WO2022005100A1 - Préparation d'anticorps anti-vegf à haute concentration et anticorps anti-vegf à utiliser dans celle-ci - Google Patents

Préparation d'anticorps anti-vegf à haute concentration et anticorps anti-vegf à utiliser dans celle-ci Download PDF

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WO2022005100A1
WO2022005100A1 PCT/KR2021/007939 KR2021007939W WO2022005100A1 WO 2022005100 A1 WO2022005100 A1 WO 2022005100A1 KR 2021007939 W KR2021007939 W KR 2021007939W WO 2022005100 A1 WO2022005100 A1 WO 2022005100A1
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seq
antibody
composition
variable region
heavy chain
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PCT/KR2021/007939
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English (en)
Korean (ko)
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김근수
전승철
안동현
김희정
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(주)메디톡스
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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
    • 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
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present disclosure relates to a high concentration antibody formulation, and more particularly, to a stable aqueous pharmaceutical composition containing a high concentration of an anti-VEGF antibody and an anti-VEGF antibody suitable for use in the composition.
  • VEGF Vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • hypoxia increases the differentiation of vascular endothelial cells and the permeability of retinal blood vessels. It is known to play an important role in the increase.
  • vascular endothelial growth factor is an important treatment in age-related macular degeneration, and several vascular endothelial growth factor antagonists are being used.
  • Representative examples include bevacizumab (Avastin®), ranibizumab (Ranibizumab, Lucentis®), and aflibercept (Aflibercept, Eylea®).
  • the concentration of therapeutic antibody in liquid pharmaceutical formulations varies greatly, for example, depending on the route of administration.
  • High concentration formulations may be desirable for intravitreal injection.
  • formulations with high antibody concentrations may have a short shelf life, and formulated antibodies may lose biological activity due to chemical and physical instability during storage. Aggregation, deamidation and oxidation are known to be the most common causes of antibody degradation. In particular, aggregation should be minimized or prevented, as it can potentially increase the patient's immune response and raise safety concerns.
  • International Patent Publication No. WO2013/063510 discloses an antibody, including bevacizumab, ranibizumab, and the like, an amino acid for stabilizing an antibody, and an additional amino acid selected from the group consisting of valine, serine, threonine, alanine and glycine; and additional amino acids selected from the group consisting of isoleucine, asparagine, glutamine and aspartic acid; and proline as an amino acid for reducing the viscosity of the formulation, a stable pharmaceutical formulation comprising an amino acid, a viscosity of less than 100 cP, and free of arginine and histidine.
  • 2020-0029374 discloses a liquid composition
  • a liquid composition comprising aflibercept, a stabilizer including at least one selected from the group consisting of polyols and sugars, and a surfactant, and having a pH of 4 to 8.
  • Korea Patent Publication No. 2017-0076781 discloses an aqueous solution comprising at least 50 mg/ml of an anti-VEGF antibody, Brolucizumab, sucrose or trehalose, citrate or histidine buffer, and polysorbate 80 as a surfactant.
  • a pharmaceutical composition is disclosed.
  • Another object of the present disclosure is to provide an anti-VEGF antibody suitable for use in the composition.
  • One aspect of the present disclosure provides a stable aqueous pharmaceutical composition
  • a stable aqueous pharmaceutical composition comprising a humanized antibody to vascular endothelial growth factor (VEGF) as an active ingredient at a concentration of 150 mg/ml or more.
  • VEGF vascular endothelial growth factor
  • Another aspect of the present disclosure provides an anti-VEGF antibody suitable for use in the composition, one or more of the following antibodies:
  • a stable aqueous pharmaceutical composition comprising a humanized antibody against vascular endothelial growth factor (VEGF) at a high concentration as an active ingredient.
  • VEGF vascular endothelial growth factor
  • aqueous pharmaceutical composition refers to a composition suitable for pharmaceutical use containing an aqueous carrier.
  • compositions suitable for pharmaceutical use may be sterile, homogeneous and/or isotonic.
  • Aqueous pharmaceutical composition means, for example, a liquid formulation or a lyophilized formulation to be reconstituted.
  • stable pharmaceutical composition refers to an agent that preserves stability, including physical stability.
  • analytical techniques are available in the art, such as visual inspection, SDS-PAGE, IEF, size exclusion chromatography, reversed phase liquid chromatography, ion exchange chromatography, capillary electrophoresis, light scattering, particle counting, turbidity, etc. It can be determined by the above method.
  • stability is assessed for aggregate formation.
  • a stable aqueous pharmaceutical composition is less than 5%, less than 4.5%, less than 4%, less than 3.5%, less than 3%, less than 2.5%, less than 2%, less than 1.5%, less than 1% as measured by gel filtration chromatography. It may be to form the following aggregates.
  • the concentration of anti-VEGF antibody in the aqueous pharmaceutical composition may be at least 150 mg/ml, for example between 150 and 300 mg/ml.
  • the aqueous pharmaceutical composition can be, for example, about 150 mg/ml, about 160 mg/ml, about 170 mg/ml, about 180 mg/ml, about 190 mg/ml, about 200 mg/ml, about 210 mg/ml, about 220 mg/ml, about 230 mg/ml, about 240 mg/ml, about 250 mg/ml, about 260 mg/ml, about 270 mg/ml, about 280 mg/ml, about 290 mg/ml or about 300 mg/ml of anti-VEGF antibody may be included.
  • the term “antibody” includes a whole antibody and any antigen-binding fragment or single chain thereof.
  • “Antibody” includes a glycoprotein comprising at least two heavy (H) chains and two light (L) chains or antigen-binding portions thereof interconnected by disulfide bonds.
  • Each heavy chain is composed of a heavy chain variable region (VH or HV) and a heavy chain constant region.
  • the heavy chain constant region consists of three regions, namely CH1, CH2 and CH3.
  • Each light chain consists of a light chain variable region (VL or LV) and a light chain constant region.
  • the light chain constant region consists of one region, namely CL.
  • VH and VL regions can be subdivided into framework regions (FRs) interspersed with more conserved regions, and hypervariable regions called complementarity determining regions (CDRs).
  • each of VH and VL is FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (for VH, HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4; for VL, LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4), consisting of 3 CDRs and 4 FRs arranged from amino terminus to carboxy terminus.
  • the variable regions of the heavy and light chains contain binding regions that interact with antigens.
  • the constant regions can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).
  • humanized antibody refers to a molecule wherein the antigen-binding portion of the molecule is substantially derived from an immunoglobulin from a non-human species, while the remaining immunoglobulin structure of the molecule is based on the structure and/or sequence of a human immunoglobulin.
  • the antigen-binding portion may comprise a complete variable region fused onto a constant region or may comprise only a complementarity determining region (CDR) grafted to an appropriate framework region within the variable region.
  • CDR complementarity determining region
  • the antigen-binding moiety may be wild-type or may be modified by one or more amino acid substitutions, eg, modified to more closely resemble human immunoglobulins.
  • Some types of humanized antibodies preserve all CDR sequences (eg, a humanized rabbit antibody containing all six CDRs from a rabbit antibody). Other types may differ in one or more CDRs from the original antibody.
  • the antibody comprises a heavy chain variable region having a heavy chain framework having one or more of Kabat numbering 13Q, 37I, 74S, 77T and 105Q as compared to human germ-line immunoglobulin; a light chain variable region having a light chain framework having at least one of 22T, 43A, 83F and 100Q; Or it may include a combination thereof.
  • the heavy chain framework may be one having one or more of 49A and 73D.
  • the antibody has a heavy chain framework of one or more of HFR1 of SEQ ID NO:39 or SEQ ID NO:53, HFR2 of SEQ ID NO:40 or SEQ ID NO:54, HFR3 of SEQ ID NO:41 or SEQ ID NO:55, and HFR4 of SEQ ID NO:42 heavy chain variable region; a light chain variable region having a light chain framework of at least one of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44 or SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46; Or it may include a combination thereof.
  • the antibody comprises a heavy chain variable region having one or more heavy chain CDRs of HCDR1 of SEQ ID NO: 47 or SEQ ID NO: 57, HCDR2 of SEQ ID NO: 48 or SEQ ID NO: 58, and HCDR3 of SEQ ID NO: 49 or SEQ ID NO: 59; a light chain variable region having at least one light chain CDR of LCDR1 of SEQ ID NO: 50 or SEQ ID NO: 60, LCDR2 of SEQ ID NO: 51 or SEQ ID NO: 61, and LCDR3 of SEQ ID NO: 52 or SEQ ID NO: 62; Or it may include a combination thereof.
  • the antibody comprises a heavy chain variable region of SEQ ID NO: 30; And it may include a light chain variable region of SEQ ID NO: 31.
  • the antibody comprises a heavy chain variable region of SEQ ID NO: 33; and a light chain variable region of SEQ ID NO: 34.
  • the antibody may have reduced or eliminated effector function.
  • the antibody may have reduced or eliminated binding ability to one or more of Fc ⁇ I, Fc ⁇ II, Fc ⁇ III, and FcRn.
  • the antibody may have one or more mutations among L234A, L235A, P331G, 1253A, and H310A.
  • the antibody may be one comprising the heavy chain of SEQ ID NO: 32 or SEQ ID NO: 38.
  • the antibody may have a binding affinity for VEGF of KD 100 pM or less.
  • the antibody has a KD for VEGF of 50 pM or less, 30 pM or less, 20 pM or less, 15 pM or less, 10 pM or less, 5 pM or less, 1-50 pM, 1-30 pM, 1-20 pM, 1 It may have a binding affinity of ⁇ 15 pM, 1 ⁇ 10 pM, 1 ⁇ 5 pM.
  • the antibody may have a thermal stability of Tm of 70 °C or higher.
  • Tm 71 °C or higher Tm 72 °C or higher, Tm 73 °C or higher, Tm 74 °C or higher, Tm 75 °C or higher, Tm 76 °C or higher, Tm 77 °C or higher, Tm 70-80 °C or higher, Tm 75- It may have a thermal stability of 80 °C, Tm 75 ⁇ 77 °C.
  • the aqueous pharmaceutical composition may include, in addition to the active ingredient, a buffer and a tonicity agent.
  • a buffer is, for example, maintaining a pH in the range of 5.0 to 7.5, for example, the aqueous pharmaceutical composition may have a pH of 5.0 to 7.5, 5.0 to 7.0, 5.0 to 6.5, 5.0 to 6.0, 5.0 to 5.5.
  • buffers are one or more of the conventional ones known in the art, and include salts of organic acids such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; tris, tromethamine hydrogloide or phosphate buffers, amino acid buffers such as histidine.
  • the buffer may be a histidine buffer.
  • the buffer may have a concentration in the range of 1-50 mM, 5-50 mM, 5-40 mM, 5-30 mM, 5-20 mM, 5-15 mM.
  • the isotonic agent may be a salt.
  • the salt may include at least one selected from the group consisting of NaCl, KCl, NaF, KBr, NaBr, Na2SO 4 , NaSCN, and K2SO 4 , and in particular may be NaCl.
  • the salt may have a concentration in the range of 50-200 mM, 75-175 mM, 100-150 mM.
  • the tonicity agent may be a sugar or sugar alcohol, or not a polyol. Examples of sugars or sugar alcohols, or polyols, may include trehalose, sucrose, mannitol, sorbitol, xylitol, glucose, glycerol, and the like.
  • the aqueous pharmaceutical composition may be free of stabilizers, surfactants, or both.
  • the surfactant may be a nonionic surfactant, for example, polysorbates (eg, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene ( 20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate);
  • the numerical value (20) of oxyethylene groups meaning the total number of oxyethylene groups (-(CH 2 CH 2 O)-)), poloxamer (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly (propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compounds (eg, polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C 1 -C 30 ), polyoxyethylene monolau
  • the surfactant may be polysorbates (eg, polysorbate 20).
  • the stabilizer may include one or more amino acids or salts thereof, for example, amino acids other than histidine or salts thereof.
  • the amino acid may include valine, serine, threonine, alanine, glycine, isoleucine, asparagine, glutamine, aspartic acid, and proline.
  • the aqueous pharmaceutical composition may be proline-free.
  • the aqueous pharmaceutical composition may comprise proline.
  • the aqueous pharmaceutical composition comprises or consists essentially of 5-50 mM histidine buffer and 50-200 mM NaCl with an anti-VEGF antibody. (consisting of) may be.
  • the aqueous pharmaceutical composition may have a viscosity of 20 cP or less at 25 °C.
  • the viscosity may be within the range of 1 to 20 cP, 2 to 18 cP, 2 to 15 cP, 3 to 15 cP, 2 to 10 cP, 3 to 10 cP, 5 to 15 cP, 5 to 10 cP at 25°C.
  • an aqueous pharmaceutical composition comprising an anti-VEGF antibody may be used to treat a variety of diseases or disorders.
  • a pharmaceutical composition comprising an anti-VEGF antibody is useful for treating an angiogenic disease, particularly an angiogenic ocular disease, in a subject or patient.
  • Angiogenic eye diseases that can be treated using the aqueous pharmaceutical composition according to the present disclosure include abnormal angiogenesis, choroidal neovascularization (CNV), retinal vascular permeability, retinal edema, diabetic retinopathy (particularly proliferative diabetic retinopathy).
  • ALD neovascular age-related macular degeneration
  • CNV associated with nAMD (neovascular age-related macular degeneration (AMD)
  • CRVO central retinal vein occlusion
  • diseases or disorders associated with ocular neovascularization including, but not limited to, posterior segment neovascularization.
  • the aqueous pharmaceutical composition may comprise additional active ingredients in addition to the anti-VEGF antibody.
  • Additional pharmacological agents may include, for example, other antibodies useful for the treatment of ocular diseases.
  • the aqueous pharmaceutical composition may be administered orally or parenterally.
  • parenteral administration eg, injection
  • intraocular administration eg, intravitreal administration
  • intravenous administration subcutaneous administration, intramuscular administration, intraperitoneal administration, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration Administration, or intratumoral administration, etc.
  • the aqueous pharmaceutical composition may be an ophthalmic composition comprising an anti-VEGF antibody, and in this case, may be an injection administered into the vitreous of the eye.
  • the pharmaceutical composition according to the present disclosure may be administered at an appropriate administration cycle and/or dose according to the judgment of an expert such as an ophthalmologist according to the patient's condition, including age, health status, disease severity, and the like. Since the composition may contain a high concentration of the anti-VEGF antibody, the amount of the active ingredient administered at one time may be increased, and thus may be administered less frequently, that is, at a longer cycle. For example, the composition may be administered at an administration cycle of once every 3 months or more, once every 4 months, once every 5 months, or once every 6 months or more. In addition, the composition can increase the amount of the active ingredient administered per volume, for example, it can be administered in a dose ranging from 2 to 30 mg.
  • the dose may be administered in a dose of about 2 mg, 5 mg, 7.5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 30 mg.
  • the dose may be administered in various volumes suitable for intraocular administration, such as 100 ⁇ l or less, 50-100 ⁇ l, 50 ⁇ l or less.
  • the aqueous pharmaceutical composition may be in the form of a solution or a lyophilized powder.
  • the composition may be contained in a vial, an ampoule, or a pre-filled syringe.
  • lyophilization is contemplated to provide an aqueous pharmaceutical composition of the present disclosure. Lyophilization techniques are known in the art. The lyophilisate must be reconstituted with an aqueous reconstitution agent prior to administration to the patient. This step makes it possible to redissolve the antibody and other components in the lyophilisate to provide a solution suitable for injection into the patient. The volume of aqueous medium used for reconstitution determines the concentration of antibody in the resulting pharmaceutical composition.
  • Reconstitution with a volume of reconstitution agent smaller than the volume prior to lyophilization provides a more concentrated composition than prior to lyophilization.
  • the reconstitution factor (volume of formulation after lyophilization: volume of formulation before lyophilization) may be 1:0.5 to 1:6.
  • the lyophilisate of the present invention can be reconstituted to provide an aqueous composition having an anti-VEGF antibody concentration of at least 150 mg/ml, for example 150-300 mg/ml, wherein the volume of the reconstituted agent is thus will be selected accordingly. If necessary, the reconstituted formulation can be diluted prior to administration to a patient when appropriate to deliver the intended dose.
  • Conventional reconstitution agents include sterile water or buffers, optionally containing a preservative.
  • the reconstitution agent may comprise an additional buffer (which may be the same or different from the buffer of the lyophilisate) or a buffer (e.g., WFI (water for injection) or physiological saline).
  • WFI water for injection
  • physiological saline e.g., physiological saline
  • excipients examples include antibacterial agents, antioxidants, antistatic agents, lipids such as phospholipids or fatty acids, steroids such as cholesterol, protein excipients such as serum albumin (human serum albumin), recombinant human albumin. , gelatin, casein, salt forming counterions such as sodium and the like.
  • Another aspect of the present disclosure provides a humanized anti-VEGF antibody suitable for use in an aqueous pharmaceutical composition containing a high concentration of anti-VEGF.
  • the antibody comprises a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 39, HFR2 of SEQ ID NO: 40, HFR3 of SEQ ID NO: 41, and HFR4 of SEQ ID NO: 42; And it may include a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 44, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46.
  • the antibody comprises a heavy chain variable region of SEQ ID NO: 30; And it may include a light chain variable region of SEQ ID NO: 31.
  • the antibody may include the heavy chain of SEQ ID NO: 32.
  • the antibody comprises a heavy chain variable region having a heavy chain framework consisting of HFR1 of SEQ ID NO: 53, HFR2 of SEQ ID NO: 54, HFR3 of SEQ ID NO: 55, and HFR4 of SEQ ID NO: 42; And it may include a light chain variable region having a light chain framework consisting of LFR1 of SEQ ID NO: 43, LFR2 of SEQ ID NO: 56, LFR3 of SEQ ID NO: 45, and LFR4 of SEQ ID NO: 46.
  • the antibody comprises a heavy chain variable region of SEQ ID NO: 33; and a light chain variable region of SEQ ID NO: 34.
  • the antibody may include the heavy chain of SEQ ID NO: 38.
  • the antibody comprises a heavy chain variable region of SEQ ID NO: 35, SEQ ID NO: 36 or SEQ ID NO: 37; and a light chain variable region of SEQ ID NO: 34.
  • Another aspect of the present disclosure is a disease or disorder mediated by VEGF comprising administering to a subject or patient in a therapeutically effective amount of the aqueous pharmaceutical composition or the humanized anti-VEGF antibody, e.g.
  • Methods are provided for treating eg angiogenic diseases, eg, neovascular ocular diseases, in particular age related macular degeneration.
  • terapéuticaally effective amount means an amount sufficient to produce a therapeutic effect when administered to a subject or patient in need of treatment.
  • treating or treatment means treating a disease or medical condition in a subject, such as a mammal, including a human, including:
  • the term "subject” or “patient” refers to human and non-human mammals including, but not limited to, primates, rabbits, pigs, horses, dogs, cats, sheep and cattle.
  • the subject or patient is a human.
  • a further aspect of the present disclosure provides a method of delivering an anti-VEGF antibody to a subject or human, comprising administering the aqueous pharmaceutical composition or the humanized anti-VEGF antibody to the subject or patient.
  • a further further aspect relates to said aqueous pharmaceutical composition or said humanized antibiotic for the manufacture of a medicament for the treatment of a disease or disorder mediated by VEGF, for example an angiogenic disease, for example an angiogenic eye disease, in particular age-related macular degeneration.
  • VEGF vascular endothelial growth factor
  • an angiogenic disease for example an angiogenic eye disease, in particular age-related macular degeneration.
  • VEGF antibody for example an angiogenic disease, for example an angiogenic eye disease, in particular age-related macular degeneration.
  • Still another aspect relates to the use of said aqueous pharmaceutical composition or said humanized anti-VEGF antibody to treat a disease or disorder mediated by VEGF, eg an angiogenic disease, eg an angiogenic eye disease, in particular age-related macular degeneration. It provides a method for preparing a medicament for VEGF.
  • a stable aqueous pharmaceutical composition having a low viscosity while containing a high concentration of the anti-VEGF antibody and an anti-VEGF antibody suitable for use in the composition are provided.
  • 1 is a diagram comparing the amino acid sequences of rabbit and humanized anti-VEGF antibody.
  • ranibizumab is a diagram comparing the amino acid sequences of ranibizumab and mutated ranibizumab.
  • Figure 3a is a graph showing the VEGF binding ability of the anti-VEGF antibody (Hz1, Hz2, RlgG1).
  • Figure 3b is a graph showing the VEGF binding ability of the anti-VEGF antibody (Lu007, F8, E9, H2, Lucentis-Ig).
  • Figure 4a is a graph showing the calcium inhibitory effect induced by VEGF in HUVEC cells of anti-VEGF antibodies (Hz1, Hz2, Aflibercept).
  • Figure 4b is a graph showing the calcium inhibitory effect induced by VEGF in HUVEC cells of the anti-VEGF antibody (Avastin, Lu007, Lucentis-Ig).
  • Figure 5a is a view showing the results of analysis of the aggregate content of the anti-VEGF antibody (Hz1).
  • Figure 5b is a view showing the results of analysis of the aggregate content of the anti-VEGF antibody (Hz2).
  • Figure 5c is a view showing the results of analysis of the aggregate content of the anti-VEGF antibody (Lu007).
  • FIG. 6 is a graph showing the results of measuring the viscosity at a high concentration of the anti-VEGF antibody.
  • Figure 7a is a diagram showing the binding ability of the anti-VEGF antibody to Fc ⁇ RI.
  • Figure 7b is a diagram showing the binding ability of the anti-VEGF antibody to Fc ⁇ RII, Fc ⁇ RIII.
  • FIG. 8 is a diagram showing the binding ability of the anti-VEGF antibody to FcRn.
  • variable region (VL and VH) genes of the rabbit antibody were amplified.
  • VL and VH variable region genes of the rabbit antibody were amplified.
  • VH heavy chain variable region
  • 5' specific primers and SEQ ID NOs: 13 and 14 of the rabbit heavy chain variable region of SEQ ID NOs: 9, 10, 11, and 12 using the cDNA as a template PCR was performed to selectively amplify the rabbit antibody heavy chain gene by using the 3'-specific primers described as a pair, respectively.
  • the 5' specific primers set forth in SEQ ID NOs: 15, 16, and 17 and the 3' specific primers set forth in SEQ ID NOs: 18, 19, 20, and 21 using pComb3XSS-scFv DNA as a template PCR was performed using each pair of red primers to selectively amplify the linker gene.
  • Extension PCR was performed using each VL, linker, and VH gene amplified in the PCR as a template.
  • 100 ng of the VL, linker, and VH genes amplified in the PCR 100 ng of the VL, linker, and VH genes amplified in the PCR, the primers shown in SEQ ID NO: 22 and the primers shown in SEQ ID NO: 23 were used. These PCR reaction conditions were repeated 30 times using Ex-Taq DNA polymerase (Takara) for 5 minutes at 95°C, pre-denaturing at 95°C, 50 seconds at 95°C, 50 seconds at 55°C, and 1 minute at 72°C. .
  • VL-Linker-VH (scFv) gene and pComb3XSS vector obtained above were each digested with restriction enzyme SfiI, purified, and the two cut DNA fragments were ligated and then the enzyme was inactivated. After eluting with distilled water using a XXPC DNA concentration column, the library DNA was transformed into E. coli ER2738 by electroporation.
  • Panning and screening were performed using the rabbit immune library phage prepared above. Bio-panning was performed three times with an Immunotube (ImmunoTMtube) coated with 5 ⁇ g/ml recombinant human VEGF fusion protein to select clones reactive to human VEGF. 96 phage clones were randomly selected from the colonies grown on the output plate, and their reactivity to human VEGF was tested through a phage enzyme immunoassay.
  • ImmunoTMtube ImmunoTMtube coated with 5 ⁇ g/ml recombinant human VEGF fusion protein
  • DNA was sequenced for the finally selected clones and classified into 6 scFv clones identified as having different complementarity determinant sequences. Afterwards, one final type of 2-19 clone was selected through an enzyme immunoassay to confirm VEGF binding ability, which is described in SEQ ID NO: 26 and SEQ ID NO: 27, which encode the amino acid sequence shown in SEQ ID NO: 24 and SEQ ID NO: 25 The nucleotide sequence was confirmed.
  • PCR assembly PCR
  • SEQ ID NO: 26 SEQ ID NO: 27 as templates.
  • TAKARA's Infusion kit for the animal expression vector pCEP6-WPRE the vector in which the antibody constant region is inserted
  • the humanized antibody light chain expression vector and heavy chain expression vector 2-19 were prepared in the same way. After that, it was confirmed through DNA sequencing.
  • SEQ ID NOs are as follows: Hz1 HV; SEQ ID NO: 28, Hz1 LV; SEQ ID NO: 29, Hz2 HV; SEQ ID NO: 30, Hz2 LV; SEQ ID NO: 31.
  • a phage library was prepared in order to replace the framework partial sequence of the heavy chain variable region of the Hz2 antibody in order to convert from the ranibizumab (Lucentis) Fab form to the whole antibody form, increase activity and improve hydrophilicity.
  • the framework portion of the heavy chain variable region based on the Hz2 sequence, Kabat Nos. A24, V27, G49, F69, L71, T73, S76, A78, K94 sequences V, I, A, F, L, T, S, A, A primer was prepared so as to be substituted with R, and library preparation and screening were performed by the method of Example 2.
  • variable region of Lu007 SEQ ID NO: 33, SEQ ID NO: 34
  • variable region of F8 SEQ ID NO: 35, SEQ ID NO: 34
  • variable region of E9 SEQ ID NO: 36, SEQ ID NO: 34
  • variable region of H2 SEQ ID NO: 37, SEQ ID NO: 34
  • Humanized antibody expression was performed using the ExpiCHO-S system (Thermo). The general culture of hamster uterine cells ExpiCHO-S was performed according to the manufacturer's instructions. After 12 days, the culture medium was recovered, centrifuged at 5000 rpm, 15 minutes, and room temperature, passed through a 0.22 ⁇ m Amicon Ultra-15 (Millipore) filter to remove cell debris, and then AKTA pure device and Mabselectsure (GE) column was used for purification.
  • ExpiCHO-S system Thermo. The general culture of hamster uterine cells ExpiCHO-S was performed according to the manufacturer's instructions. After 12 days, the culture medium was recovered, centrifuged at 5000 rpm, 15 minutes, and room temperature, passed through a 0.22 ⁇ m Amicon Ultra-15 (Millipore) filter to remove cell debris, and then AKTA pure device and Mabselectsure (GE) column was used for purification.
  • GE AKTA pure device and M
  • ELISA was performed to confirm the binding ability to VEGF.
  • 100 ⁇ l of hVEGF was added to an immunoplate at a concentration of 500 ng/ml and reacted at room temperature for 1 hour.
  • 200 ⁇ l of VEGF-coated wells were added and reacted at room temperature for 2 hours.
  • FIG. 3a it was confirmed that all of Hz1 and Hz2 and RIgG1, a rabbit antibody, bind to VEGF without any difference in VEGF binding ability.
  • FIG. 3b it was confirmed that the variant showed better binding ability than that of Lucentis-Ig or Lu007.
  • Lucentis-Ig SEQ ID NO: 65, SEQ ID NO: 66
  • an antibody constant region was inserted into Lucentis to prepare an entire IgG form.
  • Affinity determination for VEGF was performed as follows. Octet red96E was used, and human VEGF immobilized on the biosensor AR2G and RIgG1, Hz1, Hz2, Lu007, and Lucentis-Ig used as analytes interacted and the binding and dissociation values were compared and measured.
  • human VEGF recombinant human VEGF165 (Sino biological) was used, and VEGF was immobilized on the AR2G sensor chip by amine coupling. At this time, the amount of VEGF used was 10 ⁇ g/ml, and the degree of coating was confirmed with a wavelength of 5 nm.
  • the antibody concentrations were measured as 25, 12.5, 6.25, 3.12, 1.56, 0.78 nM. During the measurement, the rotation speed was set to 100, and the temperature was measured at 30 °C. The concentration of the sample was adjusted using a kinetic buffer (Molecular Device). The antibody solution of each concentration was used as a binding phase for 5 minutes, and then switched to a kinetic buffer to be a dissociated phase for 30 minutes. Measurements for each antibody are shown in Table 1. As a result, it was confirmed that RIgG1, Hz1, and Hz2 all showed excellent affinity of 5 pM or less, and Lu007 and Lucentis-Ig also showed excellent affinity at 12 and 14 pM levels.
  • HUVEC In order to compare the VEGF inhibitory efficacy of the antibody, the amount of calcium increase by VEGF in HUVEC cells was compared. Comparative tests were performed using samples Hz1, Hz2, aflibercept (Eylea), bevacizumab (Avastin), Lucentis-Ig, and Lu007.
  • HUVEC cells were seeded at 30,000 cells/well on a black 96-well plate (NUNC, 165305) coated with 0.1% collagen and cultured for one day, followed by calcium analysis. No purified material is added, or 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 or 20 nM is mixed with VEGF165 (R&D systems, 293-VE-500) 1.5 nM and incubated at room temperature for 30 minutes. Antigen binding was induced. To measure the amount of intracellular calcium, a calcium assay kit (BD, #640176) was used according to the manufacturer's instructions, and a calcium indicator dye was added to each well and incubated at 37°C for 1 hour.
  • NUNC black 96-well plate
  • the purified Hz1 and Hz2 were concentrated by an ultrafiltration membrane in a solution of 10 mM sodium phosphate, 150 mM NaCl, pH 6.2, and concentrated to a final concentration of 200 mg/ml. Comparing the time of the enrichment process, it was confirmed that Hz1 requires a longer period of enrichment than Hz2. After the completion of concentration, the presence or absence of the formation of aggregates was first confirmed by gel filtration chromatography. The conditions are as follows. A SEC-3 300A, 7.8 ⁇ 300 mm (Agilent) column was used in the Waters Alliance HPLC instrument, and 1 ⁇ PBS pH 7.4 buffer was used for the mobile phase, and the test was performed under the condition of a flow rate of 1.0 ml/min.
  • Viscosity measurements were performed as follows. An m-VROC microflow viscometer (RheoSense) was used, and after temperature equilibration at 25 °C, the viscosity was measured three times at each concentration, and the average viscosity (Viscosity, mPa ⁇ s) was calculated. After injecting a 0.3 ml sample into a 0.5 ml Hamilton syringe, the syringe was mounted on a chip enclosure and seated in a thermal jacket, and then the sample was measured. The m-VROC viscometer was equipped with a B05 chip made with a 50 micron channel and measured at multiple flow rates of approximately 20%, 40% and 60% of the maximum pressure of the chip. As a result, as shown in FIG. 6 , it was also confirmed that the viscosity of Hz2 was 12 cP, twice lower than that of Hz1.
  • Hz2 at high concentration has the advantage of low aggregation formation and low viscosity, which can be attributed to the difference in the sequence of the framework part between the two antibodies, and the sequence of Hz2 is responsible for the formation of aggregates and It was thought to contribute to lowering the viscosity.
  • thermo stability As an evaluation method of stability, evaluation of the heat denaturation intermediate temperature (Tm value) was performed, and in general, it is preferable that the heat denaturation intermediate temperature is high.
  • the thermal stability of each antibody is determined by measuring the fluorescence of the dye by temperature increase as the hydrophobic portion located inside the protein is exposed in the folded state of the protein and a dye having a binding force with the hydrophobic portion is introduced. ) was used. PTS assay was performed according to the manufacturer's method using a Protein Thermal Shift Dye Kit (Catalog No 4461146, Applied Biosystems).
  • RT-PCR C1000 thermal cycler equipped with a CFX96 optical reaction module, Bio-Rad
  • the ROX signal was monitored up to 95 °C with increasing temperature.
  • the melting point (melting temperature, Tm) of each antibody was determined based on the melting curve, and the melting point of the antibody is shown in Table 2.
  • variable region sequence heavy chain SEQ ID NO: 30 and light chain SEQ ID NO: 31 were used, and mutations of L234A, L235A, P331G were introduced to reduce Fc ⁇ receptor binding to the Fc portion, and mutations of I253A, H310A were introduced to inhibit FcRn binding.
  • An Hz2-F expression vector was prepared. The expression vector was prepared by the method of Example 3, the heavy chain using Hz2-F HC (SEQ ID NO: 32), and the light chain using Hz2 LV (SEQ ID NO: 31), and CL (SEQ ID NO: 64) of Example 5 Culture and purification were performed by the method. In the same manner, Lu007-F was prepared using heavy chain (SEQ ID NO: 38), light chain LV (SEQ ID NO: 34), and light chain CL (SEQ ID NO: 64).
  • Binding evaluation to Fc ⁇ RI, II, III was performed as follows. Octet red96E was used, and 100 mM Fc ⁇ RII (R&D system, 1257-FC), 100mM Fc ⁇ RII (R&D system, 1330-CD), 100mM Fc ⁇ RIII (R&D system, 4325-FC) for the anti-penta his biosensor, respectively was loaded and a signal of about 5 nm was confirmed.
  • Hz2 antibody (heavy chain variable region (SEQ ID NO: 30), and light chain variable region (SEQ ID NO: 31)) against Fc ⁇ RI, Fc ⁇ RII, Fc ⁇ RIII and Hz2-F antibody (heavy chain (SEQ ID NO: 32), light chain variable region (SEQ ID NO: 31)) , and the light chain constant region (SEQ ID NO: 64) in order to confirm the reactivity of the antibody concentration of 100 nM as an analyte, the binding force was measured. During the measurement, the rpm was set to 100, and the temperature was measured at 30 °C. The sample was diluted using a kinetic buffer (Molecular Device).
  • a kinetic buffer (Molecular Device).
  • the antibody solution of each concentration was used as a bound phase for 2 minutes, and then switched to a kinetic buffer to be a dissociated phase for 2 minutes.
  • the binding force between the Fc ⁇ I receptor and the antibody is shown in FIG. 7A, and the binding force between the Fc ⁇ II and Fc ⁇ III receptors and the antibody is shown in FIG. 7B.
  • Hz2 seems to normally bind to Fc ⁇ receptors, and in the case of Hz2-F and Lu007-F, which weakened binding to Fc ⁇ receptors, it was confirmed that binding was weak as expected.
  • FcRn binding evaluation to FcRn was performed as follows. Octet red96E was used, and a signal of about 5 nm was confirmed by loading an anti-Fab 2nd generation (FAB2G) biosensor with 10 ⁇ g/ml of Hz2 and Hz2-F antibodies.
  • FcRn binding buffer 100 mM sodium phosphate, 150 mM NaCl, 0.05% Tween20, pH 6.0
  • Formulation 1 Form 2
  • Form 3 Histidine 10 mM 10 mM 10 mM NaCl 135 mM 135 mM 135 mM Proline - 200 mM 200 mM pH 5.5 5.0 5.5
  • concentration was performed using an ultrafiltration membrane using Formulations 1 and 2 at concentrations of 150, 180, and 200 mg/ml.
  • concentration was performed in the same manner as above at concentrations of 150, 180, 200, 230, and 300 mg/ml.
  • concentration was performed to 150, 180, and 200 mg/ml using Formulation 3, and in the case of Lu007, 150, 180, 200, 230, and 300 mg/ml were obtained using Formulations 2 and 3 concentrated to a concentration.
  • the viscosity measurement was performed as follows.
  • An m-VROC microflow viscometer (RheoSense) was used, and after temperature equilibrium at 25 °C, the viscosity was measured three times at each concentration, and the average viscosity (Viscosity, mPa ⁇ s) was calculated.
  • the syringe was mounted on a chip enclosure, and the sample was measured after being seated in a thermal jacket.
  • the m-VROC viscometer was equipped with a B05 chip fabricated with a 50 micron channel and measured at multiple flow rates approximately 20%, 40% and 60% of the maximum pressure of the chip. The results are shown in Table 4 and Table 5.
  • Viscosity confirmation test after high concentration for Lucentis-Ig and Lu007-F Sample name Antibody concentration (mg/ml) Viscosity of Formulation 2 (cP) Viscosity of Formulation 3 (cP) Lucentis-Ig 150 - 4.6 180 - 7.8 200 - 11.0 Lu007-F 150 3.0 3.3 180 4.4 4.2 200 5.3 7.8 230 6.9 9.0

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Abstract

L'invention concerne une composition pharmaceutique aqueuse stable qui contient une concentration élevée d'anticorps anti-VEGF, et des anticorps anti-VEGF à utiliser dans la composition.
PCT/KR2021/007939 2020-06-29 2021-06-24 Préparation d'anticorps anti-vegf à haute concentration et anticorps anti-vegf à utiliser dans celle-ci WO2022005100A1 (fr)

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KR20170076781A (ko) * 2014-11-07 2017-07-04 노파르티스 아게 고농도의 항-vegf 항체를 포함하는 안정한 단백질 용액 제제
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KR20170076781A (ko) * 2014-11-07 2017-07-04 노파르티스 아게 고농도의 항-vegf 항체를 포함하는 안정한 단백질 용액 제제
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