 293 expression culture medium (Gibco, 12338-018) at a ratio of 1: 1, and mix at a ratio of PEI: mutant gene = 4: 1 at room temperature. After being allowed to stand for 20 minutes, Expi293F cells subcultured the previous day at a density of 2x10 6 cells/ml were transfected. Thereafter, CO 2 was cultured for 7 days under conditions of 37 °C, 125 rpm and 8% CO 2 in a shaking incubator, and only the supernatant was taken by centrifugation. The supernatant was equilibrated with 25x PBS and filtered through a 0.2 μm filter (Corning, 430513) using a bottle top filter. Add 500 μl of Protein A resin to the filtered culture medium, stir at 4 ° C for 16 hours, recover the resin through the column, wash with 5 ml PBS, elute with 3 ml of 100 mM glycine buffer at pH 2.7, and Neutralized using Tris-HCl pH 8.0. Size and purity of highly purified aglycosylated antibody trastuzumab Fc variants (aFc44, aFc44-ITKE and aFc44-ATKE) after buffer replacement using Amicon Ultra-4 centrifugal filter units 3K (Merck Millipore, UFC800324) was analyzed through SDS-PAGE (FIG. 3).
실시예 4. 무당화 트라스트주맙Example 4. Aglycosylated Trastuzumab Fc 변이체의 FcγRⅢa 결합력 분석Analysis of FcγRIIIa binding affinity of Fc variants
상기 실시예 3에서 발현 및 정제한 3종의 무당화 트라스트주맙 Fc 변이체들 (aFc44, aFc44-ITKE 및 aFc44-ATKE)의 FcγRⅢa 결합력을 측정하기 위해, BLItz(Fortebio)를 이용하여 BLI(Biolayer Interferometry) 분석을 수행하였다. 구체적으로, AR2G(Amine reactive 2nd-generation) 바이오센서(biosensor)를 20 mM EDC 및 10 mM s-NHS로 5 분간 활성화하고, 20 μg/ml의 3종의 무당화 트라스트주맙 Fc 변이체들을 각각 고정화하였다. 그 후, 1 M 에탄올아민(ethanolamine)으로 5 분간 바이오센서를 소광(quenching)시킨 뒤 각각 60 초씩의 기준선(baseline), FcγRⅢa-158V-His 결합(association) 및 해리(dissociation) 과정을 거쳐 결합력을 확인하였다. 이 때, FcγRⅢa-158V-His는 40 μM부터 1/2씩 희석하여 농도별 결합력을 분석하였고, 각 측정 간 바이오센서의 재생(regeneration)은 10 mM 글라이신(glycine) (pH 1.5) 버퍼 및 제조사에서 제공하는 키네틱(kinetics) 버퍼를 5 초씩 번갈아 반응시켜 총 5 사이클 진행하였다. 측정 완료 후 평형 반응(equilibrium response) 값을 이용해 정상-상태(steady-state) 방법으로 각 무당화 트라스트주맙 Fc 변이체들의 FcγRⅢa-158V-His에 대한 결합 상수를 계산하였다. 그 결과, 상기 실시예 3에서 발현 및 정제한 3 종의 무당화 트라스트주맙 Fc 변이체들 중 aFc44-ITKE 및 aFc44-ATKE가 야생형 당화 트라스트주맙 항체 (임상적으로 사용되는 Herceptin)와 비교하여 FcγRⅢa에 대해 유사하거나 더 높은 결합력을 가지는 것으로 나타났다 (도 4).In order to measure the FcγRIIIa binding affinity of the three aglycosylated trastuzumab Fc variants (aFc44, aFc44-ITKE and aFc44-ATKE) expressed and purified in Example 3, BLI (Biolayer Interferometry) using BLItz (Fortebio) analysis was performed. Specifically, an amine reactive 2nd-generation (AR2G) biosensor was activated with 20 mM EDC and 10 mM s-NHS for 5 minutes, and 20 μg/ml of three aglycosylated trastuzumab Fc variants were immobilized, respectively. . After that, the biosensor was quenched with 1 M ethanolamine for 5 minutes, followed by baseline, FcγRIIIa-158V-His association and dissociation for 60 seconds each to increase binding force. Confirmed. At this time, FcγRIIIa-158V-His was diluted by 1/2 from 40 μM and the binding ability was analyzed by concentration, and the regeneration of the biosensor between each measurement was performed in 10 mM glycine (pH 1.5) buffer and manufacturer. A total of 5 cycles were performed by alternately reacting the provided kinetics buffer for 5 seconds each. After completion of the measurement, the binding constant for FcγRIIIa-158V-His of each aglycosylated trastuzumab Fc variant was calculated by a steady-state method using the equilibrium response value. As a result, among the three aglycosylated trastuzumab Fc variants expressed and purified in Example 3, aFc44-ITKE and aFc44-ATKE were significantly better against FcγRIIIa compared to the wild-type glycosylated trastuzumab antibody (Herceptin used clinically). It was found to have similar or higher binding strength (FIG. 4).
실시예 5. 무당화 Fc 변이체의 Fc 감마 수용체에 대한 결합력 분석Example 5. Analysis of binding affinity of aglycosylated Fc variants to Fc gamma receptors
3종의 무당화 트라스트주맙 Fc 변이체들 (aFc44, aFc44-ITKE 및 aFc44-ATKE)의 Fc 감마 수용체(FcγRs)에 대한 결합력을 확인하기 위해 ELISA 분석을 진행하였다. 구체적으로, 0.05 M Na2CO3 pH 9.6에 4 μg/ml로 희석한 FcγRs-GST (FcγRⅢa-158V-GST 및 FcγRⅡb-GST)를 각각 50 μl씩 flat bottom polystyrene high bind 96웰 마이크로플레이트 (Costar, 3590)에 4℃에서 16 시간 동안 고정화한 후 100 μl의 4% 스킴밀크 (GenomicBase) (in PBS)로 상온에서 2 시간 동안 블로킹하였다. 0.05% PBST 180 μl로 4 회씩 세척한 뒤 1% 스킴밀크 (in PBS)로 연속 희석한 무당화 트라스트주맙 Fc 변이체들 (aFc44, aFc44-ITKE 및 aFc44-ATKE)을 각각 50 μl씩 각 웰에 분주하여 상온에서 1 시간 동안 반응시켰다. 세척 과정 후 50 μl의 HRP-Protein L (GenScript, M00098)과 상온에서 1 시간 동안 각각 항체 반응을 진행하고 세척하였다. 그 후 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028)를 50 μl씩 첨가해 발색한 뒤 50 μl의 2 M H2SO4를 첨가하여 반응을 종료시킨 다음 Epoch Microplate Spectrophotometer (BioTek)을 이용해 흡광도를 분석하였다 (도 5). 또한, 해당 ELISA 결과를 바탕으로 FcγRⅢa 및 FcγRⅡb에 대한 결합 경향성을 야생형 당화 항체 트라스트주맙(trastuzumab)과 분석하였다. 그 결과, 본 발명에서 발굴한 무당화 트라스트주맙-aFc44-ITKE 및 무당화 트라스트주맙-aFc44-ATKE가 야생형 트라스트주맙보다 높은 FcγRⅢa 결합력 및 월등히 감소한 FcγRⅡb 결합력을 가지는 것으로 나타났으며, 야생형 무당화 Fc가 도입된 트라스트주맙-aFc와 비교하면 FcγRⅡb에 결합하지 않는 특성을 유지하면서 동시에 월등히 향상된 FcγRⅢa 결합력을 가지는 것으로 나타나 매우 우수한 FcγRⅢa 선택적 결합력을 가짐을 확인하였다 (도 6). ELISA analysis was performed to confirm the binding ability of the three aglycosylated trastuzumab Fc variants (aFc44, aFc44-ITKE and aFc44-ATKE) to Fc gamma receptors (FcγRs). Specifically, 50 μl each of FcγRs-GST (FcγRIIIa-158V-GST and FcγRIIb-GST) diluted to 4 μg/ml in 0.05 M Na 2 CO 3 pH 9.6 was placed in a flat bottom polystyrene high bind 96-well microplate (Costar, 3590) at 4°C for 16 hours, and then blocked with 100 μl of 4% skim milk (GenomicBase) (in PBS) for 2 hours at room temperature. After washing four times with 180 μl of 0.05% PBST, 50 μl each of aglycosylated trastuzumab Fc variants (aFc44, aFc44-ITKE and aFc44-ATKE) serially diluted with 1% skim milk (in PBS) was dispensed into each well. and reacted at room temperature for 1 hour. After the washing process, the antibody reaction was carried out with 50 μl of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, respectively, and then washed. Then, 50 μl of 1-Step Ultra TMB-ELISA substrate solution (Thermo Fisher Scientific, 34028) was added to develop color, and 50 μl of 2 MH 2 SO 4 was added to terminate the reaction, followed by Epoch Microplate Spectrophotometer (BioTek). The absorbance was analyzed using (FIG. 5). In addition, based on the ELISA results, the binding tendency to FcγRIIIa and FcγRIIb was analyzed with the wild-type glycosylation antibody trastuzumab. As a result, aglycosylated trastuzumab-aFc44-ITKE and aglycosylated trastuzumab-aFc44-ATKE discovered in the present invention were found to have higher FcγRIIIa binding ability and significantly reduced FcγRIlb binding affinity than wild-type trastuzumab, and wild-type aglycosylated Fc Compared to the introduced trastuzumab-aFc, it was found to have significantly improved FcγRIIIa binding ability while maintaining the non-binding properties to FcγRIIb, confirming that it had very good FcγRIIIa selective binding ability (FIG. 6).

Claims (19)

  1. 야생형(Wild type) 인간 항체 Fc 도메인에서, 카밧 넘버링 시스템(Kabat numbering system)에 따라 넘버링된 225, 247, 264, 299, 330, 334 및 366 위치의 아미노산으로 이루어진 군으로부터 선택되는 어느 하나 이상의 위치의 아미노산이 야생형의 아미노산과 다른 서열로 치환된, Fc 감마 수용체(FcγR)와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.In the wild type human antibody Fc domain, at least one position selected from the group consisting of amino acids at positions 225, 247, 264, 299, 330, 334 and 366 numbered according to the Kabat numbering system A human antibody Fc domain variant in which the amino acid is substituted with a sequence different from that of the wild-type amino acid, and the binding force with an Fc gamma receptor (FcγR) is increased.
  2. 제 1항에 있어서, P247L, V264E, T299A 및 A330I로 이루어진 군으로부터 선택된 어느 하나 이상의 아미노산 치환을 포함하는, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.According to claim 1, P247L, V264E, T299A and A330I, including any one or more amino acid substitutions selected from the group consisting of, human antibody Fc domain variant with increased binding force to the Fc gamma receptor.
  3. 제 2항에 있어서, T225I, K334E 또는 T366A의 아미노산 치환을 추가로 포함하는, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 2, further comprising an amino acid substitution of T225I, K334E or T366A, with increased binding force to the Fc gamma receptor.
  4. 제 2항에 있어서, T225I, P247L, V264E, T299A, A330I 및 T366A의 아미노산 치환을 포함하는, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 2, comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I and T366A, with increased binding affinity to Fc gamma receptors.
  5. 제 2항에 있어서, P247L, V264E, T299A, A330I, K334E 및 T366A의 아미노산 치환을 포함하는, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 2, comprising amino acid substitutions of P247L, V264E, T299A, A330I, K334E and T366A, with increased binding force to Fc gamma receptors.
  6. 제 2항에 있어서, T225I, P247L, V264E, T299A, A330I 및 K334E의 아미노산 치환을 포함하는, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 2, comprising amino acid substitutions of T225I, P247L, V264E, T299A, A330I and K334E, with increased binding affinity to Fc gamma receptors.
  7. 제 1항에 있어서, Fc 감마 수용체 FcγRⅢa에 대한 선택적 결합력이 향상된, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체.The human antibody Fc domain variant according to claim 1, wherein the selective binding ability to the Fc gamma receptor FcγRIIIa is improved and the binding force to the Fc gamma receptor is increased.
  8. 제 1항의 Fc 도메인 변이체를 포함하는 Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편.An aglycosylated antibody specific for an Fc gamma receptor comprising the Fc domain variant of claim 1 or a fragment having immunological activity thereof.
  9. 제 8항에 있어서, 서열번호 1 내지 3의 아미노산 서열로 이루어지는 군으로부터 선택되는 어느 하나를 포함하는 중쇄 불변 영역 도메인 2(CH2) 및 서열번호 4 또는 5의 아미노산 서열을 포함하는 중쇄 불변 영역 도메인 3(CH3)를 포함하는, Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편.The method of claim 8, wherein the heavy chain constant region domain 2 (C H 2) comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 3 and the heavy chain constant region comprising the amino acid sequence of SEQ ID NOs: 4 or 5 An aglycosylated antibody specific for an Fc gamma receptor or an immunologically active fragment thereof, comprising domain 3 ( CH 3).
  10. 제 1항의 Fc 도메인 변이체, 또는 제 8항의 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 8 or a fragment having immunological activity thereof.
  11. 제 10항의 핵산분자를 포함하는 벡터.A vector comprising the nucleic acid molecule of claim 10.
  12. 제 11항의 벡터를 포함하는 숙주세포.A host cell comprising the vector of claim 11.
  13. 제 1항의 Fc 도메인 변이체, 또는 제 8항의 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 8 or a fragment having immunological activity thereof as an active ingredient.
  14. 제 13항에 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.14. The method of claim 13, 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.
  15. 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, Fc 감마 수용체와의 결합력이 증대된 인간 항체 Fc 도메인 변이체의 제조방법.b) a method for producing a human antibody Fc domain variant with increased binding affinity to an Fc gamma receptor, comprising the step of recovering a polypeptide expressed by a host cell.
  16. a) 제 8항의 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 8 or a fragment having immunological activity thereof; and
    b) 숙주세포로부터 발현된 항체를 정제하는 단계를 포함하는 Fc 감마 수용체에 특이적인 무당화 항체의 제조방법.b) a method for preparing an aglycosylated antibody specific for an Fc gamma receptor comprising the step of purifying an antibody expressed from a host cell.
  17. 항체 치료제의 제조에 사용하기 위한, Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편의 용도.Use of an aglycosylated antibody specific for an Fc gamma receptor or an immunologically active fragment thereof for use in the manufacture of an antibody therapeutic.
  18. Fc 감마 수용체에 특이적인 무당화 항체 또는 이의 면역학적 활성을 가진 단편의 암의 예방 또는 치료 용도.Use of an aglycosylated antibody specific for an Fc gamma receptor or a fragment having immunological activity for the prevention or treatment of cancer.
  19. Fc 감마 수용체에 특이적인 무당화 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 to a subject suffering from cancer a pharmaceutically effective amount of an Fc gamma receptor-specific aglycosylated antibody or an immunologically active fragment thereof.
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US20130202606A1 (en) * 2003-01-09 2013-08-08 Macrogenics, Inc. Identification and Engineering of Antibodies with Variant Fc Regions and Methods of Using Same
KR20180051100A (en) * 2016-11-08 2018-05-16 국민대학교산학협력단 Aglycosylated Antibody Fc Region Exhibiting Enhanced Binding Specificity to an Fcγ Receptor
KR20190015583A (en) * 2010-11-05 2019-02-13 자임워크스 인코포레이티드 Stable heterodimeric antibody design with mutations in the fc domain
KR20190044348A (en) * 2017-10-20 2019-04-30 국민대학교산학협력단 An Antibody Fc Variant for Enhancing ADCC Activity
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US20130202606A1 (en) * 2003-01-09 2013-08-08 Macrogenics, Inc. Identification and Engineering of Antibodies with Variant Fc Regions and Methods of Using Same
KR20190015583A (en) * 2010-11-05 2019-02-13 자임워크스 인코포레이티드 Stable heterodimeric antibody design with mutations in the fc domain
KR20180051100A (en) * 2016-11-08 2018-05-16 국민대학교산학협력단 Aglycosylated Antibody Fc Region Exhibiting Enhanced Binding Specificity to an Fcγ Receptor
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