WO2023043125A1 - Variant de fc présentant une liaison améliorée à divers récepteurs de fc gamma - Google Patents

Variant de fc présentant une liaison améliorée à divers récepteurs de fc gamma Download PDF

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WO2023043125A1
WO2023043125A1 PCT/KR2022/013489 KR2022013489W WO2023043125A1 WO 2023043125 A1 WO2023043125 A1 WO 2023043125A1 KR 2022013489 W KR2022013489 W KR 2022013489W WO 2023043125 A1 WO2023043125 A1 WO 2023043125A1
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cancer
antibody
present
fragment
domain
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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

Definitions

  • the present invention relates to Fc variants with increased binding ability to various Fc gamma receptors, and relates to aglycosylated Fc variants with improved binding affinity to Fc ⁇ RIIa, Fc ⁇ RIIIa and Fc ⁇ RIIb.
  • 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 a receptor for immunoglobulins on cells that are differentially expressed by all immunocompetent cells (Fc receptor or FcR).
  • Fc receptor Fc receptor
  • 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.
  • 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 binding ability to various Fc gamma receptors (Fc ⁇ Rs).
  • an object of the present invention is to provide an aglycosylated antibody with improved binding ability to various Fc gamma receptors 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 binding ability to various Fc gamma receptors.
  • 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.
  • the present invention provides human antibody Fc domain variants with increased binding ability with various Fc gamma receptors.
  • the present invention provides an aglycosylated antibody having increased binding ability to various Fc gamma receptors 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 producing an aglycosylated antibody having increased binding ability to various Fc gamma receptors.
  • 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 human antibody Fc domain variants of the present invention have improved binding ability to Fc ⁇ RIIa and Fc ⁇ RIIIa, which are activating receptors, as well as Fc ⁇ RIIb, an inhibitory receptor among Fc gamma receptors. It can be used as an antibody drug useful for killing target cells, and since it is an aglycosylated variant, there is no problem of glycan heterogeneity of protein therapeutics, and it is easy to mass-produce inexpensively even in bacteria, There is an effect that makes it possible to manufacture biopharmaceuticals without problems.
  • Figure 2 shows the aglycosylated Fc variants aFc-S1 to 12 prepared by a combination of mutations and the aglycosylated Fc variants aFc34, aFc43, aFc46 and aFc67 selected in Example 1 for binding to Fc ⁇ RIIIa (A) and for Fc ⁇ RIIIa. This is the result of analyzing the selective binding force (B) by FACS.
  • Figure 3 is a diagram confirming by SDS-PAGE after expression and purification of trastuzumab Fc variants including aglycosylated Fc variants in Expi293F cells.
  • the present invention provides, in a wild type human antibody Fc domain, 239, 262, 264, 292, 299, 300, 350, 358, 382, 390, numbered according to the Kabat numbering system; 392, 396, 399, 401, 414 and 428 amino acids selected from the group consisting of amino acids at any one or more positions are substituted with a sequence different from the amino acid of the wild type, human antibody with increased binding affinity to Fc gamma receptor (Fc ⁇ R) It relates to Fc domain variants.
  • Fc ⁇ R Fc gamma receptor
  • the Fc domain variant of the present invention is selected from the group consisting of S239D, V262A, V264E, R292P, T299A, Y300L, T350A, L358P, E382V, N390S/N390G, K392E, P396L, D399G, D401N, K414R and M428L Any one or more amino acid substitutions may be included.
  • an Fc domain variant of the present invention may include amino acid substitutions of V264E, T299A and E382V.
  • the Fc domain variant of the present invention may be aFc46 comprising amino acid substitutions of S239D, V264E, R292P, T299A, Y300L, E382V, D399G, K414R and M428L.
  • the Fc domain variant of the present invention may be aFc67 comprising amino acid substitutions of V262A, V264E, T299A, T350A, E382V, N390S and P396L.
  • an Fc domain variant of the present invention comprises aFc34 comprising amino acid substitutions of V264E, T299A, T350A, L358P, E382V and N390G; or aFc43 comprising amino acid substitutions of V264E, R292P, T299A, Y300L, T350A, E382V, K392E, P396L and D401N.
  • 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: 11 and may be encoded by the nucleic acid molecule of SEQ ID NO: 17.
  • the Fc domain variant of the present invention may include any one selected from the group consisting of amino acid sequences of SEQ ID NOs: 12 to 16.
  • 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).
  • 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 variants in the present invention are S239D, V262A, V264E, R292P, T299A, Y300L, T350A, L358P, E382V, N390S/N390G, K392E, P396L, D399G, D401N, K414R and M428L (the above numbering is described in Kabat according to the EU index) to increase selective binding to Fc gamma receptors compared to wild-type antibody Fc domains (regions).
  • 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 is from the group consisting of a heavy chain constant region domain 2 (CH2) comprising any one selected from the group consisting of the amino acid sequences of SEQ ID NOs: 1 to 5 and the amino acid sequences of SEQ ID NOs: 6 to 10 It may include a heavy chain constant region domain 3 (C H 3) including any one selected.
  • CH2 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.
  • the antibody or fragment having immunological activity comprising the Fc domain variant of the present invention can increase the effector action, and the binding ability to the activating receptors Fc ⁇ RIIa and Fc ⁇ RIIIa as well as the inhibitory receptor Fc ⁇ RIIb is improved
  • the binding affinity to Fc ⁇ RIIb is improved, effects such as inducing apoptosis and T cell activation of Fc ⁇ RIIb binding mediation including ADCC (antibody dependent cellular cytotoxicity) and ADCP (Antibody-dependent cellular phagocytosis) (agonistic effect ) by inducing a complex immune response, the therapeutic efficiency of antibody drugs can be dramatically improved.
  • 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 present invention relates to a vector containing the nucleic acid molecule and a host cell containing the vector.
  • 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 18 to 22.
  • 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 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 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 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.
  • aFc-S1 to 12 (S1: V264E/T299A/E382V, S2: V264E/T299A/E382V/T350A, S3: V264E/T299A/E382V/Y300L, S4: V264E/T299A/E382V/R292P, S5: V264E/T299A/E382V/P396L, S6: V264E/T299A/E382V/T350A/Y300L, S7: V264E/T299A/E382V/T350A/R292P, S8: V294E/T E382V/T350A/P396L; S9: V264E/T299A/E382V/Y300L/R292P; S10: V264E/T299A/E382V/Y300L/P396L; T350
  • the 12 types of aglycosylated Fc variants did not show a high binding affinity to Fc ⁇ RIIIa compared to the aglycosylated Fc variants selected in Example 1, in particular, aFc64 and aFc67
  • the binding force for Fc ⁇ RIIIa was remarkably high (FIG. 2A).
  • the aFc46 variant and the aFc67 variant were analyzed to have a low A/I ratio because Fc ⁇ RIIIa binding ability was significantly reduced in a state masked with Fc ⁇ RIIb (FIG. 2B).
  • aFc46 variants and aFc67 variants were finally selected as aglycosylated Fc variants analyzed to show a low A / I ratio (Fc ⁇ RIIIa binding force / Fc ⁇ RIIb binding force) while maintaining excellent Fc ⁇ RIIIa binding force (Table 1).
  • trastuzumab Fc variants including the aglycosylated Fc variants selected in the above example
  • 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.
  • ELISA analysis was performed to confirm the binding ability of the aglycosylated trastuzumab Fc variants (aFc, aFc46 and aFc67) expressed and purified in Example 3 to Fc gamma receptors (Fc ⁇ Rs).
  • Fc ⁇ Rs-GST Fc ⁇ RIIa-131H-GST, Fc ⁇ RIIa-131R-GST, Fc ⁇ RIIb-GST and Fc ⁇ RIIIa-158V-GST
  • 50 ⁇ l each of Fc ⁇ Rs-GST diluted to 4 ⁇ g/ml in 0.05 M Na 2 CO 3 pH 9.6
  • the cells were blocked with 100 ⁇ l of 4% skim milk (GenomicBase, SKI400) (in PBS) at room temperature for 2 hours.
  • aglycosylated trastuzumab Fc variants (aFc, aFc46 and aFc67) serially diluted with 1% skim milk (in PBS) was dispensed into each well and incubated at room temperature for 1 reacted over time.
  • the antibody reaction was performed with 50 ⁇ l of HRP-Protein L (GenScript, M00098) at room temperature for 1 hour, respectively, and then washed.
  • trastuzumab variants including aglycosylated Fc variants (aFc46 and aFc67) to Fc ⁇ RIIa-131H, Fc ⁇ RIIa-131R, Fc ⁇ RIIb and Fc ⁇ RIIIa was similar to or higher than that of wild-type glycosylated trastuzumab, and in particular, aFc46 It was confirmed that trastuzumab into which the aglycosylated Fc variant was introduced had significantly increased binding affinity to Fc ⁇ RIIb, Fc ⁇ RIIIa-158V, and Fc ⁇ RIIa-131R compared to wild-type glycosylated trastuzumab (FIG. 4).
  • trastuzumab into which the aFc67 aglycosylated Fc variant was introduced
  • the binding affinity to Fc ⁇ RIIb was significantly higher than that of wild-type glycosylated trastuzumab and trastuzumab (trastuzumab-aFc46) into which an aglycosylated Fc variant of aFc46 was introduced, and also in Fc ⁇ RIIIa-131R. It showed increased binding ability than wild-type glycosylated trastuzumab (FIG. 4).

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Abstract

La présente invention concerne un variant de Fc aglycosylé présentant une capacité de liaison améliorée à divers variants de Fc-gamma, c'est-à-dire, FcγRⅡa, FcγRⅢa, et FcγRⅡb. Des variants du domaine Fc d'anticorps humain de la présente Invention ont une capacité de liaison améliorée non seulement à FcγRⅡa et FcγRⅢa qui sont des récepteurs d'activation, mais à FcγRⅡb qui est un récepteur Fc inhibiteur, parmi les récepteurs Fc-gamma, et en particulier, ont une capacité de liaison significativement accrue à FcγRⅡb, et peuvent donc être utilisés en tant que médicaments à base d'anticorps utiles pour l'apoptose de cellules cibles qui nécessitent un faible rapport A/I. En tant que variant aglycosylé, il n'a pas de problème avec l'hétérogénéité glycane d'un agent thérapeutique protéique, et une production en masse du variant n'est pas coûteuse et est facile même dans des bactéries, ce qui a pour effet de permettre la fabrication de produits biopharmaceutiques sans problèmes provoqués par des lignées cellulaires, des processus de culture et des processus de purification.
PCT/KR2022/013489 2021-09-17 2022-09-07 Variant de fc présentant une liaison améliorée à divers récepteurs de fc gamma WO2023043125A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099249A2 (fr) * 2003-05-02 2004-11-18 Xencor, Inc. Variants fc optimises et leurs procedes de generation
KR20180046179A (ko) * 2016-10-27 2018-05-08 국민대학교산학협력단 암 치료용 무당화 항체 Fc 영역
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

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004099249A2 (fr) * 2003-05-02 2004-11-18 Xencor, Inc. Variants fc optimises et leurs procedes de generation
KR20180046179A (ko) * 2016-10-27 2018-05-08 국민대학교산학협력단 암 치료용 무당화 항체 Fc 영역
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

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* Cited by examiner, † Cited by third party
Title
DATABASE PROTEIN ANONYMOUS : "Chain A, Ig gamma-1 chain C region", XP093048520, retrieved from NCBI *

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