WO2023043127A1 - Variant de fc présentant une affinité accrue pour la liaison à des récepteurs gamma fc - Google Patents

Variant de fc présentant une affinité accrue pour la liaison à des récepteurs gamma fc 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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WO2023043127A9 (fr
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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

La présente invention concerne un variant de Fc aglycosylé présentant une liaison sélective améliorée pour les récepteurs gamma Fc, en particulier FcγRⅢa. Les variants de domaine Fc d'anticorps humain de la présente invention présentent une liaison sélective améliorée à FcγRⅢa parmi les récepteurs Fc gamma en présentant ainsi un rapport A/I accru, et peuvent donc être efficacement utilisés en tant que médicaments à base d'anticorps ayant un effet ADCC amélioré, et les variants sont des variants Fc aglycosylés, et ne posent donc pas de problèmes en termes d'hétérogénéité de glycosylation (hétérogénéité de glycane) des agents thérapeutiques protéiques, sont faciles à produire en masse à faible coût même dans des bactéries, et permettent de produire des produits biologiques ne posant aucun problème en termes de lignées cellulaires, de culture et de purification.
PCT/KR2022/013492 2021-09-17 2022-09-07 Variant de fc présentant une affinité accrue pour la liaison à des récepteurs gamma fc WO2023043127A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (ko) * 2016-11-08 2018-05-16 국민대학교산학협력단 Ｆｃγ 수용체에 대한 결합 특이성이 향상된 무당화 항체 Ｆｃ 영역
KR20190015583A (ko) * 2010-11-05 2019-02-13 자임워크스 인코포레이티드 Fc 도메인 내의 돌연변이를 갖는 안정한 이종이량체 항체 디자인
KR20190044348A (ko) * 2017-10-20 2019-04-30 국민대학교산학협력단 ADCC 향상을 위한 항체 Fc 변이체
US20200190200A1 (en) * 2018-12-18 2020-06-18 Janssen Biotech, Inc. Methods of Producing Heterodimeric Antibodies

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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 (ko) * 2010-11-05 2019-02-13 자임워크스 인코포레이티드 Fc 도메인 내의 돌연변이를 갖는 안정한 이종이량체 항체 디자인
KR20180051100A (ko) * 2016-11-08 2018-05-16 국민대학교산학협력단 Ｆｃγ 수용체에 대한 결합 특이성이 향상된 무당화 항체 Ｆｃ 영역
KR20190044348A (ko) * 2017-10-20 2019-04-30 국민대학교산학협력단 ADCC 향상을 위한 항체 Fc 변이체
US20200190200A1 (en) * 2018-12-18 2020-06-18 Janssen Biotech, Inc. Methods of Producing Heterodimeric Antibodies

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