WO2022135441A1 - 抗il-4r抗体或其抗原结合片段的复合物及医药用途 - Google Patents

抗il-4r抗体或其抗原结合片段的复合物及医药用途 Download PDF

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WO2022135441A1
WO2022135441A1 PCT/CN2021/140310 CN2021140310W WO2022135441A1 WO 2022135441 A1 WO2022135441 A1 WO 2022135441A1 CN 2021140310 W CN2021140310 W CN 2021140310W WO 2022135441 A1 WO2022135441 A1 WO 2022135441A1
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seq
antibody
amino acid
variable region
set forth
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PCT/CN2021/140310
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English (en)
French (fr)
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王欢
林�源
唐昱澄
柯可
林侃
廖成
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江苏恒瑞医药股份有限公司
上海盛迪医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海盛迪医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to US18/268,104 priority Critical patent/US20240075158A1/en
Priority to CA3202875A priority patent/CA3202875A1/en
Priority to CN202180079299.2A priority patent/CN116583539A/zh
Priority to MX2023007178A priority patent/MX2023007178A/es
Priority to EP21909437.2A priority patent/EP4269447A1/en
Priority to JP2023561420A priority patent/JP2023554557A/ja
Publication of WO2022135441A1 publication Critical patent/WO2022135441A1/zh

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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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Definitions

  • the present application relates to a complex of an anti-IL-4R antibody or an antigen-binding fragment thereof, and its use as an anticancer drug.
  • GBM Glioblastoma
  • IL-4R is an interleukin-4 receptor expressed on glioblastoma tumor cells and infiltrating immunosuppressive cells (eg, tumor-associated macrophages (TAM), myeloid-derived immunosuppressive cells (MDSC), etc.).
  • immunosuppressive cells eg, tumor-associated macrophages (TAM), myeloid-derived immunosuppressive cells (MDSC), etc.
  • TAM tumor-associated macrophages
  • MDSC myeloid-derived immunosuppressive cells
  • MDNA55 developed by Medicenna is a fusion protein of IL-4 and bacterial toxin Pseudomonas exotoxin A (PE38KDEL), which has shown positive efficacy in clinical development experiments (see WO9527732, Biochem.J. (1995) 307, 29 -37, CANCER RESEARCH 55, 3357-3363, August 1, 1995).
  • Antibody-drug conjugates aim to combine the selectivity of monoclonal antibodies (mAbs) with the cytotoxic potential of chemotherapeutic drugs.
  • Antibody-drug conjugates consist of three main components: “antibody”, “linker” and “drug”. Compared with traditional fully or partially humanized antibodies or antibody fragments, antibody-drug conjugates have theoretically higher efficacy because they can release highly active cytotoxins in tumor tissues; compared with fusion proteins, they have higher efficacy. tolerance or lower side effects.
  • Antibodies that lack high specificity and cross-react with other antigens may not perform as intended, for example, causing off-target toxicity by interacting with healthy tissue, or being cleared prematurely by the body before reaching the tumor site (see: “Introduction to Antibody” –Drug Conjugates(ADCs)”, Ilona Pysz, Paul J.M. Jackson and David E. Thurston, CHAPTER 1: Introduction to Antibody–Drug Conjugates (ADCs), in Cytotoxic Payloads for Antibody–Drug Conjugates, 2019).
  • IL-4R-directed antibody-drug conjugates are rarely reported explicitly, such as WO2015188934, WO2014124227, WO2018217227, etc., all only generally mention that antibodies in antibody-drug conjugates can selectively target IL-4R, but none of them discloses Specific anti-IL-4R antibody-drug conjugates.
  • the present disclosure utilizes a high-affinity anti-IL-4R antibody to prepare a second-generation immunotoxin drug for targeted delivery of drugs, so as to achieve the purpose of effectively killing cancer cells, and is used to meet the needs of cancer treatment or delaying cancer progression.
  • the present disclosure relates to complexes of anti-IL-4R antibodies or antigen-binding fragments thereof, preparation methods and medical uses.
  • the present disclosure provides a complex of anti-IL-4R antibodies or antigen-binding fragments thereof, comprising:
  • An anti-IL-4R antibody or antigen-binding fragment thereof and one or more toxin molecules is provided.
  • the anti-IL-4R antibody or antigen-binding fragment thereof in the complex comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises:
  • the antibody light chain variable region comprises:
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
  • the anti-IL-4R antibody or antigen-binding fragment thereof is covalently or non-covalently linked to the toxin.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one of the following (I) to (IV):
  • a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively;
  • a light chain variable region comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:39 and SEQ ID NO:8, respectively.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region contains:
  • the light chain variable region comprises:
  • hu25G7-A LCVR (hu25G7-A light chain variable region)
  • the anti-IL-4R antibody or antigen-binding fragment in the anti-IL-4R antibody or antigen-binding fragment:
  • the heavy chain variable region is shown in the sequence SEQ ID NO: 1, and the light chain variable region is shown in the sequence SEQ ID NO: 2; or
  • the heavy chain variable region sequence is shown in SEQ ID NO: 9, and the light chain variable region sequence is shown in SEQ ID NO: 10; or
  • the heavy chain variable region sequence is shown in SEQ ID NO: 43, and the light chain variable region sequence is shown in SEQ ID NO: 37; or
  • the heavy chain variable region sequence is shown in SEQ ID NO:43, and the light chain variable region sequence is shown in SEQ ID NO:41; or
  • the heavy chain variable region sequence is shown in SEQ ID NO:47, and the light chain variable region sequence is shown in SEQ ID NO:48.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region contains:
  • the light chain variable region comprises:
  • the heavy chain variable region is set forth in one of the sequences of SEQ ID NOs: 25-27, and the light chain variable region is set forth in one of the sequences of SEQ ID NOs: 28-30; or
  • the heavy chain variable region sequence is set forth in one of SEQ ID NOs: 31-33, and the light chain variable region sequence is set forth in one of SEQ ID NOs: 34-36.
  • amino acid at position 44 (VH-44) of the heavy chain variable region and position 100 (VL-100) of the light chain variable region are optionally mutated to cysteine.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein the heavy chain comprises:
  • the heavy chain sequence is set forth in SEQ ID NO: 17, and the light chain sequence is set forth in SEQ ID NO: 18; or
  • the heavy chain sequence is set forth in SEQ ID NO: 19, and the light chain sequence is set forth in SEQ ID NO: 20; or
  • the heavy chain sequence is set forth in SEQ ID NO:44, and the light chain sequence is set forth in SEQ ID NO:45; or
  • the heavy chain sequence is set forth in SEQ ID NO:44, and the light chain sequence is set forth in SEQ ID NO:46.
  • the anti-IL-4R antibody or antigen-binding fragment is a murine antibody, a chimeric antibody, a fully human antibody, a humanized antibody, or a fragment thereof. In some specific embodiments, the anti-IL-4R antibody or antigen-binding fragment is humanized.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from a human germline light chain template IGKV3-11*01 (SEQ ID NO: 22 for antibody 25G7) or a backmutated sequence that is at least 95% identical to it.
  • the back mutation is selected from one or more of 46P, 47W, 71Y.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from a human germline heavy chain template IGHV3-48*01 (SEQ ID NO: 21 for antibody 25G7) or a backmutated sequence that is at least 95% identical to it.
  • the back mutation is selected from one or more of 94A, 67S, 93T.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from a human germline light chain template IGKV2D-29*01 (SEQ ID NO: 24 for antibody 7B10) or a backmutated sequence that is at least 95% identical to it.
  • the back mutation is selected from 4L and/or 58I.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a FR region sequence derived from a human germline heavy chain template IGHV1-2*02 (SEQ ID NO: 23 for antibody 7B10) or a backmutated sequence that is at least 95% identical to it.
  • the back mutation is selected from one or more of 69L, 71I, 73K, 94K.
  • the anti-IL-4R antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from human IgGl, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the heavy chain constant region of human IgG1 or a variant thereof is included. In some embodiments, the anti-IL-4R antibody or antigen-binding fragment thereof comprises the constant region of a human kappa, lambda chain or variant thereof.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 17 or has at least 85% sequence identity thereto
  • the light chain sequence is set forth in SEQ ID NO: 18 or has at least 85% sequence identity thereto.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 19 or has at least 85% sequence identity thereto
  • the light chain sequence is set forth in SEQ ID NO: 20 or has at least 85% sequence identity thereto.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity thereto
  • the light chain sequence is set forth in SEQ ID NO: 45 or has at least 85% sequence identity thereto.
  • the anti-IL-4R antibody or antigen-binding fragment thereof wherein the antibody is a humanized antibody
  • the heavy chain sequence is as shown in SEQ ID NO: 44 or has at least 85% sequence identity thereto
  • the light chain sequence is set forth in SEQ ID NO: 46 or has at least 85% sequence identity thereto.
  • an isolated anti-IL-4R antibody or antigen-binding fragment thereof characterized by competing with any of the anti-IL-4R antibodies or antigen-binding fragments thereof described above for binding to human IL-4R or its gauge.
  • a bispecific or multispecific antibody comprising the light chain variable region and/or heavy chain variable region of any of the anti-IL-4R antibodies or antigen-binding fragments thereof as described above Area.
  • a single chain antibody comprising the light chain variable region and/or the heavy chain variable region of any of the anti-IL-4R antibodies or antigen-binding fragments thereof as described above.
  • the humanized anti-IL-4R antibody, or antigen-binding fragment thereof further comprises a heavy chain constant region of human IgGl, IgG2, IgG3, or IgG4, or a variant thereof.
  • human IgG2 or IgG4 heavy chain constant regions are included since IgG2 or IgG4 are not ADCC toxic.
  • IgG1 without ADCC antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity
  • the variant comprises a heavy chain constant region mutation with reduced or absent ADCC effector function, such as, but not limited to, 297A, 234A, 235A of IgGl.
  • the anti-IL-4R antibody or antigen-binding fragment thereof may be an antibody variant having 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes, and/or 1 to 10 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid changes in the heavy chain.
  • the aforementioned variants have the same or similar biological function or effect as the parental anti-IL-4R antibody or fragment thereof.
  • anti-IL-4R antibodies or antigen-binding fragments thereof may be known, such as anti-IL-4R antibodies as described in, eg, WO2010053751, WO2001092340, WO2008054606, WO2014031610, WO2020038454 (each of which is incorporated herein by reference). 4R antibody or antigen-binding fragment thereof.
  • anti-IL-4R antibodies or antigen-binding fragments thereof include, but are not limited to, Dupixent, PRS-060, AK-120, 63 IgG1, CBP201, AMG-317, or antigen-binding fragments thereof.
  • the anti-IL-4R antibody or antigen-binding fragment thereof is an anti-human IL-4R antibody or antigen-binding fragment thereof.
  • antigen binding fragments include, but are not limited to, Fab, Fab', Fv, F(ab')2, linear antibody, scFv (single chain Fv antibody), tandem di-scFv, tandem tri-scFv, double chain diabody, triabody, tetrabody, sdAb (single domain antibody or nanobody), sdFv, peptibody, domain antibody, multispecific antibody (e.g. bispecific antibody, trispecific or tetraspecific), dsFv (disulfide stabilized Fv), ScdsFv (disulfide stabilized single chain Fv antibody).
  • the anti-IL-4R antibody or antigen-binding fragment introduces mutations that stabilize the structure of the antibody, eg, mutation of some amino acid residues to cysteine residues.
  • the mutation occurs in the framework region; in some specific embodiments, the mutation occurs in the heavy chain variable region and/or the light chain variable region; in some specific embodiments, the Mutations occur in the heavy or light chain variable and framework regions.
  • amino acid residues located at one or more of the following positions are mutated to cysteine residues: VL100 and VH44; VL101 and VH44; VL34 and VH100; VL43 and VH91; VL43 and VH103 ; VL49 and H100; VL87 and VH45; VL91 and VH98; VL91 and VH99; VL96 and VH47; VL98 and VH45.
  • amino acid residues at positions selected from one or more of the following are mutated to cysteine residues: VL100 and VH44; VL43 and VH91; VL43 and VH103; VL98 and VH45.
  • amino acids at positions VH44 and VL100 are mutated to cysteine.
  • amino acid sequences in the anti-IL-4R antibodies or antigen-binding proteins thereof of the present disclosure are encoded according to Kabat.
  • the anti-IL-4R antibody or antigen-binding fragment is a scFv or ScdsFv, and the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linking peptide L2.
  • the linking peptide L2 is greater than 12 amino acid residues in length and is rich in small, non-polar amino acid residues, polar amino acid residues, and/or hydrophilic amino acid residues, eg, Glycine, serine and threonine.
  • the linking peptide L2 may be selected from a variety of linkers known in the art, including "GS” linkers, eg, (GxS)n, (SxG)n, (GGGGS)n, (G)n, where x is an integer from 1-6 and n is an integer from 1-30.
  • GS linkers known in the art, including "GS” linkers, eg, (GxS)n, (SxG)n, (GGGGS)n, (G)n, where x is an integer from 1-6 and n is an integer from 1-30.
  • linker peptide L2 include GKSSGSGSESKS (SEQ ID NO: 54), EGKSSGSGSESKEF (SEQ ID NO: 55), GTSGSGKSSEGKG (SEQ ID NO: 56), GTSGSGKSSEGS GSTKG (SEQ ID NO: 57), GSTSGSGKPGSGEGSTKG (SEQ ID NO:58), SRSSG (SEQ ID NO:59) and SGSSC (SEQ ID NO:60).
  • the linker peptide L2 consists of a repeating GGGGS amino acid sequence (SEQ ID NO: 61) or a variant thereof, such as (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5, or More, eg n is 2, 3, 4, eg n is 3.
  • the ScFv is constructed in the form of VH-L2-VL, meaning that L2 is linked to the C-terminus of VH and to the N-terminus of VL;
  • the ScFv is constructed in the form of VL-L2-VH, meaning that L2 is attached to the C-terminus of VL and to the N-terminus of VH;
  • the ScdsFv is constructed in the form of VH-L2-VL, meaning that L2 is linked to the C-terminus of VH and to the N-terminus of VL;
  • the ScdsFv is constructed as VL-L2-VH, meaning that L2 is linked to the C-terminus of VL and to the N-terminus of VH.
  • the toxin is selected from bacterial toxins, animal toxins, or plant toxins.
  • toxins include truncated toxins or toxin variants comprising amino acid deletions, insertions, substitutions, or combinations thereof.
  • the toxin includes a pore-forming toxin.
  • the pore-forming toxin comprises Aeromonas hydrophila aerolysin or proaerolysin.
  • the toxin includes bouganin, ricin, pseudomonas exotoxin (PE), cholera toxin, or diphtheria toxin.
  • toxins include, but are not limited to, cytotoxic fragments or cytotoxic variants of Pseudomonas exotoxin.
  • the toxin includes PE38KDEL as set forth in SEQ ID NO: 49, but similarly functional variants of PE38DKEL, PE38RDEL and PE38KNEL can also be used.
  • the complex of the anti-IL-4R antibody or antigen-binding fragment thereof of the present disclosure optionally further comprises a linker (linker) L1.
  • Linker L1 connects the toxin and the anti-IL-4R antibody or antigen-binding fragment thereof.
  • a suitable linker L1 generally allows each component of the present disclosure to fold in a three-dimensional structure that closely resembles the structure formed by the components in the absence of any linkers or other components.
  • the linker L1 is extracellularly stable such that the ADC remains intact when present in the extracellular environment, but can be cleaved when internalized in cells such as cancer cells.
  • suitable linkers L1 can include, for example, protease-sensitive, ambient redox-potential-sensitive, pH-sensitive, acid-cleavable, photo-cleavable, and/or heat-sensitive linkers.
  • linker L1 can be non-proteinaceous, eg, a chemical linker.
  • non-proteinaceous chemical linkers include, but are not limited to: N-succinimidyl(4-iodoacetyl)-aminobenzoate, S-(N-succinimidyl)thioacetate ( SATA), N-succinimidyl-oxycarbonyl-cu-methyl-a-(2-pyridyldithio)toluene (SMPT), N-succinimidyl 4-(2-pyridyl) dithio)-valerate (SPP), succinimidyl 4-(N-maleimidomethyl)cyclohexanecarboxylate (SMCC or MCC), sulfosuccinimidyl (4-Iodoacetyl)-aminobenzoate, 4-succinimidyl-oxycarbonyl- ⁇ -(2-pyridyldi
  • SATA
  • the complex formed by the anti-IL-4R antibody or antigen-binding fragment thereof and the toxin is in the form of a fusion protein.
  • linker L1 is a proteinaceous linker, including but not limited to 1 or more amino acids or polypeptides.
  • Linker L1 typically comprises about 2-50 amino acid residues, eg, about 5-30 amino acid residues.
  • proteinaceous linkers contain most amino acid residues with polar, uncharged and/or charged residues, eg, threonine, proline, glutamine, glycine, and alanine Wait.
  • Non-limiting examples of proteinaceous linkers include alanine-serine-glycine-glycine-proline-glutamic acid (ASGGPE) (SEQ ID NO: 50), valine-methionine (VM), Alanine-Methionine (AM), AM(G2-4S)xAM, where G is glycine, S is serine, and x is an integer from 1-10.
  • ASGGPE alanine-serine-glycine-glycine-proline-glutamic acid
  • VM valine-methionine
  • AM Alanine-Methionine
  • AM(G2-4S)xAM AM(G2-4S)xAM
  • linker L1 is selected from the following peptides listed in standard one-letter codes: ASGCGPE (SEQ ID NO:62), ASGCCGPE (SEQ ID NO:63), ASGCGSCPE (SEQ ID NO:64), ASCGTTGCPE ( SEQ ID NO: 65), KASGKKYGCKKGPE (SEQ ID NO: 66), KGGGCAGGPE (SEQ ID NO: 67).
  • the anti-IL-4R antibody or antigen-binding fragment is linked or fused to each other via linker L1 to the toxin.
  • the C-terminus of the anti-IL-4R antibody or antigen-binding fragment is linked or fused to linker L1.
  • the N-terminus of the anti-IL-4R antibody or antigen-binding fragment is linked or fused to linker L1.
  • the anti-IL-4R antibody or antigen-binding fragment is attached to or fused to the same or different linker L1 at the C-terminus and N-terminus, respectively.
  • the toxin is a Pseudomonas exotoxin, a truncated fragment or a toxin variant of a Pseudomonas exotoxin.
  • the toxin is a truncated fragment PE38 of Pseudomonas exotoxin.
  • the toxin is PE38KDEL set forth in SEQ ID NO: 49, although similarly functional variants of PE38DKEL, PE38RDEL and PE38KNEL can also be used.
  • L1 is a proteinaceous linker.
  • L1 is selected from ASGGPE (SEQ ID NO:50), ASGCCGPE (SEQ ID NO:63), ASGCGSCPE (SEQ ID NO:64), ASCGTTGCPE (SEQ ID NO:65), KASGKKYGCKKGPE (SEQ ID NO:65) : 66), KGGGCAGGPE (SEQ ID NO: 67), AM(G 2-4 S) x AM, where x is an integer from 1-10.
  • the ScFv specifically binds IL-4R, wherein the VH and VL are linked by a linker peptide L2, and the VH and VL have introduced mutations in the framework regions that stabilize the antibody structure.
  • the amino acid at position VH-44, VL-100 is mutated to cysteine.
  • the ScFv is constructed in the form of VH-L2-VL or VL-L2-VH.
  • L2 consists of repeated GGGGS amino acid sequences or variants thereof, eg (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5 or more, eg n is 2, 3, 4, eg n is 3.
  • the VH in the above ScFv comprises:
  • VL contains:
  • LCDR1, LCDR2 and LCDR3 having amino acid sequences as shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
  • the above-mentioned ScFv comprises any one selected from the following (I) to (IV):
  • VH comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively;
  • VL comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively;
  • VH comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13, respectively;
  • VL comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively;
  • VL comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO: 38, SEQ ID NO: 7 and SEQ ID NO: 40, respectively;
  • VH comprising HCDR1, HCDR2 and HCDR3 whose amino acid sequences are set forth in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively;
  • VL comprising LCDR1, LCDR2 and LCDR3 whose amino acid sequences are shown in SEQ ID NO:42, SEQ ID NO:39 and SEQ ID NO:8, respectively.
  • VH contains:
  • VL contains:
  • VH and VL introduce mutations in the framework regions that stabilize the antibody structure, in some embodiments, amino acids at positions VH-44 and VL-100 are mutated to cysteine.
  • the VH sequence in the ScFv is shown in SEQ ID NO: 1, and the VL sequence is shown in SEQ ID NO: 2; or
  • VH sequence is shown in SEQ ID NO: 9 and the VL sequence is shown in SEQ ID NO: 10; or
  • VH sequence is set forth in SEQ ID NO:43 and the VL sequence is set forth in SEQ ID NO:37; or
  • VH sequence is shown in SEQ ID NO: 43
  • VL sequence is shown in SEQ ID NO: 41
  • VH and VL introduce mutations in the framework regions that stabilize the antibody structure.
  • the amino acids at positions VH-44 and VL-100 are mutated to cysteine.
  • the VH sequence is shown in SEQ ID NO:47
  • the VL sequence is shown in SEQ ID NO:48.
  • the ScFv wherein:
  • VH contains:
  • VL contains:
  • VH and VL introduce mutations in the framework regions that stabilize the antibody structure.
  • the amino acids at positions VH-44 and VL-100 are mutated to cysteine.
  • VH is set forth in one of the sequences of SEQ ID NOs: 25-27
  • VL is set forth in one of the sequences of SEQ ID NOs: 28-30, and the amino acid break at positions VH-44 and VL-100 becomes cysteine; or
  • VH sequence is set forth in one of SEQ ID NOs: 31-33
  • VL sequence is set forth in one of SEQ ID NOs: 34-36, with the amino acids at positions VH-44 and VL-100 mutated to cysteine.
  • VH and VL introduce mutations in the framework regions that stabilize the antibody structure.
  • the amino acids at positions VH-44 and VL-100 are mutated to cysteine.
  • the ScFv comprises a heavy chain and a light chain, wherein the heavy chain comprises:
  • VH and VL introduce mutations in the framework regions that stabilize the antibody structure.
  • the amino acids at positions VH-44 and VL-100 are mutated to cysteine.
  • the heavy chain variable region VH sequence is set forth in SEQ ID NO: 17, and the light chain variable region VL sequence is set forth in SEQ ID NO: 18; or
  • VH sequence is set forth in SEQ ID NO: 19, and the VL sequence is set forth in SEQ ID NO: 20, wherein the amino acids at positions VH-44 and VL-100 are mutated to cysteine; or
  • VH sequence is set forth in SEQ ID NO:44
  • VL sequence is set forth in SEQ ID NO:45, wherein the amino acids at positions VH-44 and VL-100 are mutated to cysteine; or
  • VH sequence is set forth in SEQ ID NO:44
  • VL sequence is set forth in SEQ ID NO:46, wherein the amino acids at positions VH-44 and VL-100 are mutated to cysteine.
  • the complex of anti-IL-4R antibody or antigen-binding fragment thereof has the sequence set forth in SEQ ID NO:52 or SEQ ID NO:53.
  • the present disclosure also provides a complex of an anti-IL-4R antibody or an antigen-binding fragment thereof, comprising: an anti-IL-4R antibody or an antigen-binding fragment thereof and one or more toxins; wherein the toxins are selected from pore-forming Toxin, Aeromonas hydrophila aerolysin, Aeromonas lysinogen, bujanin, ricin, Pseudomonas exotoxin, cholera toxin or diphtheria toxin; preferably, the toxin is selected from PE - LR, PE-LO10R456A, PE-T20, PE-T20-KDEL, PE4E, PE40, PE38, PE24, PE25, PE38QQR, PE35, PE38KDEL, PE38DKEL, PE38RDEL, PE38KNEL; more preferably, the toxin is as in SEQ ID NO: PE38KDEL shown in 49.
  • the toxins are selected from pore-forming Toxin
  • the present disclosure provides a polynucleotide encoding a complex of any one of the anti-IL-4R antibodies or antigen-binding fragments thereof described in the present disclosure.
  • the polynucleotide is DNA or RNA.
  • the present disclosure provides a vector containing the above-mentioned polynucleotide, which is a eukaryotic expression vector, a prokaryotic expression vector or a viral vector.
  • the present disclosure provides a host cell comprising the vector as described above, the host cell being selected from prokaryotic cells or eukaryotic cells.
  • the prokaryotic cells are selected from bacteria, such as E. coli.
  • the eukaryotic cells are selected from yeast or mammalian cells, such as Pichia or CHO cells or human embryonic kidney (HEK) 293 cells.
  • the present disclosure provides a method for preparing a complex of an anti-IL-4R antibody or an antigen-binding fragment thereof, comprising the steps of:
  • the present disclosure further provides a pharmaceutical composition whose active ingredient contains the complex of the anti-IL-4R antibody or antigen-binding fragment thereof as described above, which is used as a medicine.
  • compositions of the present disclosure may contain, in addition to the active ingredient, one or more pharmaceutically acceptable excipients, diluents or vehicles.
  • the pharmaceutical composition may contain from 0.1 to 99% by weight of the active ingredient.
  • the present disclosure further provides the use of any one or a combination selected from the group consisting of: an anti-IL-4R antibody or an antigen-binding fragment thereof according to the present disclosure, a complex of an anti-IL-4R antibody or an antigen-binding fragment thereof, an anti-IL-4R antibody or an antigen-binding fragment thereof according to the present disclosure
  • the pharmaceutical composition of the present disclosure wherein, the antibody or antigen-binding fragment or complex thereof is used for treating and/or preventing proliferative diseases or delaying the progression of proliferative diseases.
  • the proliferative disorder may be a cancer or tumor; eg, wherein the cancer or tumor is a cancer or tumor associated with IL-4R expression.
  • the cancer or tumor is selected from prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma (eg, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma), lung cancer, kidney cancer, liver cancer (eg, small cell lung cancer and non-small cell lung cancer), colorectal cancer (eg colon cancer), pancreatic cancer, stomach cancer, leukemia (eg, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia) and brain cancer, tumors of the central nervous system.
  • lymphoma eg, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma
  • lung cancer eg, kidney cancer, liver cancer (e
  • the central nervous system tumor comprises glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, Hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, meningioma, neuroblastoma, retinoblastoma, adult blastoma.
  • the central nervous system tumor is a glioblastoma.
  • the glioblastoma is relapsed or refractory glioblastoma.
  • the glioblastoma is positive or negative for O6-methylguanine-DNA methyhransferase (MGMT) expression.
  • MGMT O6-methylguanine-DNA methyhransferase
  • the present disclosure provides a method of treating and/or preventing a proliferative disease or delaying the progression of a proliferative disease, the method comprising administering to a subject in need thereof an amount of an anti-IL-4R antibody or antigen thereof effective to treat or delay the disease according to the present disclosure
  • an anti-IL-4R antibody or antigen thereof effective to treat or delay the disease according to the present disclosure
  • a binding fragment, or a pharmaceutical composition according to the present disclosure, or a complex of an anti-IL-4R antibody or antigen-binding fragment thereof according to the present disclosure; wherein the proliferative disorder may be cancer or tumor.
  • the present disclosure provides methods of enhancing immune function in a subject having, suspected of having, or susceptible to a cell proliferative disorder.
  • the cell proliferative disorder is cancer or a tumor.
  • the cancer or tumor is a cancer or tumor associated with IL-4R expression.
  • the cancer or tumor is selected from prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma (eg, Hodgkin lymphoma, non-Hodgkin lymphoma, or recurrent anaplastic large cell lymphoma), lung cancer, kidney cancer, liver cancer (eg, small cell lung cancer and non-small cell lung cancer), colorectal cancer (eg colon cancer), pancreatic cancer, stomach cancer, leukemia (eg, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia) and brain cancer, tumors of the central nervous system.
  • leukemia eg, acute lymphoblastic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic
  • the central nervous system tumor comprises glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, Hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, meningioma, neuroblastoma, retinoblastoma, adult blastoma.
  • the central nervous system tumor is a glioblastoma.
  • the glioblastoma is relapsed or refractory glioblastoma.
  • the glioblastoma is positive or negative for O6-methylguanine-DNA methyhransferase (MGMT) expression.
  • MGMT O6-methylguanine-DNA methyhransferase
  • a suitable unit dose may range from 0.1 mg to 1000 mg.
  • the dosage of a drug administered depends on a variety of factors, including but not limited to the following factors: the activity of the particular compound used, the age of the subject, the weight of the subject, the The health condition, the behavior of the subject, the diet of the subject, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, etc., can be verified according to well-known treatment regimens.
  • Figures 1A and 1B Schematic representations of the structures of the exemplary complexes; antibody scdsFv of antibody 25G7 and antibody-drug conjugates 25G7-scdsFv-PE38KDEL ( Figure 1A) and 25G7-scFv-PE38KDEL ( Figure 1B) of toxin PE38KDEL.
  • FIG. 1 SDS-PAGE profiles of complexes 25G7-scdsFv-PE38KDEL and 25G7-scFv-PE38KDEL after purification, where M is a molecular weight marker (Marker).
  • FIG. 3 Detection of IL-4R expression levels in glioblastoma (GBM) cell lines by flow cytometry.
  • the LN229, U251, and U87 glioma cell lines stably transfected with human IL-4R were constructed by lentivirus system, and the overexpression level of IL-4R was detected by flow cytometry. corresponding cells.
  • FIGS 4A-4C Flow cytometry detection of complex binding on the surface of glioblastoma cells. Flow cytometry was used to determine the binding capacity of 25G7-scdsFv-PE38KDEL and MDNA55 on the cell surface of IL-4R stably transfected cell lines LN229-IL4R, U251-IL4R and U87-IL4R, respectively.
  • FIG. 5A to Figure 5C Endocytosis efficiency of complexes in glioblastoma cells; flow cytometry was used to determine 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55 in LN229-IL4R, U251-IL4R, Endocytosis efficiency in U87-IL4R cells from 0 to 24 hours.
  • Figures 6A to 6F In vitro cytotoxicity of the complexes against glioblastoma cells; 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55 were used to measure LN229-IL4R, U251-IL4R, U87-IL4R, respectively, by CTG assay and in vitro cytotoxicity results of control cells.
  • Figures 7A to 7D Comparison of in vitro cytotoxicity results of 25G7-scFv-PE38KDEL and 25G7-scdsFv-PE38KDEL, MDNA55 in LN229-IL4R, U251-IL4R and control glioma cells by CTG method, respectively.
  • Figure 8A to Figure 8C The results of the in vivo efficacy comparison of the complex in the nude mouse tumor model subcutaneously inoculated with U87-MG; ), intratumoral administration (i.t.).
  • Figure 9A to Figure 9C Results of the in vivo efficacy comparison of the complex in a nude mouse tumor model subcutaneously inoculated with LN229-IL4R (LN229 cells overexpressing IL-4R); 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55 drug administration
  • the doses were both 0.25mpk (i.t.), or 25G7-scdsFv-PE38KDEL 0.25mpk (i.v.).
  • FIG 10A to Figure 10C The in vivo efficacy results of the complex in a nude mouse tumor model subcutaneously inoculated with LN229-IL4R; 0.05mpk), 0.3mpk (reduced to 0.15mpk for the second time and later), MDNA55 drug administration at a dose of 0.1mpk (reduced to 0.05mpk for the second time and later), intratumoral administration (i.t.).
  • Human IL-4R means a human cytokine receptor that specifically binds to interleukin-4 (IL-4), IL-4R ⁇ .
  • hIL-4R is intended to encompass various forms of molecules of IL-4R at various stages in vivo, such as, but not limited to, molecules produced by the IL-4R gene during amplification, replication, transcription, splicing, processing, translation, modification For example, precursor IL-4R, mature IL-4R, naturally occurring IL-4R splice variants, modified IL-4R, or fragments thereof.
  • Antibody refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by interchain disulfide bonds.
  • the amino acid composition and sequence of the immunoglobulin heavy chain constant region are different, so their antigenicity is also different.
  • immunoglobulins can be divided into five classes, or isotypes called immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding heavy chains are ⁇ , ⁇ , ⁇ chains, respectively , alpha chains and epsilon chains.
  • IgG can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of disulfide bonds in the heavy chain.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified into kappa chains or lambda chains by the difference in the constant region.
  • Each of the five classes of Ig can have a kappa chain or a lambda chain.
  • the antibody light chain may further comprise a light chain constant region comprising human or murine kappa, lambda chains or variants thereof.
  • the antibody heavy chain may further comprise a heavy chain constant region comprising human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • variable region The sequence of about 110 amino acids near the N-terminus of the antibody heavy and light chains varies greatly, which is the variable region (V region); the remaining amino acid sequences near the C-terminus are relatively stable and are the constant region (C region).
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions.
  • the sequence from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to the light chain complementarity determining region 1 (LCDR1), the light chain complementarity determining region 2 (LCDR2), and the light chain complementarity determining region 3 (LCDR3);
  • the three CDR regions of the heavy chain refer to the heavy chain complementarity Determining region 1 (HCDR1), heavy chain complementarity determining region 2 (HCDR2) and heavy chain complementarity determining region 3 (HCDR3).
  • Antibodies include murine antibodies, chimeric antibodies, humanized antibodies, and fully human antibodies, which may be obtained recombinantly, eg, may be recombinant fully human antibodies obtained by affinity maturation.
  • Recombinant fully human antibodies include fully human antibodies prepared, expressed, created or isolated by recombinant methods, the techniques and methods involved are well known in the art, such as (1) from transgenic, transgenic human immunoglobulin genes Antibodies isolated from chromosomal animals (eg, mice) or hybridomas prepared therefrom; (2) antibodies isolated from host cells transformed to express antibodies, such as transfectomas; (3) from recombinant combinatorial human antibody libraries isolated antibodies; and (4) antibodies prepared, expressed, created or isolated by methods such as splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant fully human antibodies contain variable and constant regions that utilize specific human germline immunoglobulin sequences encoded by germline genes, but also include subsequent rearrangements and mutations such as those that occur during antibody maturation.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells can include bacterial, microbial, plant or animal cells.
  • Bacteria susceptible to transformation include members of the enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
  • a "murine antibody” is herein a monoclonal antibody to human IL-4R prepared according to the knowledge and skill in the art. In preparation, test subjects are injected with IL-4R antigen (or epitope-containing polypeptide), and hybridomas expressing antibodies with the desired sequence or functional properties are isolated.
  • the murine antibody or antigen-binding fragment thereof that binds to human IL-4R may further comprise a light chain constant region of a murine ⁇ , ⁇ chain or a variant thereof, or further comprise a murine IgG1, Heavy chain constant regions of IgG2, IgG3 or IgG4 or variants thereof.
  • Fully human antibodies include antibodies having variable and constant regions of human germline immunoglobulin sequences. Fully human antibodies herein may include amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “fully human antibody” does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human backbone sequences (ie, "humanized antibodies”). ).
  • Humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody)
  • CDR-grafted antibody refers to an antibody produced by grafting non-human CDR sequences into the framework of human antibody variable regions.
  • the strong immune response induced by chimeric antibodies can be overcome because they carry a large number of heterologous protein components.
  • the variable regions of the human antibody may be subjected to minimal reverse mutations to maintain activity.
  • a “chimeric antibody” is an antibody obtained by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To build a chimeric antibody select a hybridoma that secretes mouse-specific monoclonal antibodies, then clone the variable region genes from the mouse hybridoma cells, and then clone the constant region genes of the human antibody as needed.
  • the region gene and the human constant region gene are connected to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or its variants, for example comprising the heavy chain constant region of human IgG2 or IgG4, or without ADCC (antibody-dependent) after amino acid mutation. cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxicity of IgG1.
  • Antigen-binding fragment refers to Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments that bind to human IL-4R, scFv fragments, and, polypeptides or proteins comprising such fragments, which have antigen-binding activity.
  • the "antigen-binding fragment” comprises one or more CDR regions of the antibodies described herein. Fv fragments contain antibody heavy and light chain variable regions, but no constant regions, and are the smallest antibody fragments with all antigen-binding sites. Typically, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Different linkers can also be used to link the two antibody variable regions into a single polypeptide chain, called a single chain antibody (single chain antibody) or a single chain Fv (scFv).
  • single-chain antibody is intended to comprise a heavy chain variable domain (or region; VH) and a light chain variable domain (or region) linked by a linker (or linking peptide) ; VL) molecules.
  • linker or linking peptide
  • Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof, eg variants comprising 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) .
  • linkers useful in the present disclosure are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56: 3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
  • antibody framework refers to the portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.
  • Binds to IL-4R means capable of interacting with human IL-4R (or an epitope, fragment thereof).
  • antigen-binding site refers to a site in three-dimensional space recognized by an antibody or antigen-binding fragment herein.
  • Epitope refers to the site on an antigen to which an immunoglobulin or antibody specifically binds. Epitopes may be formed by adjacent amino acids, or non-adjacent amino acids by tertiary folding. Epitopes formed by adjacent amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically include at least 3-15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody are well known in the art and include immunoblotting and immunoprecipitation assays, among others. Methods for determining the spatial conformation of epitopes include techniques in the art and those described herein, such as X-ray crystallography and two-dimensional nuclear magnetic resonance, among others.
  • binds means that an antibody binds to an epitope on a predetermined antigen.
  • the antibody dissociates at equilibrium approximately below 10-7M or even less when measured by surface plasmon resonance (SPR) techniques in the instrument
  • SPR surface plasmon resonance
  • KD constant binds to a predetermined antigen with at least twice the affinity for binding to the predetermined antigen or non-specific antigens other than closely related antigens (eg, BSA, etc.).
  • antibody that recognizes an antigen is used interchangeably herein with the term “antibody that specifically binds”.
  • Cross-reactivity refers to the ability of an antibody herein to bind IL-4R from a different species.
  • an antibody herein that binds human IL-4R can also bind IL-4R of another species.
  • Cross-reactivity is measured by detecting specific reactivity with purified antigen in binding assays (eg SPR and ELISA), or binding or functional interaction with cells that physiologically express IL-4R. Methods to determine cross-reactivity include standard binding assays as described herein, such as surface plasmon resonance (SPR) analysis, or flow cytometry.
  • SPR surface plasmon resonance
  • a “neutralizing” or “blocking” antibody means an antibody whose binding to hIL-4R results in inhibition of the biological activity of hIL-4 and/or hIL-13.
  • This inhibition of hIL-4 and/or IL-13 biological activity can be achieved by measuring one or more indicators of hIL-4/or hIL-13 biological activity well known in the art, such as hIL-4/or hIL-13 induced cells Activation and binding of hIL-4 to hIL-4R were assessed. See for example in CN103739711A.
  • “Inhibition of growth” (eg, in relation to a cell) is intended to include any measurable reduction in cell growth.
  • Inducing an immune response and “enhancing an immune response” are used interchangeably and refer to the stimulation (ie, passive or adaptive) of an immune response to a specific antigen.
  • the term “induction” with respect to induction of CDC or ADCC refers to stimulation of a specific mechanism of direct cell killing.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • Fc receptors directly kill antibody-coated target cells by recognizing the Fc segment of the antibody.
  • the ADCC effector function of the antibody can be reduced or eliminated by modification of the Fc region of IgG. Said modification refers to mutation in the constant region of the heavy chain of the antibody, such as N297A, L234A, L235A of IgG1; IgG2/4 chimera, F235E of IgG4, or L234A/E235A mutation.
  • a fusion protein is a protein product obtained by co-expression of two genes through DNA recombination.
  • anti-IL-4R antibody-PE38KDEL fusion protein is a fusion protein that co-expresses anti-IL-4R antibody and toxin molecule PE38KDEL through DNA recombination.
  • Methods of producing and purifying antibodies and antigen-binding fragments are well known and can be found in the art, eg, Cold Spring Harbor's Technical Guide to Antibody Assays, Chapters 5-8 and 15.
  • mice can be immunized with human IL-4R or a fragment thereof, and the resulting antibody can be renatured, purified, and amino acid sequenced by conventional methods.
  • Antigen-binding fragments can likewise be prepared by conventional methods.
  • the antibodies or antigen-binding fragments described herein are genetically engineered to add one or more human FRs to CDR regions of non-human origin.
  • Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website, or from the Journal of Immunoglobulins, 2001 ISBN012441351.
  • Engineered antibodies or antigen-binding fragments can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • the sequence of the humanized antibody in this paper is inserted into the corresponding expression vector by molecular cloning technology, and the corresponding humanized antibody can be obtained by expressing and producing by HEK293 cell expression system.
  • mammalian-like expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminus of the Fc region.
  • Stable clones are obtained by expressing antibodies that specifically bind to human antigens. Positive clones were expanded in serum-free medium in bioreactors for antibody production. The antibody-secreted culture medium can be purified and collected by conventional techniques. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, and ion exchange. The obtained product should be frozen immediately, eg -70°C, or lyophilized.
  • An antibody may be a monoclonal antibody (mAb), which refers to an antibody obtained from a single clonal cell strain, which is not limited to eukaryotic, prokaryotic or phage cloned cell strains.
  • mAb monoclonal antibody
  • Monoclonal antibodies or antigen-binding fragments can be obtained by recombinant techniques such as hybridoma technology, recombinant technology, phage display technology, synthetic technology (such as CDR-grafting), or other existing techniques.
  • Antibodies can be monospecific, bispecific or multispecific. Multispecific antibodies may exhibit specificity for different epitopes of the target peptide, or may also contain antigen-binding domains that exhibit specificity for more than one target peptide.
  • Human anti-IL-4R antibodies can be linked to, or co-expressed with, another functional molecule (eg, another peptide or protein).
  • another functional molecule eg, another peptide or protein.
  • an antibody or fragment thereof can be functionally linked (eg, by chemical conjugation, genetic fusion, non-covalent association, or otherwise) to one or more other molecules (eg, another antibody or antigen-binding fragment) to produce a At least one bispecific or multispecific antibody of binding specificity.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refer to exogenous drugs, therapeutic agents, diagnostic agents, or compositions that interact with the animal. , contact of humans, subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of cells includes contact of reagents with cells, and contact of reagents with fluids, wherein the fluids are in contact with cells.
  • administering also mean the in vitro and ex vivo treatment of, for example, cells by an agent, diagnosis, composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering an internal or external therapeutic agent, such as any of the complexes or compositions herein, to a subject having (suspected of having, susceptible to) one or more symptoms of a disease, In turn, the therapeutic agent has a therapeutic effect on these symptoms.
  • a therapeutic agent is administered in an amount effective to alleviate one or more symptoms of a disease in a subject or population to be treated, whether by inducing regression of such symptoms or inhibiting progression of such symptoms to any clinically measured degree.
  • the amount of a therapeutic agent effective to alleviate symptoms of any particular disease may vary depending on a variety of factors, such as the subject's disease state, age, and weight, and the level of the drug that produces the desired effect in the subject. ability. Whether symptoms of a disease have been alleviated can be assessed by any clinical test commonly used by doctors or other health care professionals to assess the severity or progression of the symptoms.
  • embodiments of the present invention may be ineffective in alleviating symptoms of a target disease in a single subject
  • any statistical test known in the art such as Student's t-test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determine that it should reduce target disease symptoms in a statistically significant number of subjects.
  • naturally occurring refers to the fact that the object can be found in nature.
  • a polypeptide sequence or a polynucleotide sequence that is present in an organism (including a virus) that can be isolated from natural sources and has not been intentionally modified by humans is naturally occurring.
  • an “effective amount” includes an amount sufficient to ameliorate or prevent the symptoms or conditions of the medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate diagnosis.
  • the effective amount for a particular subject or veterinary subject may vary depending on factors such as the condition being treated, the general health of the subject, the method, route and dosage of administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Exogenous refers to a substance produced outside an organism, cell, or human body depending on the context.
  • Endogenous refers to a substance produced in an organism, cell or human body depending on the context.
  • “Homology” or “identity” refers to the sequence similarity between two polynucleotide sequences or between two polypeptides.
  • Two DNA molecules are homologous when a position in the two compared sequences is occupied by the same base or amino acid monomer subunit, for example if each position is occupied by an adenine, then the molecules are homologous at that position .
  • the percent homology between the two sequences is a function of the number of matches or homologous positions shared by the two sequences divided by the number of positions compared x 100%. For example, when sequences are optimally aligned, two sequences are 60% homologous if 6 of 10 positions in the two sequences are matched or homologous.
  • At least 85% sequence identity means that the two sequences have at least 85% homology, in some aspects, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology; in some specific schemes, it has 90%, 95% or 99% % or more; in other specific schemes, it has at least 95% sequence homology.
  • Said amino acid sequences having at least 85% sequence identity include those obtained by mutating one or more amino acid deletions, insertions or substitutions into the parental sequence.
  • the terms “cell”, “cell line”, “cell strain” and “cell culture” are used interchangeably and all such terms include progeny thereof.
  • the terms “transformants” and “transformed cells” include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that, due to intentional or unintentional mutations, progeny and parental cells cannot be exactly identical in DNA content. The term includes mutant progeny that have the same function or biological activity as the parent cell selected from the initially transformed cell.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a "plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors disclosed herein are capable of autonomous replication in the host cell into which they have been introduced (eg, bacterial vectors and episomal mammalian vectors having a bacterial origin of replication) or may integrate into the host cell's genome upon introduction into the host cell, thereby following The host genome replicates together (eg, a non-episomal mammalian vector).
  • “Pharmaceutical composition” means a mixture comprising one or more of the complexes described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other components, eg, physiologically/pharmaceutically acceptable salts or prodrugs The vehicle or excipient used.
  • the purpose of the pharmaceutical composition is to facilitate the administration to the organism, facilitate the absorption of the active ingredient and then exert the biological activity.
  • excipient is an addition other than the active ingredient in a pharmaceutical formulation, which may also be referred to as an adjuvant.
  • adjuvant such as binders, fillers, disintegrants, lubricants in tablets; matrix parts in semi-solid preparations ointments and creams; preservatives, antioxidants, flavoring agents, fragrances in liquid preparations Agents, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc. can all be called excipients.
  • the term "diluent”, also known as filler, is primarily used to increase the weight and bulk of the tablet.
  • the addition of the diluent not only ensures a certain volume, but also reduces the dosage deviation of the main components and improves the compression molding of the drug.
  • an absorbent to absorb the oily substances, so as to keep the "dry” state, so as to facilitate the tableting.
  • Immunohistochemical scoring is a histological scoring method for processing immunohistochemical results. The number of positive cells in each section and their staining intensity are converted into corresponding values, so as to achieve the purpose of semi-quantitative tissue staining. A score of 0 means no staining or membrane staining is observed in less than 10% of tumor cells; a score of +1 means weak/barely perceptible membrane staining is detected in greater than 10% of tumor cells; for a score of +2, at Moderate complete membrane staining was observed in greater than 10% of tumor cells; a score of +3 indicates strong complete membrane staining was observed in greater than 10% of tumor cells. In the present disclosure, those samples with a score of 0 or +1 for IL-4R expression can be considered as overexpressing IL-4R, while those with a score of +2 or +3 can be considered as overexpressing IL-4R.
  • the humanized IL-4R monoclonal antibody hu25G7 (heavy chain variable region sequence shown in SEQ ID NO: 43, hu25G7-VH; light chain variable region sequence shown in SEQ ID NO: 37, hu25G7-A LCVR ) single-chain variable segment (scFv) expressed through the linker ASGGPE (SEQ ID NO: 50) and mutated and truncated Pseudomonas exotoxin (PE38KDEL) (SEQ ID NO: 49) fusion expression; wherein the hu25G7 antibody Amino acids at positions VH-44 and VL-100 were mutated to cysteines (eg, SEQ ID NOs: 47-48), and VH and VL were linked by a linking peptide (GGGGS) 3 (SEQ ID NO: 51); thus obtained
  • the immunotoxin 25G7-(scdsFv)-PE38KDEL (hereinafter referred to as 25G7
  • 1A and 1B are schematic diagrams of the structures of two immunotoxins.
  • 25G7-IT was expressed in E. coli BL21, and the protein was purified and renatured from the inclusion bodies; then endotoxin was removed to obtain purified 25G7-IT protein with a molecular weight of about 63KDa (SDS-PAGE results are shown in Figure 2A and Figure 2B). Show).
  • the heavy chain sequence of the humanized anti-human IL-4R antibody 25G7 is shown in SEQ ID NO: 17, and the light chain sequence is shown in SEQ ID NO: 18.
  • Antibodies can be expressed and purified according to routine antibody expression and purification methods in the art, eg according to the methods described in patent application WO2020038454A1. The aforementioned patent applications are incorporated herein by reference in their entirety.
  • Biacore biomacromolecule interaction instrument bias 8K, GE
  • surface plasmon resonance Surface Plasmon Resonance, SPR
  • 25G7-IT and reference sample MDNA55 were determined to interact with hrIL- Affinity kinetics of 4R-Fc (ILR-H5253, Acro) interaction.
  • Anti-human IgG antibody (29234600, GE) was covalently coupled to a CM5 chip (29149603, GE), and then rhIL-4R-Fc was used as a ligand to capture on the chip, and then 25G7-IT and the reference sample MDNA55 were injected as analytes for affinity analysis and calculation.
  • Complete medium was prepared by adding 10% FBS and 1% penicillin/streptomycin to DMEM medium, and U87-, U251-, LN229-IL4R stably transfected cell lines were cultured at 37°C in a 5% CO 2 incubator.
  • Cells in T75 flasks were digested with 1.5 ml of TrpLE (Gibco, #12605-010) at 37°C for 3-5 minutes, then neutralized with FBS-containing DMEM medium, and centrifuged to prepare a concentration of 2 ⁇ 10 6 cells/ ml of cell suspension was added to a 96-well round bottom plate in a volume of 50 ⁇ l per well.
  • the 25G7-IT, MDNA55, and hIgG1 isotype controls were diluted in DMEM medium to 8 concentration points (three-fold gradient: 20; 6.67; 2.22; 0.74; 0.25; 0.08; 0.03; 0 ⁇ g/ml), respectively.
  • a volume of 50 ⁇ l was added to a 96-well plate to mix well with the cells and incubated on ice for 40 minutes.
  • MFI mean fluorescence intensity
  • the final concentration of 2 ⁇ g/ml was selected as the concentration for detecting the endocytic efficiency, because the endocytic efficiency in the three cell samples basically reached a saturation state at this concentration.
  • 25G7-IT, MDNA55 and hIgG1 isotype control were diluted to 2 ⁇ g/ml in DMEM medium. Add 50 ⁇ l per well to the 96-well plate to mix well with cells and incubate for 40 min on ice.
  • DMEM complete medium After washing three times with DMEM complete medium, resuspend the cells with 100 ⁇ l DMEM complete medium, transfer 100 ⁇ l of cells in the 96-well plate marked as 0h to another 96-well round bottom plate, and add 100 ⁇ l of 4% paraformaldehyde fixative, Fixed at 4°C. The remaining cells were cultured in a 37°C incubator for 1 hour, 2 hours, 4 hours, and 24 hours, respectively, and then the corresponding cells were taken out and transferred to another 96-well plate to fix at 4°C in the same way.
  • Complete medium was prepared by adding 10% FBS and 1% penicillin/streptomycin to DMEM medium.
  • U87-IL4R, U251-IL4R, LN229-IL4R stably transfected cell lines and U87, U251, LN229 control cells were cultured in a 37°C incubator with 5% CO 2 .
  • Plates were performed at a density of 4000 cells per well and 130 ⁇ l of medium in 96-well plates. After overnight, when the cells were completely adherent, 20 ⁇ l of gradient concentration drugs containing 25G7-IT or MDNA55 were added (final concentrations: 100nM, 20nM, 4nM, 0.8nM, 0.16nM, 0.032nM, 0.0064nM, 0.00128nM, 0.000256nM, 0 nM) for 3 days in the incubator.
  • CTG reagent CellTiter-Glo Luminescent, Promega, #G7573
  • Cell viability was detected using the Luminescence mode built into the EnVision2105 Multimode Plate Reader (PerkinElmer). Absorbance results were converted into percentages and analyzed with Prism 8.
  • Example 6 In vivo efficacy of drugs targeting IL-4R in GBM tumor models
  • the experimental mice were subcutaneously inoculated with 2 ⁇ 10 6 U87-MG cells on the right back, and the cells were resuspended in 1:1 PBS and Matrigel (0.1 ml/mice), and tumor growth was observed regularly.
  • groupings were performed with 8 mice per group.
  • Multi-site intratumoral administration the drug 25G7-scdsFv-PE38KDEL (25G7-IT) and MDNA55 were administered at a dose of 0.5 mg/kg (mpk) once a week for a total of four doses.
  • the body weight and tumor size of the mice were measured twice a week.
  • s.c. subcutaneous
  • mpk mg/kg (mg per kilogram of body weight)
  • i.t. intratumoral injection
  • i.v. intravenous injection
  • QW once a week. ** means p ⁇ 0.01, *** means p ⁇ 0.001.
  • Example 7 In vivo efficacy of drugs targeting IL-4R in GBM tumor model (IL-4R 3+)
  • mice were subcutaneously inoculated with 1 ⁇ 10 7 LN229-IL4R cells on the right back, and the cells were resuspended in 1:1 PBS (0.1 ml/mice), and tumor growth was observed regularly.
  • groupings were performed with 6-7 mice per group.
  • MDNA55 was administered at a dose of 0.25mpk (it)
  • 25G7-scdsFv-PE38KDEL 25G7-IT
  • 0.25mpk it or iv
  • the dose of immunotoxin was further reduced, and the dose of 25G7-scdsFv-PE38KDEL (25G7-IT) was 0.1 (p.
  • the dose of MDNA55 is 0.1 (reduced to 0.05 for the second time and later), 0.3 (reduced to 0.15 for the second time and later) mpk, and intratumoral administration.
  • the tumor growth inhibitory effect of low concentration of 25G7-IT is better than that of MDNA55 at the same dose, and the high dose of 25G7-IT drug can make tumor-bearing mice completely Relief, showing a significant dose effect.
  • s.c. subcutaneous
  • mpk mg/kg (milligrams per kilogram)
  • i.t. intratumoral injection
  • i.v. intravenous injection
  • QW once a week. ** means p ⁇ 0.01, *** means p ⁇ 0.001.
  • the use and welfare of laboratory animals in this disclosure complied with the International Association for the Assessment and Accreditation of Laboratory Animals (AAALAC).
  • AALAC International Association for the Assessment and Accreditation of Laboratory Animals
  • the health status and death of the animals are monitored daily. Routine inspections include observing the effects of the test substances and drugs on the animals' daily behaviors, such as behavioral activities, weight changes, and physical signs.

Abstract

提供了抗IL-4R抗体或其抗原结合片段的复合物及医药用途。具体而言，提供特异性结合IL-4R的抗体或其抗原结合片段与毒素共价连接的复合物，含有所述复合物的药物组合物，及其在制备治疗IL-4R介导的疾病或病症的药物中的用途，尤其在用于制备抗癌药物中的用途。

Description

抗IL-4R抗体或其抗原结合片段的复合物及医药用途
本申请要求2020年12月22日提交的中国专利申请202011529235.1的优先权。
技术领域
本申请涉及一种抗IL-4R抗体或其抗原结合片段的复合物，以及其作为抗癌药物的用途。
背景技术
恶性肿瘤(癌症)是全球第二位死因，仅排在心脏病之后。神经胶质母细胞瘤(GBM)是最常见的恶性原发性脑瘤。经历标准疗法治疗的胶质母细胞瘤患者的中位生存期小于15个月。尽管在肿瘤生物学方面的研究取得进展，但并未转化成使患者受益的产品或疗法。因此，有迫切需求去开发有效的方法用来治疗神经胶质母细胞瘤。
IL-4R是白介素4受体，在脑胶质母细胞瘤肿瘤细胞以及浸润的免疫抑制细胞(如肿瘤相关巨噬细胞(TAM)、髓系来源免疫抑制细胞(MDSC)等)上表达。靶向IL-4R的治疗不仅可以杀伤肿瘤细胞，还可以改善肿瘤微环境。在脑胶质母细胞瘤临床前动物模型以及早期临床实验中，靶向IL-4R的治疗均显示优于传统治疗的效果。
Medicenna公司开发的MDNA55是IL-4与细菌毒素假单胞菌外毒素A(PE38KDEL)形成的融合蛋白，在临床开发实验中显示了积极疗效(参见WO9527732、Biochem.J.(1995)307，29-37、CANCER RESEARCH 55，3357-3363，August 1，1995)。
但是第一代的免疫毒素治疗存在与靶标亲和力不够强等问题。提高免疫毒素的靶向性和亲和力是肿瘤免疫毒素治疗研究的重点与难点之一(参见“中国肿瘤临床年鉴2009”，赵平主编，中国协和医科大学出版社，2010年07月，第61页)。
抗体-药物偶联物(ADC)旨在将单克隆抗体(mAbs)的选择性与化疗药物的细胞毒性潜力结合起来。抗体-药物偶联物由“抗体(antibody)”、“接头(linker)”和“药物分子(drug)”三个主要组件构成。和传统的完全或部分人源化抗体或抗体片段相比，抗体-药物偶联物因为能在肿瘤组织内释放高活性的细胞毒素从而理论上疗效更高；和融合蛋白相比，具有更高的耐受性或较低的副作用。抗体-药物偶联物与靶抗原的亲和力影响抗体-药物偶联物渗透到肿瘤组织与靶抗原结合过程，并直接影响抗体-药物偶联物的黏合效率(参见“生物技术制药”，冯美卿主编，中国医药科技出版社，2016年01月，第209-211页)。
设计抗体-药物偶联物时，最关键的因素之一就是抗体的选择，重要的是对抗原的高特异性。缺乏高特异性并与其他抗原发生交叉反应的抗体可能无法发挥预 期作用，例如，通过与健康组织相互作用而引起脱靶毒性，或在到达肿瘤部位之前被机体过早清除(参见：“Introduction to Antibody–Drug Conjugates(ADCs)”，Ilona Pysz，Paul J.M.Jackson and David E.Thurston，CHAPTER 1：Introduction to Antibody–Drug Conjugates(ADCs)，in Cytotoxic Payloads for Antibody–Drug Conjugates，2019)。
目前各国已有多家制药公司正在研发针对IL-4R的单克隆抗体，相关专利申请如WO2010053751、WO2001092340、WO2008054606、WO2014031610等。发明人的在先申请WO2020038454也涉及一类新的抗IL-4R抗体。但是，IL-4R定向的抗体-药物偶联物鲜有明确报道，如WO2015188934、WO2014124227、WO2018217227等均仅泛泛提及抗体-药物偶联物中抗体可选靶向IL-4R，但均未披露具体的抗IL-4R抗体-药物偶联物。
本公开利用高亲和力的抗IL-4R抗体制备靶向递送药物的第二代免疫毒素药物，达到强效杀伤癌细胞的目的，用于满足癌症治疗或延缓癌症进展的需求。
发明内容
本公开涉及抗IL-4R抗体或其抗原结合片段的复合物、制备方法及医药用途。
复合物
本公开提供了一种抗IL-4R抗体或其抗原结合片段的复合物，其包含：
抗IL-4R抗体或其抗原结合片段以及一个或多个毒素分子。
一些实施方案中，复合物中抗IL-4R抗体或其抗原结合片段包含重链可变区和轻链可变区，其中重链可变区包含：
(I)氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；或
(II)氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；
和/或，抗体轻链可变区包含：
(I)氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；或
(II)氨基酸序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；或
(III)氨基酸序列分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；或
(IV)氨基酸序列分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
其中，所述抗IL-4R抗体或其抗原结合片段与所述毒素共价或非共价连接。
表1抗IL-4R抗体或其抗原结合片段的CDR序列
名称 序列 编号
HCDR1 GFTFSDYGMH SEQ ID NO：3
HCDR2 FISSGSSIIYYADIVKG SEQ ID NO：4
HCDR3 GNKRGFFDY SEQ ID NO：5
LCDR1 NASSSVSYMY SEQ ID NO：6
LCDR2 LTSNLAS SEQ ID NO：7
LCDR3 QQWRSNPPMLT SEQ ID NO：8
HCDR1 GYTFTSYWMH SEQ ID NO：11
HCDR2 LIHPNSDTTKFSENFKT SEQ ID NO：12
HCDR3 SKIITTIVARHWYFDV SEQ ID NO：13
LCDR1 KASQSVDYGGDSYMN SEQ ID NO：14
LCDR2 AASNLES SEQ ID NO：15
LCDR3 QHSNENPPT SEQ ID NO：16
LCDR1 RASSSVPYMY SEQ ID NO：38
LCDR2 LASSRPS SEQ ID NO：39
LCDR3 QQWRAYPPMLT SEQ ID NO：40
LCDR1 RASPGVPPLA SEQ ID NO：42
在一些实施方案中，抗IL-4R抗体或其抗原结合片段包含选自以下(I)至(IV)中的任一项：
(I)重链可变区，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
(II)重链可变区，其包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含氨基酸序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
(III)重链可变区，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含氨基酸序列分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；
(IV)重链可变区，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
轻链可变区，其包含氨基酸序列分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
重链可变区包含：
(I)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：43所示或与SEQ ID NO：43具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
和/或，轻链可变区包含：
(I)如SEQ ID NO：2所示或与SEQ ID NO：2具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：10所示或与SEQ ID NO：10具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(IV)如SEQ ID NO：41所示或与SEQ ID NO：41具有至少70％、80％、90％、95％、98％、99％同一性的序列。
25G7 HCVR(25G7重链可变区)
Figure PCTCN2021140310-appb-000001
25G7 LCVR(25G7轻链可变区)
Figure PCTCN2021140310-appb-000002
7B10 HCVR(7B10重链可变区)
Figure PCTCN2021140310-appb-000003
7B10 LCVR(7B10轻链可变区)
Figure PCTCN2021140310-appb-000004
hu25G7-A LCVR(hu25G7-A轻链可变区)
Figure PCTCN2021140310-appb-000005
Figure PCTCN2021140310-appb-000006
hu25G7-B LCVR(hu25G7-B轻链可变区)
Figure PCTCN2021140310-appb-000007
hu25G7-VH(hu25G7重链可变区)
Figure PCTCN2021140310-appb-000008
在至少一个实施方案中，所述抗IL-4R抗体或抗原结合片段中：
重链可变区如序列SEQ ID NO：1所示，轻链可变区如序列SEQ ID NO：2所示；或
重链可变区序列如SEQ ID NO：9所示，轻链可变区序列如SEQ ID NO：10所示；或
重链可变区序列如SEQ ID NO：43所示，轻链可变区序列如SEQ ID NO：37所示；或
重链可变区序列如SEQ ID NO：43所示，轻链可变区序列如SEQ ID NO：41所示；或
重链可变区序列如SEQ ID NO：47所示，轻链可变区序列如SEQ ID NO：48所示。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
重链可变区包含：
(I)如SEQ ID NO：25-27之一所示或与SEQ ID NO：25-27之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：31-33之一所示或与SEQ ID NO：31-33之一具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或，轻链可变区包含：
(I)如SEQ ID NO：28-30之一所示或与SEQ ID NO：28-30之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：34-36之一所示或与SEQ ID NO：34-36之一具有至少70％、80％、90％、95％、98％或99％同一性的序列。
hu25G7-VH-a：
Figure PCTCN2021140310-appb-000009
Figure PCTCN2021140310-appb-000010
hu25G7-VH-b：
Figure PCTCN2021140310-appb-000011
hu25G7-VH-c：
Figure PCTCN2021140310-appb-000012
hu25G7-VL-a：
Figure PCTCN2021140310-appb-000013
hu25G7-VL-b：
Figure PCTCN2021140310-appb-000014
hu25G7-VL-c：
Figure PCTCN2021140310-appb-000015
hu7B10-VH-a：
Figure PCTCN2021140310-appb-000016
hu7B10-VH-b：
Figure PCTCN2021140310-appb-000017
hu7B10-VH-c：
Figure PCTCN2021140310-appb-000018
hu7B10-VL-a：
Figure PCTCN2021140310-appb-000019
hu7B10-VL-b：
Figure PCTCN2021140310-appb-000020
hu7B10-VL-c：
Figure PCTCN2021140310-appb-000021
在一些具体的实施方案中，重链可变区如序列SEQ ID NO：25-27之一所示，和轻链可变区如序列SEQ ID NO：28-30之一所示；或
重链可变区序列如SEQ ID NO：31-33之一所示，和轻链可变区序列如SEQ ID NO：34-36之一所示。
在一些实施方案中，重链可变区第44位(VH-44)和轻链可变区第100位(VL-100)位置的氨基酸可选地突变成半胱氨酸。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段的包含重链和轻链，其中重链包含：
(I)如SEQ ID NO：17所示或与SEQ ID NO：17具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少70％、80％、90％、95％、98％、或99％同一性的序列；或
(III)如SEQ ID NO：44所示或与SEQ ID NO：44具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或轻链包含：
(I)如SEQ ID NO：18所示或与SEQ ID NO：18具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：20所示或与SEQ ID NO：20具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(III)如SEQ ID NO：45所示或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的序列；或
(IV)如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的序列。
hu25G7 HC
Figure PCTCN2021140310-appb-000022
hu25G7 LC
Figure PCTCN2021140310-appb-000023
hu7B10 HC
Figure PCTCN2021140310-appb-000024
hu7B10 LC
Figure PCTCN2021140310-appb-000025
hu25G7 HC
Figure PCTCN2021140310-appb-000026
Figure PCTCN2021140310-appb-000027
hu25G7-A LC
Figure PCTCN2021140310-appb-000028
hu25G7-B LC
Figure PCTCN2021140310-appb-000029
在至少一个实施方案中，重链序列如SEQ ID NO：17所示，和轻链序列如SEQ ID NO：18所示；或
重链序列如SEQ ID NO：19所示，和轻链序列如SEQ ID NO：20所示；或
重链序列如SEQ ID NO：44所示，和轻链序列如SEQ ID NO：45所示；或
重链序列如SEQ ID NO：44所示，和轻链序列如SEQ ID NO：46所示。
在一些实施方案中，所述的抗IL-4R抗体或抗原结合片段是鼠源抗体、嵌合抗体、全人抗体、人源化抗体或其片段。在一些具体的实施方案中，所述抗IL-4R抗体或抗原结合片段是人源化的。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，包含来源于人种系轻链模板IGKV3-11*01(SEQ ID NO：22，用于抗体25G7)的FR区序列或与其至少有95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自46P，47W，71Y中的一个或多个。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，包含来源于人种系重链模板IGHV3-48*01(SEQ ID NO：21，用于抗体25G7)的FR区序列或与其至少有95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自94A，67S，93T中的一个或多个。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，包含来源于人种系轻链模板IGKV2D-29*01(SEQ ID NO：24，用于抗体7B10)的FR区序列或与其至少有95％同一性的回复突变序列。在一些具体实施方案中，所述的回复 突变选自4L和/或58I。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，包含来源于人种系重链模板IGHV1-2*02(SEQ ID NO：23，用于抗体7B10)的FR区序列或与其至少有95％同一性的回复突变序列。在一些具体实施方案中，所述的回复突变选自69L，71I，73K，94K中的一个或多个。
人种系重链模版IGHV3-48*01：
Figure PCTCN2021140310-appb-000030
人种系轻链模板IGKV3-11*01：
Figure PCTCN2021140310-appb-000031
人种系重链模版IGHV1-2*02：
Figure PCTCN2021140310-appb-000032
人种系轻链模板IGKV2D-29*01：
Figure PCTCN2021140310-appb-000033
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段包含选自人源IgG1、IgG2、IgG3、IgG4的重链恒定区或其变体。一些具体的方案中，包含人源IgG1的重链恒定区或其变体。在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段包含人源κ、λ链或其变体的恒定区。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：17所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：18所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：19所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：20所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：44所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：45所示或与其具有至少85％序列同一性。
在一些实施方案中，所述的抗IL-4R抗体或其抗原结合片段，其中所述抗体为人源化抗体，重链序列如SEQ ID NO：44所示或与其具有至少85％序列同一性；轻链序列如SEQ ID NO：46所示或与其具有至少85％序列同一性。
在一些实施方案中，提供一种分离的抗IL-4R抗体或其抗原结合片段，其特征在于与如上所述的任一抗IL-4R抗体或其抗原结合片段竞争结合人IL-4R或其表位。
在一些实施方案中，提供一种双特异性抗体或多特异性抗体，其含有如上所述的任一抗IL-4R抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在另一些实施方案中，提供一种单链抗体，其含有如上所述的任一抗IL-4R抗体或其抗原结合片段的轻链可变区和/或重链可变区。
在一些实施方案中，人源化抗IL-4R抗体或其抗原结合片段，还包含人源IgG1，IgG2，IgG3或IgG4的重链恒定区、或其变体。在至少一个实施方案中，包含的是人源IgG2或IgG4重链恒定区，因为IgG2或IgG4没有ADCC毒性。在另一个实施方案中，使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。在至少一个实施方案中，变体包含ADCC效应功能降低或缺失的重链恒定区突变，例如但不限于IgG1的297A、234A、235A。
在一些实施方案中，所述抗IL-4R抗体或其抗原结合片段可以为抗体变体，所述变体在轻链有1至10个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸变化，和/或重链有1至10个(例如1、2、3、4、5、6、7、8、9、10个)氨基酸变化。
在一些实施方案中，上述变体具有与亲本抗IL-4R抗体或其片段相同或相似的生物学功能或效果。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段可以是已知的，如在例如WO2010053751、WO2001092340、WO2008054606、WO2014031610、WO2020038454(其各自通过引用并入本文)中所描述的抗IL-4R抗体或其抗原结合片段。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段包括但不限于Dupixent、PRS-060、AK-120、63 IgG1、CBP201、AMG-317或其抗原结合片段。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段是抗人IL-4R的抗体或其抗原结合片段。
在一些实施方案中，抗原结合片段包括但不限于Fab、Fab’、Fv、F(ab’)2、线性抗体、scFv(单链Fv抗体)、串联二-scFv、串联三-scFv、双链抗体(diabody)、三链抗体(triabody)、四链抗体(tetrabody)、sdAb(单域抗体或纳米抗体)、sdFv、肽抗体(peptibody)、结构域抗体、多特异性抗体(例如双特异性抗体、三特异性抗体或四特异性抗体)、dsFv(二硫键稳定化的Fv)、ScdsFv(二硫键稳定化的单链Fv抗体)。
在一些实施方案中，抗IL-4R抗体或抗原结合片段引入稳定抗体结构的突变，例如一些氨基酸残基突变为半胱氨酸残基。在一些具体实施方案中，所述突变发 生在框架区；在一些具体实施方案中，所述突变发生在重链可变区和\或轻链可变区；在一些具体实施方案中，所述突变发生在重链或轻链可变区和框架区。
在一些实施方案中，位于选自以下一组或多组位置上的氨基酸残基突变成半胱氨酸残基：VL100和VH44；VL101和VH44；VL34和VH100；VL43和VH91；VL43和VH103；VL49和H100；VL87和VH45；VL91和VH98；VL91和VH99；VL96和VH47；VL98和VH45。
在一些具体的实施方案中，位于选自以下一组或多组位置上的氨基酸残基突变成半胱氨酸残基：VL100和VH44；VL43和VH91；VL43和VH103；VL98和VH45。
在一个具体的实施方案中，VH44和VL100位置的氨基酸突变成半胱氨酸。
除非另有说明，本公开的抗IL-4R抗体或其抗原结合蛋白中的氨基酸序列根据Kabat编码。
在一些实施方案中，抗IL-4R抗体或抗原结合片段是scFv或ScdsFv，重链可变区(VH)和轻链可变区(VL)间用连接肽(linking peptide)L2连接。
在一些实施方案中，连接肽L2大于12个氨基酸残基的长度，且富含小的、非极性的氨基酸残基、极性的氨基酸残基和/或亲水的氨基酸残基，例如，甘氨酸、丝氨酸和苏氨酸。
在一些实施方案中，连接肽L2可以选自多种现有技术已知的接头，包括“GS”接头，例如，(GxS)n、(SxG)n、(GGGGS)n、(G)n，其中x是1-6的整数，且n是1-30的整数。
在一些实施方案中，连接肽L2非限制性例子包括GKSSGSGSESKS(SEQ ID NO：54)、EGKSSGSGSESKEF(SEQ ID NO：55)、GSTSGSGKSSEGKG(SEQ ID NO：56)、GSTSGSGKSSEGSGSTKG(SEQ ID NO：57)、GSTSGSGKPGSGEGSTKG(SEQ ID NO：58)、SRSSG(SEQ ID NO：59)和SGSSC(SEQ ID NO：60)。
在一些实施方案中，连接肽L2由重复的GGGGS氨基酸序列(SEQ ID NO：61)或其变体组成，例如(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多，例如n为2、3、4，例如n为3。
在一些实施方案中，ScFv的构建形式是VH-L2-VL，表示L2与VH的C端连接并与VL的N端连接；
在一些实施方案中，ScFv的构建形式是VL-L2-VH，表示L2与VL的C端连接并与VH的N端连接；
在一些实施方案中，ScdsFv的构建形式是VH-L2-VL，表示L2与VH的C端连接并与VL的N端连接；
在一些实施方案中，ScdsFv的构建形式是VL-L2-VH，表示L2与VL的C端连接并与VH的N端连接。
在一些实施方案中，毒素选自：细菌毒素、动物毒素或植物毒素。
在一些实施方案中，毒素包括截短型毒素或毒素变体，所述毒素变体包含氨基酸缺失、***、替换或其组合。
在一些实施方案中，毒素包括成孔毒素。
在一些实施方案中，成孔毒素包括嗜水气单胞菌气溶素(aerolysin)或气单胞菌溶素原(proaerolysin)。
在一些实施方案中，毒素包括布加宁(bouganin)、蓖麻毒素、假单胞菌外毒素(pseudomonas exotoxin、PE)、霍乱毒素(cholera toxin)或白喉毒素(diphtheria toxin)。
在一些实施方案中，毒素包括但不限于假单胞菌外毒素的细胞毒性片段或细胞毒性变体。在例如美国专利第4,892,827号；第5,512,658号；第5,602,095号；第5,608,039号；第5,821,238号；第5,854,044号；第8,871,906号；第8,907,060号；第8,936,792号；第9,346,859号；第9,206,240号；和第9,388,222号(将其各自通过引用并入本文)中所述的PE-LR、PE-LO10R456A、PE-T20、PE-T20-KDEL、PE4E、PE40、PE38、PE24、PE25、PE38QQR、PE38KDEL和PE35中的任一种或多种。
在一些实施方案中毒素包括如SEQ ID NO：49所示的PE38KDEL，也可以使用类似功能的PE38DKEL、PE38RDEL和PE38KNEL等变体。
本公开的抗IL-4R抗体或其抗原结合片段的复合物，可选的进一步包含接头(linker)L1。
接头L1连接毒素和抗IL-4R抗体或其抗原结合片段。
合适的接头L1通常允许本公开的每个组分以三维结构进行折叠，这种三维结构非常类似于各组分在没有任何接头或其它组分的情况下所形成的结构。
在一些实施方案中，接头L1在细胞外是稳定的，使得ADC在存在于细胞外环境中时保持完整，但在例如癌细胞的细胞中内化时能够被裂解。
在一些实施方案中，合适的接头L1可以包括，例如，蛋白酶敏感的、环境氧化还原电位敏感的、pH敏感的、酸可切割的、光可切割的和/或热敏感的接头。
在一些实施方案中，接头L1可以是非蛋白性的，例如，化学接头。非蛋白性化学接头的例子包括但不限于：N-琥珀酰亚胺基(4-碘乙酰基)-氨基苯甲酸酯，S-(N-琥珀酰亚胺基)硫代乙酸酯(SATA)，N-琥珀酰亚胺基-氧基羰基-cu-甲基-a-(2-吡啶基二硫基)甲苯(SMPT)，N-琥珀酰亚胺基4-(2-吡啶基二硫基)-戊酸酯(SPP)，琥珀酰亚胺基4-(N-马来酰亚胺基甲基)环己烷甲酸酯(SMCC或MCC)，磺基琥珀酰亚胺基(4-碘乙酰基)-氨基苯甲酸酯，4-琥珀酰亚胺基-氧基羰基-α-(2-吡啶基二硫基)甲苯，磺基琥珀酰亚胺基-6-(α-甲基-α-(吡啶基二硫醇)-甲苯酰氨基)己酸酯，N-琥珀酰亚胺基-3-(-2-吡啶基二硫基)-丙酸酯(SPDP)，琥珀酰亚胺基6(3(-(-2-吡啶基二硫基)-丙酰氨基)己酸酯，磺基琥珀酰亚胺 基6(3(-(-2-吡啶基二硫基)-丙酰氨基)己酸酯，马来酰亚胺基己酰基(MC)，马来酰亚胺基己酰基-缬氨酸-瓜氨酸-p-氨基苄氧基羰基(MC-vc-PAB)，3-马来酰亚胺基苯甲酸N-羟基琥珀酰亚胺酯(MBS)，α-烷基衍生物，磺基NHS-ATMBA(磺基琥珀酰亚胺基N-[3-(乙酰基硫基)-3-甲基丁酰基-β-丙氨酸])，磺基二氯苯酚，2-亚氨基硫杂环戊烷(2-iminothiolane)，3-(2-吡啶基二硫基)-丙酰基酰肼，Ellman氏试剂，二氯三嗪酸和S-(2-硫代吡啶基)-L-半胱氨酸。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段与毒素形成的复合物是融合蛋白的形式。
在一些实施方案中，接头L1是蛋白性接头，包括但不限定于1个或多个氨基酸或多肽。接头L1典型地包含约2-50个氨基酸残基，例如约5-30个氨基酸残基。通常，蛋白性的接头包含大多数具有极性的、不带电荷的和/或带电荷的残基的氨基酸残基，例如，苏氨酸、脯氨酸、谷氨酰胺、甘氨酸和丙氨酸等。蛋白性的接头的非限制性例子包括丙氨酸-丝氨酸-甘氨酸-甘氨酸-脯氨酸-谷氨酸(ASGGPE)(SEQ ID NO：50)、缬氨酸-甲硫氨酸(VM)、丙氨酸-甲硫氨酸(AM)、AM(G2-4S)xAM，其中G是甘氨酸，S是丝氨酸，且x是1-10的整数。
在一些实施方案中，接头L1选自以下以标准单字母代码列出的肽：ASGCGPE(SEQ ID NO：62)、ASGCCGPE(SEQ ID NO：63)、ASGCGSCPE(SEQ ID NO：64)、ASCGTTGCPE(SEQ ID NO：65)、KASGKKYGCKKGPE(SEQ ID NO：66)、KGGGCAGGPE(SEQ ID NO：67)。
本公开复合物的各个组分，例如抗IL-4R抗体或抗原结合片段、接头L1和毒素，可以通过本领域众所周知的和/或本文描述的方式适当地彼此连接或融合。
在一些实施方案中，抗IL-4R抗体或抗原结合片段通过接头L1与毒素彼此连接或融合。
在一些实施方案中，抗IL-4R抗体或抗原结合片段的C端与接头L1连接或融合。在一些实施方案中，抗IL-4R抗体或抗原结合片段的N端与接头L1连接或融合。在一些实施方案中，抗IL-4R抗体或抗原结合片段C端和N端分别连接或融合相同或不同的接头L1。
在一些实施方案中，毒素是假单胞菌外毒素、假单胞菌外毒素的截短型片段或毒素变体。
在一些实施方案中，毒素是假单胞菌外毒素的截短型片段PE38。
在一些实施方案中，毒素是SEQ ID NO：49所示的PE38KDEL，也可以使用类似功能的PE38DKEL、PE38RDEL和PE38KNEL等变体。
在一些实施方案中，L1是蛋白性接头。
在一些实施方案中，L1选自ASGGPE(SEQ ID NO：50)、ASGCCGPE(SEQ ID NO：63)、ASGCGSCPE(SEQ ID NO：64)、ASCGTTGCPE(SEQ ID NO：65)、KASGKKYGCKKGPE(SEQ ID NO：66)、KGGGCAGGPE(SEQ ID NO：67)、 AM(G 2-4S) xAM，其中x是1-10的整数。
在一些实施方案中，ScFv特异性结合IL-4R，其中VH和VL间用连接肽L2连接，且VH和VL在框架区引入稳定抗体结构的突变。在一些实施方案中，VH-44、VL-100位置的氨基酸突变成半胱氨酸。
在一些实施方案中，ScFv的构建形式是VH-L2-VL或VL-L2-VH。
L2由重复的GGGGS氨基酸序列或其变体组成，例如为(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多，例如n为2、3、4，例如n为3。
在一些实施方案中，上述ScFv中所述VH包含：
(I)氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；或
(II)氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；
和/或所述VL包含：
(I)氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；或
(II)氨基酸序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；或
(III)分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；或
(IV)分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，上述ScFv包含选自以下(I)至(IV)中的任一项：
(I)VH，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
VL，其包含氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
(II)VH，其包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
VL，其包含氨基酸序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
(III)VH，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
VL，其包含氨基酸序列分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；
(IV)VH，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
VL，其包含氨基酸序列分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
在一些实施方案中，上述ScFv中：
VH包含：
(I)如SEQ ID NO：1所示或与SEQ ID NO：1具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：9所示或与SEQ ID NO：9具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：43所示或与SEQ ID NO：43具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
和/或，VL包含：
(I)如SEQ ID NO：2所示或与SEQ ID NO：2具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(II)如SEQ ID NO：10所示或与SEQ ID NO：10具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(III)如SEQ ID NO：37所示或与SEQ ID NO：37具有至少70％、80％、90％、95％、98％、99％同一性的序列；或
(IV)如SEQ ID NO：41所示或与SEQ ID NO：41具有至少70％、80％、90％、95％、98％、99％同一性的序列。
任选地，VH和VL在框架区引入稳定抗体结构的突变，在一些实施方案中，VH-44和VL-100位置的氨基酸突变成半胱氨酸。
在至少一个实施方案中，所述ScFv中VH序列如SEQ ID NO：1所示，VL序列如SEQ ID NO：2所示；或
VH序列如SEQ ID NO：9所示，VL序列如SEQ ID NO：10所示；或
VH序列如SEQ ID NO：43所示，VL序列如SEQ ID NO：37所示；或
VH序列如SEQ ID NO：43所示，VL序列如SEQ ID NO：41所示，
任选地，VH和VL在框架区引入稳定抗体结构的突变。
在一些实施方案中，VH-44和VL-100位置的氨基酸突变成半胱氨酸。VH序列如SEQ ID NO：47所示，VL序列如SEQ ID NO：48所示。
在一些实施方案中，所述ScFv，其中：
VH包含：
(I)如SEQ ID NO：25-27之一所示或与SEQ ID NO：25-27之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：31-33之一所示或与SEQ ID NO：31-33之一具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或VL包含：
(I)如SEQ ID NO：28-30之一所示或与SEQ ID NO：28-30之一具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：34-36之一所示或与SEQ ID NO：34-36之一具有至少70％、80％、90％、95％、98％或99％同一性的序列，
任选地，VH和VL在框架区引入稳定抗体结构的突变。在一些实施方案中，VH-44和VL-100位置的氨基酸突变成半胱氨酸。
在一些具体的实施方案中，VH如序列SEQ ID NO：25-27之一所示，和VL如序列SEQ ID NO：28-30之一所示，VH-44和VL-100位置的氨基酸突变成半胱氨酸；或
VH序列如SEQ ID NO：31-33之一所示，和VL序列如SEQ ID NO：34-36之一所示，VH-44和VL-100位置的氨基酸突变成半胱氨酸。
任选地，VH和VL在框架区引入稳定抗体结构的突变。在一些实施方案中，VH-44和VL-100位置的氨基酸突变成半胱氨酸。
在一些实施方案中，所述ScFv包含重链和轻链，其中重链包含：
(I)如SEQ ID NO：17所示或与SEQ ID NO：17具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：19所示或与SEQ ID NO：19具有至少70％、80％、90％、95％、98％、或99％同一性的序列；或
(III)如SEQ ID NO：44所示或与SEQ ID NO：44具有至少70％、80％、90％、95％、98％、或99％同一性的序列；
和/或轻链包含：
(I)如SEQ ID NO：18所示或与SEQ ID NO：18具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(II)如SEQ ID NO：20所示或与SEQ ID NO：20具有至少70％、80％、90％、95％、98％或99％同一性的序列；或
(III)如SEQ ID NO：45所示或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的序列；或
(IV)如SEQ ID NO：46所示或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的序列；
任选地，VH和VL在框架区引入稳定抗体结构的突变。在一些实施方案中，VH-44和VL-100位置的氨基酸突变成半胱氨酸。
在至少一个实施方案中，重链可变区VH序列如SEQ ID NO：17所示，和轻链可变区VL序列如SEQ ID NO：18所示；或
VH序列如SEQ ID NO：19所示，和VL序列如SEQ ID NO：20所示，其中VH-44和VL-100位置的氨基酸突变成半胱氨酸；或
VH序列如SEQ ID NO：44所示，和VL序列如SEQ ID NO：45所示，其中 VH-44和VL-100位置的氨基酸突变成半胱氨酸；或
VH序列如SEQ ID NO：44所示，和VL序列如SEQ ID NO：46所示，其中VH-44和VL-100位置的氨基酸突变成半胱氨酸。
在一些实施方案中，抗IL-4R抗体或其抗原结合片段的复合物具有如SEQ ID NO：52或SEQ ID NO：53所示的序列。
本公开还提供了一种抗IL-4R抗体或其抗原结合片段的复合物，其包含：抗IL-4R抗体或其抗原结合片段以及一个或多个毒素；其中，所述毒素选自成孔毒素、嗜水气单胞菌气溶素、气单胞菌溶素原、布加宁、蓖麻毒素、假单胞菌外毒素、霍乱毒素或白喉毒素；优选地，所述毒素选自PE-LR、PE-LO10R456A、PE-T20、PE-T20-KDEL、PE4E、PE40、PE38、PE24、PE25、PE38QQR、PE35、PE38KDEL、PE38DKEL、PE38RDEL、PE38KNEL；更优选地，所述毒素是如SEQ ID NO：49所示的PE38KDEL。
本公开提供一种多核苷酸，其编码本公开所述的任一种抗IL-4R抗体或其抗原结合片段的复合物。在其中一些实施方案中，多核苷酸为DNA或RNA。
本公开提供一种含有如上所述多核苷酸的载体(vector)，其为真核表达载体、原核表达载体或病毒载体。
本公开提供一种包含如上所述的载体的宿主细胞，所述宿主细胞选自原核细胞或真核细胞。在至少一个实施方案中，原核细胞选自细菌，例如大肠杆菌。在至少一个实施方案中，真核细胞选自酵母菌或哺乳动物细胞，例如毕赤酵母或CHO细胞或人胚肾(HEK)293细胞。
本公开提供一种用于制备抗IL-4R抗体或其抗原结合片段的复合物的方法，包括如下步骤：
在上述宿主细胞中表达抗IL-4R抗体或其抗原结合片段的复合物，以及从所述宿主细胞中分离所述抗IL-4R抗体或其抗原结合片段的复合物；以及任选地，纯化所述抗IL-4R抗体或其抗原结合片段的复合物。
药物组合物
本公开进一步提供一种药物组合物，其活性成分含有如上所述的抗IL-4R抗体或其抗原结合片段的复合物，其用作药物。
本公开的药物组合物除活性成分外，可含有一种或多种可药用的赋形剂、稀释剂或载体(vehicle)。药物组合物可含有0.1至99重量％的活性成分。
本公开进一步提供选自以下的任一项或组合在制备药物中的用途：根据本公开的抗IL-4R抗体或其抗原结合片段、抗IL-4R抗体或其抗原结合片段的复合物、根据本公开的药物组合物；其中，所述抗体或其抗原结合片段或其复合物用于治疗和/或预防增殖性疾病或延缓增殖性疾病进展。所述增殖性病症可以是癌症或肿瘤；例如其中所述的癌症或肿瘤为与IL-4R表达相关的癌症或肿瘤。所述癌症或肿瘤选自***癌、卵巢癌、乳腺癌、子宫内膜癌、多发性骨髓瘤、黑素瘤、淋 巴瘤(例如，霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)、肺癌、肾癌、肝癌(例如，小细胞肺癌和非小细胞肺癌)、大肠癌(例如结肠癌)、胰腺癌、胃癌、白血病(例如，急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)和脑癌、中枢神经***肿瘤。
在一些实施方案中，所述中枢神经***肿瘤包括神经胶质瘤、神经胶质母细胞瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质瘤、血管瘤、脑膜瘤、神经母细胞瘤、视网膜母细胞瘤、成年母细胞瘤。
在一些实施方案中，所述中枢神经***肿瘤是神经胶质母细胞瘤。
在一些实施方案中，所述神经胶质母细胞瘤是复发性或难治性胶质母细胞瘤。
在一些实施方案中，所述神经胶质母细胞瘤中O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyhransferase，MGMT)表达为阳性或阴性。
治疗方法
本公开提供一种治疗和/或预防增殖性疾病或延缓增殖性疾病进展的方法，该方法包括给予需要的受试者治疗或延缓疾病有效量的根据本公开的抗IL-4R抗体或其抗原结合片段、或根据本公开的药物组合物、或根据本公开的抗IL-4R抗体或其抗原结合片段的复合物；其中，所述增殖性病症可以是癌症或肿瘤。
本公开提供在患有、疑似患有、易感于细胞增殖性病症的受试者中增强免疫功能的方法。一些实施方案中，所述细胞增殖性病症为癌症或肿瘤。
例如，所述的癌症或肿瘤为与IL-4R表达相关的癌症或肿瘤。所述癌症或肿瘤选自***癌、卵巢癌、乳腺癌、子宫内膜癌、多发性骨髓瘤、黑素瘤、淋巴瘤(例如，霍奇金淋巴瘤、非霍奇金淋巴瘤或复发性间变性大细胞淋巴瘤)、肺癌、肾癌、肝癌(例如，小细胞肺癌和非小细胞肺癌)、大肠癌(例如结肠癌)、胰腺癌、胃癌、白血病(例如，急性淋巴细胞白血病、急性髓细胞白血病、急性早幼粒细胞白血病、慢性髓细胞白血病、慢性淋巴细胞白血病)和脑癌、中枢神经***肿瘤。
在一些实施方案中，所述中枢神经***肿瘤包括神经胶质瘤、神经胶质母细胞瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质瘤、血管瘤、脑膜瘤、神经母细胞瘤、视网膜母细胞瘤、成年母细胞瘤。
在一些实施方案中，所述中枢神经***肿瘤是神经胶质母细胞瘤。
在一些实施方案中，所述神经胶质母细胞瘤是复发性或难治性胶质母细胞瘤。
在一些实施方案中，所述神经胶质母细胞瘤中O6-甲基鸟嘌呤-DNA甲基转移酶(O6-methylguanine-DNA methyhransferase,MGMT)表达阳性或阴性。
本公开治疗和/或预防方法中所用化合物(如本公开的复合物或组合物)的剂 量通常将随疾病的严重性、受试者的体重和化合物的相对功效而改变。作为一般性指导，合适的单位剂量可以是0.1mg至1000mg。
如本领域技术人员所熟知的，药物的给药剂量依赖于多种因素，包括但并非限定于以下因素：所用具体化合物的活性、受试者的年龄、受试者的体重、受试者的健康状况、受试者的行为、受试者的饮食、给药时间、给药方式、***的速率、药物的组合等，可以根据公知的治疗方案来验证。
附图说明
图1A和图1B.示例性的复合物的结构示意图；抗体25G7的scdsFv与毒素PE38KDEL的抗体偶联药物25G7-scdsFv-PE38KDEL(图1A)和25G7-scFv-PE38KDEL(图1B)。
图2A和图2B.复合物25G7-scdsFv-PE38KDEL和25G7-scFv-PE38KDEL纯化后的SDS-PAGE图，其中M为分子量标记(Marker)。
图3.流式细胞法检测神经胶质母细胞瘤(GBM)细胞系中的IL-4R表达水平。利用慢病毒***构建人IL-4R稳转的LN229、U251、U87脑胶质瘤细胞株，用流式细胞法检测IL-4R的过表达水平，对照为未转染人IL-4R构建体的对应细胞。
图4A至图4C.流式细胞法检测复合物在神经胶质母细胞瘤细胞表面的结合。利用流式细胞法测定25G7-scdsFv-PE38KDEL和MDNA55分别在IL-4R稳转细胞株LN229-IL4R、U251-IL4R、U87-IL4R细胞表面的结合能力。
图5A至图5C.复合物在神经胶质母细胞瘤细胞中的内吞效率；利用流式细胞法测定25G7-scdsFv-PE38KDEL(25G7-IT)和MDNA55分别在LN229-IL4R、U251-IL4R、U87-IL4R细胞中0至24小时的内吞效率。
图6A至图6F.复合物对神经胶质母细胞瘤细胞的体外细胞毒性；用CTG法测定25G7-scdsFv-PE38KDEL(25G7-IT)和MDNA55分别对LN229-IL4R、U251-IL4R、U87-IL4R以及对照细胞的体外细胞毒性结果。
图7A至图7D.用CTG法比较25G7-scFv-PE38KDEL与25G7-scdsFv-PE38KDEL、MDNA55分别在LN229-IL4R、U251-IL4R以及对照神经胶质瘤细胞的体外细胞毒性结果。
图8A至图8C.复合物在U87-MG皮下接种的裸鼠肿瘤模型中体内药效比较结果；25G7-scdsFv-PE38KDEL(25G7-IT)和MDNA55的给药剂量均为0.5mg/kg(mpk)，瘤内给药(i.t.)。
图9A至图9C.复合物在LN229-IL4R(过表达IL-4R的LN229细胞)皮下接种裸鼠肿瘤模型中的体内药效比较结果；25G7-scdsFv-PE38KDEL(25G7-IT)和MDNA55药物给药剂量均为0.25mpk(i.t.)，或25G7-scdsFv-PE38KDEL 0.25mpk(i.v.)。
图10A至图10C.复合物在LN229-IL4R皮下接种裸鼠肿瘤模型中的体内药效 结果；25G7-scdsFv-PE38KDEL(25G7-IT)药物给药剂量为0.1mpk(第二次及以后降为0.05mpk)、0.3mpk(第二次及以后降为0.15mpk)、MDNA55药物给药剂量为0.1mpk(第二次及以后降为0.05mpk)，瘤内给药(i.t.)。
具体实施方式
术语
为了更容易理解本申请，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本申请所属领域的一般技术人员通常理解的含义。
本公开将专利文本WO2020/038454A1的内容全部引入本申请。
本申请所用氨基酸三字母代码和单字母代码如J.Biol.Chem，243，p3558(1968)中所述。
“人IL-4R”(hIL-4R)意指与白细胞介素-4(IL-4)、IL-4Rα特异性结合的人细胞因子受体。hIL-4R旨在涵盖IL-4R在体内各阶段中的各种形式的分子，例如但不限于IL-4R基因在扩增、复制、转录、剪接、加工、翻译、修饰过程中所产生的分子，例如前体IL-4R、成熟IL-4R、天然存在的IL-4R剪接变体、修饰的IL-4R、或其片段。
“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM，IgD，IgG，IgA和IgE，其相应的重链分别为μ链，δ链，γ链，α链和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1，IgG2，IgG3，IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ链或λ链。
抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1，IgG2，IgG3，IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(V区)；靠近C端的其余氨基酸序列相对稳定，为恒定区(C区)。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指轻链互补决定区1(LCDR1)，轻链互补决定区2(LCDR2)，和轻链互补决定区3(LCDR3)；重链的3个CDR区指重链互补决定区1(HCDR1)，重链互补决定区2(HCDR2)和重链互补决定 区3(HCDR3)。
抗体包括鼠源抗体，嵌合抗体，人源化抗体，全人抗体，其可以是重组获得的，例如可以是亲和力成熟得到的重组全人抗体。
“重组全人抗体”包括通过重组方法制备、表达、创建或分离的全人抗体，所涉及的技术和方法在本领域中是熟知的，诸如(1)从人免疫球蛋白基因的转基因、转染色体动物(例如小鼠)或由其制备的杂交瘤中分离的抗体；(2)从经转化以表达抗体的宿主细胞如转染瘤中分离的抗体；(3)从重组组合人抗体文库中分离的抗体；以及(4)通过将人免疫球蛋白基因序列剪接到其他DNA序列等方法制备、表达、创建或分离的抗体。此类重组全人抗体包含可变区和恒定区，这些区域利用特定的由种系基因编码的人种系免疫球蛋白序列，但也包括随后诸如在抗体成熟过程中发生的重排和突变。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)和NS0细胞。
“鼠源抗体”在本文中为根据本领域知识和技能制备的对人IL-4R的单克隆抗体。制备时用IL-4R抗原(或包含表位的多肽)注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。在一些实施方案中，所述的鼠源结合人IL-4R的抗体或其抗原结合片段，可进一步包含鼠源κ、λ链或其变体的轻链恒定区，或进一步包含鼠源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区。
“全人抗体”包括具有人种系免疫球蛋白序列的可变和恒定区的抗体。本文的全人抗体可包括不由人种系免疫球蛋白序列编码的氨基酸残基(如通过体外随机或位点特异性诱变或通过体内体细胞突变所引入的突变)。然而，术语“全人抗体”不包括这样的抗体，即其中已将衍生自另一种哺乳动物物种(诸如小鼠)种系的CDR序列移植到人骨架序列上(即“人源化抗体”)。
“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将非人CDR序列移植到人的抗体可变区框架中产生的抗体。可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的强烈的免疫应答反应。为避免在免疫原性下降的同时引起活性的下降，可对所述的人抗体可变区可进行最少反向突变，以保持活性。
“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要选建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区 基因，再要据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。人抗体的恒定区可选自人源IgG1，IgG2，IgG3或IgG4或其变体的重链恒定区，例如包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG1。
“抗原结合片段”指具有抗原结合活性的Fab片段，Fab’片段，F(ab’)2片段，与人IL-4R结合的Fv片段、scFv片段，以及，包含所述片段的多肽或蛋白。所述“抗原结合片段”包含本文所述抗体的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(scFv)。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头(或连接肽)连接的重链可变结构域(或区域；VH)和轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成，例如包含1-4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA90：6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8：725-731，Choi等人(2001)，Eur.J.Immuno l.31：94-106，Hu等人(1996)，Cancer Res.56：3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293：41-56和Roovers等人(2001)，Cancer Immunol.描述。
术语“抗体框架”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
“与IL-4R结合”，指能与人IL-4R(或其表位、片段)相互作用。本文的术语“抗原结合位点”指由本文抗体或抗原结合片段识别的三维空间位点。
“表位”是指抗原上与免疫球蛋白或抗体特异性结合的位点。表位可以由相邻的氨基酸形成、或不相邻的氨基酸通过三级折叠而形成。由相邻的氨基酸形成的表位通常在暴露于变性溶剂后保持，而通过三级折叠形成的表位通常在变性溶剂处理后丧失。表位通常以独特的空间构象包括至少3-15个氨基酸。确定什么表位被给定抗体所结合的方法在本领域中是熟知的，包括免疫印迹和免疫沉淀检测分析等。确定表位的空间构象的方法包括本领域中的技术和本文所述的技术，例如X射线晶体分析法和二维核磁共振等。
“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体与预定的抗原上的表位结合。通常，当使用重组人IL-4R作为分析物并使用抗体作为配体，在仪器中通过表面等离子体共振(SPR)技术测定时，抗体以大约低于 10-7M或甚至更小的平衡解离常数(KD)与预定的抗原结合，并且其与预定抗原结合的亲和力是其与预定抗原或紧密相关的抗原之外的非特异性抗原(如BSA等)结合的亲和力的至少两倍。术语“识别抗原的抗体”在本文中可以与术语“特异性结合的抗体”互换使用。
“交叉反应”是指本文的抗体与来自不同物种的IL-4R结合的能力。例如，结合人IL-4R的本文的抗体也可以结合另一物种的IL-4R。交叉反应性是通过在结合测定(例如SPR和ELISA)中检测与纯化抗原的特异性反应性，或与生理表达IL-4R的细胞的结合或功能性相互作用来测量。确定交叉反应性的方法包括如本文所述的标准结合测定，例如表面等离子体共振(SPR)分析，或流式细胞术。
“中和”或“阻断”抗体意指其与hIL-4R的结合造成对hIL-4和/或hIL-13生物活性抑制的抗体。这种hIL-4和/或IL-13生物活性的抑制可通过测量本领域众所周知的一个或多个hIL-4/或hIL-13生物活性指标，如hlL-4/或hIL-13诱导的细胞活化和hIL-4与hIL-4R的结合等指标来评估。例如参见CN103739711A中的。“抑制生长”(例如涉及细胞)旨在包括细胞生长任何可测量的降低。
“诱导免疫应答”和“增强免疫应答”可互换使用，并指免疫应答对特定抗原的刺激(即，被动或适应性的)。针对诱导CDC或ADCC的术语“诱导”是指刺激特定的直接细胞杀伤机制。
“ADCC(antibody-dependent cell-mediated cytotoxicity)”，抗体依赖的细胞介导的细胞毒作用，是指表达Fc受体的细胞通过识别抗体的Fc段直接杀伤被抗体包被的靶细胞。可通过对IgG上Fc段的修饰，降低或消除抗体的ADCC效应功能。所述的修饰指在抗体的重链恒定区进行突变，如选自IgG1的N297A，L234A，L235A；IgG2/4嵌合体，IgG4的F235E，或L234A/E235A突变。
融合蛋白是一种通过DNA重组得到的两个基因共表达的蛋白产物。例如抗IL-4R抗体-PE38KDEL融合蛋白通过DNA重组，把抗IL-4R抗体和毒素分子PE38KDEL共表达的融合蛋白。生产和纯化抗体和抗原结合片段的方法在现有技术中熟知和能找到，如冷泉港的抗体实验技术指南，5-8章和15章。如，小鼠可以用人IL-4R或其片段免疫，所得到的抗体能被复性，纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本文所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人FR。人FR种系序列可以从ImMunoGeneTics(IMGT)的网站得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
工程化的抗体或抗原结合片段可用常规方法制备和纯化。作为一个示例，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。利用分子克隆技术将本文的人源化抗体的序列***到相应的表达载体中，利用HEK293细胞表达***表达生产，即可获得相应的人源化抗体。作为一个示例，哺乳动物类表达***会导致抗体的糖基化，特 别是在Fc区的高度保守N端。通过表达与人源抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化、收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛，离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
抗体可以是单克隆抗体(mAb)，是指由单一的克隆细胞株得到的抗体，所述的细胞株不限于真核的，原核的或噬菌体的克隆细胞株。单克隆抗体或抗原结合片段可以用如杂交瘤技术、重组技术、噬菌体展示技术，合成技术(如CDR-grafting)，或其它现有技术进行重组得到。
抗体可以是单特异、双特异或多特异抗体。多特异性抗体可对靶标肽的不同表位显示特异性，或也可包含对一个以上靶标肽显示特异性的抗原结合域。人抗IL-4R抗体可被连接到另一个功能分子(如另一个肽或蛋白质)上，或与之共表达。例如，抗体或其片段可功能性地(如通过化学偶联、基因融合、非共价结合或其他方式)与一个或多个其他分子(如另一个抗体或抗原结合片段)连接，以产生具有至少一种结合特异性的双特异或多特异抗体。
“施用”、“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“施用”、“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“施用”、“给予”和“处理”还意指通过试剂、诊断、组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施、研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，诸如本文的任一种复合物或组合物，所述受试者具有(疑似具有、易感于)一种或多种疾病症状，而所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，无论是通过诱导这类症状退化还是抑制这类症状发展到任何临床右测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本本的实施方案(例如治疗方法或制品)在缓解单个受试者的目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
应用于某个对象的术语“天然存在的”是指这样的事实，即该对象可在自然界中发现。例如存在于可从自然界来源分离得到的生物体(包括病毒)、且未经人工有意修饰的多肽序列或多核苷酸序列即是天然存在的。
“有效量”包含足以改善或预防医学病症的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定受试者或兽医学受试者的有效量可依据以下因素而变化：如待治疗的病症、受试者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“外源性”指根据背景在生物、细胞或人体外产生的物质。
“内源性”指根据背景在生物、细胞或人体内产生的物质。
“同源性”或“同一性”是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100％的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源。一般而言，当比对两个序列而得到最大的同源性百分率时进行比较。本文所述“至少85％序列同一性”是指变体与亲本序列相比，两序列具有至少85％同源，在一些方案中，其具有至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同源；在一些具体的方案中，其具有90％、95％或99％以上；在另一些具体的方案中，其具有至少95％序列同源。所述具有至少85％序列同一性的氨基酸序列包括通过对亲本序列进行一个或者多个氨基酸缺失、***或替换突变获得。
本文使用的术语“细胞”、“细胞系”、“细胞株”和“细胞培养物”可互换使用，并且所有这类术语都包括其后代。因此，术语“转化体”和“转化细胞”包括原代受试细胞和由其衍生的培养物，而不考虑传代的次数。还应当理解的是，由于有意或非有意的突变，后代和亲本细胞在DNA含量方面不可能精确相同。该术语包括具有与亲本细胞相同的功能或生物学活性的突变后代，其中亲本细胞是从初始转化的细胞中筛选所得。
“任选”或“任选地”意味着随后所描述的事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。例如，“任选包含1-3个抗体重链可变区”意味着抗体重链可变区可以但不必须存在。
术语“载体(vector)”是指能够运输已与其连接的另一个核酸的核酸分子。在一个实施方案中，载体是“质粒”，其是指可将另外的DNA区段连接至其中的环状双链DNA环。在另一个实施方案中，载体是病毒载体，其中可将另外的DNA区段连接至病毒基因组中。本文中公开的载体能够在已引入它们的宿主细胞中自 主复制(例如，具有细菌的复制起点的细菌载体和附加型哺乳动物载体)或可在引入宿主细胞后整合入宿主细胞的基因组，从而随宿主基因组一起复制(例如，非附加型哺乳动物载体)。
“药物组合物”表示含有一种或多种本文所述复合物或其生理学上/可药用的盐或前体药物与其他组分的混合物，其中所述其他组分是例如生理学/可药用的载体(vehicle)或赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“赋形剂”是在药物制剂中除活性成分以外的附加物，也可称为辅料。如片剂中的黏合剂、填充剂(filler)、崩解剂、润滑剂；半固体制剂软膏剂、霜剂中的基质部分；液体制剂中的防腐剂、抗氧剂、矫味剂、芳香剂、助溶剂、乳化剂、增溶剂、渗透压调节剂、着色剂等均可称为赋形剂。
术语“稀释剂(diluent)”又称填充剂(filler)，其主要用途是增加片剂的重量和体积。稀释剂的加入不仅保证一定的体积大小，而且减少主要成分的剂量偏差，改善药物的压缩成型性等。当片剂的药物含有油性组分时，需加入吸收剂吸收油性物，使保持“干燥”状态，以利于制成片剂。如淀粉、乳糖、钙的无机盐、微晶纤维素等。
“免疫组织化学评分”是处理免疫组化结果的一种组织学评分方法，将每张切片内阳性的细胞数量及其染色强度转化为相应的数值，达到对组织染色半定量的目的。评分0表示未观察到染色或在小于10％的肿瘤细胞中观察到膜染色；评分+1表示在大于10％的肿瘤细胞中检测到微弱/勉强可觉察的膜染色；对于评分+2，在大于10％的肿瘤细胞中观察到中等完全膜染色；评分+3表示在大于10％的肿瘤细胞中观察到强的完全膜染色。在本公开中，IL-4R表达为0或+1评分的那些样品可以视为不过表达IL-4R，而+2或+3评分的那些样品可以视为过表达IL-4R。
以下结合实施例用于进一步描述本申请，但这些实施例并非限制保护范围。
实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.示例性免疫毒素的制备
将人源化IL-4R单克隆抗体hu25G7(重链可变区序列如SEQ ID NO：43所示，hu25G7-VH；轻链可变区序列如SEQ ID NO：37所示，hu25G7-A LCVR)的单链可变区段(scFv)通过接头ASGGPE(SEQ ID NO：50)与突变且截短的假单胞菌外毒素(PE38KDEL)(SEQ ID NO：49)融合表达；其中将hu25G7抗体VH-44和VL-100位置的氨基酸突变成半胱氨酸(如SEQ ID NO：47-48)，VH与VL由 连接肽(GGGGS) 3(SEQ ID NO：51)连接；据此获得靶向IL-4R的免疫毒素25G7-(scdsFv)-PE38KDEL(以下称25G7-IT)，完整序列如SEQ ID NO：52所示。
图1A和图1B是两种免疫毒素的结构示意图。在大肠杆菌BL21中表达25G7-IT，从包涵体中进行蛋白纯化和复性；然后去除内毒素，获得纯化的25G7-IT蛋白，分子量约为63KDa(SDS-PAGE结果如图2A和图2B所示)。
人源化抗人IL-4R的抗体25G7的重链序列如SEQ ID NO：17所示，轻链序列如SEQ ID NO：18所示。可以根据本领域常规的抗体表达和纯化方法，例如根据专利申请WO2020038454A1中描述的方法表达和纯化抗体。前述专利申请通过引用将其全文并入本文中。
hu25G7-VH-44序列
Figure PCTCN2021140310-appb-000034
hu25G7-VL-100序列
Figure PCTCN2021140310-appb-000035
PE38KDEL序列：
Figure PCTCN2021140310-appb-000036
接头L1序列：
ASGGPE         SEQ ID NO：50；
连接肽L2序列：
GGGGSGGGGSGGGGS         SEQ ID NO：51；
25G7-(scdsFv)-PE38KDEL(25G7-IT)序列：
Figure PCTCN2021140310-appb-000037
Figure PCTCN2021140310-appb-000038
此外，我们还开发了没有额外引入链间二硫键形式的免疫毒素融合蛋白25G7-(scFv)-PE38KDEL，其氨基酸序列如SEQ ID NO：53所示。
25G7-(scFv)-PE38KDEL序列：
Figure PCTCN2021140310-appb-000039
实施例2.相互作用的亲和动力学测定
使用Biacore生物大分子相互作用仪(biacore 8K,GE)，通过表面等离子共振(Surface Plasmon Resonance，SPR)技术，测定25G7-IT和参照样品MDNA55(在大肠杆菌***中进行表达纯化)分别与hrIL-4R-Fc(ILR-H5253，Acro)相互作用的亲和动力学。
本实验采用捕获法，将抗人IgG抗体(29234600，GE)共价偶联到CM5芯片(29149603，GE)上，然后将rhIL-4R-Fc作为配体捕获于芯片上，随后将25G7-IT和参照样品MDNA55作为分析物分别进样，进行亲和力分析和计算。
实验结果显示，25G7-IT和参照样品MDNA55与rhIL-4R-Fc具有强亲和力，KD分别为1.01E-10和4.39E-10M(表2)。25G7-IT与rhIL-4R-Fc亲和力显著强于参照样品MDNA55与rhIL-4R-Fc亲和力。
表2.Biacore测定的复合物的动力学亲和力
Figure PCTCN2021140310-appb-000040
实施例3.与GBM靶细胞的结合能力测定
GBM细胞系U87、U251、LN229中内源性IL-4R表达比较低，因此利用慢病毒***在这三株GBM细胞系中过表达人源IL-4R。经过G418筛选后，利用流式细胞技术鉴定IL-4R过表达的效果(图3)，结果显示IL-4R在这三株细胞系中稳定过表达。
在DMEM培养基中加入10％FBS和1％青霉素/链霉素制备成完全培养基，在37℃含5％CO 2培养箱中培养U87-、U251-、LN229-IL4R稳转细胞株。T75培养瓶里的细胞用1.5ml TrpLE(Gibco，#12605-010)在37℃消化3-5分钟，然后用含有FBS的DMEM培养基中和，离心后制备成浓度为2×10 6个/ml的细胞悬液，以每孔50μl的体积加入96孔圆底板。将25G7-IT、MDNA55以及hIgG1同种型对照分别用DMEM培养基稀释成8个浓度点(三倍梯度：20；6.67；2.22；0.74；0.25；0.08；0.03；0μg/ml)，分别以每孔50μl的体积加入96孔板与细胞混合均匀，并在冰上共孵育40分钟。孵育结束后，用200μl的流式检测缓冲液洗涤三次，用流式检测缓冲液1：300稀释抗PE38抗体(Sigma-aldrich，Cat：P2318-1ML)，并以每孔50μl的体积加入96孔板重悬细胞，让抗体和细胞在冰上共孵育40分钟。接下来用200μl的流式检测缓冲液洗涤三次，用流式检测缓冲液1：500稀释抗兔IgG Fab2 Alexa Fluor 647(Cell Signaling Technology，#02/2020)，并以每孔50μl的体积加入96孔板重悬细胞，让抗体和细胞在冰上共孵育30分钟。离心并用200μl的流式检测缓冲洗涤三次后重悬细胞，用BD FACSCelestaTM流式细胞仪检测每孔单细胞Alexa Fluor647的信号。
分析平均荧光强度(MFI)，实验结果如表3和图4A至图4C显示，25G7-IT与参照样品MDNA55均能与IL-4R阳性GBM细胞系结合，而且25G7-IT与U87-、U251-、LN229-IL4R稳转细胞株的结合能力强于MDNA55。
表3.复合物与GBM靶细胞的结合能力测试结果
Figure PCTCN2021140310-appb-000041
Figure PCTCN2021140310-appb-000042
实施例4.GBM靶细胞中的内吞效率测定
为测定25G7-IT与参照样品MDNA55在GBM靶细胞中的内吞效率，比较了25G7-IT与MDNA55分别在上述三株IL-4R稳转脑胶质瘤细胞中的内吞效率。
根据实施例3中的实验结果，选择终浓度2μg/ml作为检测内吞效率的浓度，因为在该浓度时三种细胞样品中的内吞效率基本到达饱和状态。将25G7-IT、MDNA55以及hIgG1同种型对照用DMEM培养基稀释成2μg/ml。以每孔50μl的体积加入96孔板与细胞混合均匀，并在冰上共孵育40分钟。利用DMEM完全培养基洗涤三次后，用100μlDMEM完全培养基重悬细胞，将96孔板中标注为0h的100μl细胞转移到另一块96孔圆底板，并加入100μl的4％多聚甲醛固定液，4℃固定。将剩下的细胞放入37℃培养箱中分别培养1小时、2小时、4小时和24小时后，取出相应的细胞转移到另一块96孔板中以同样的方法4℃固定。
0小时至4小时内吞的细胞在当天进行流式抗体染色；24小时内吞后的细胞在孵育至实验结束时，即进行流式抗体染色和流式分析。当4小时内吞的细胞被固定达半小时以后，将所有固定的细胞离心并用流式检测缓冲洗涤两次。用流式检测缓冲液1：300稀释抗PE38抗体(Sigma-aldrich，Cat：P2318-1ML)，并以每孔50μl的体积加入96孔板重悬细胞，接下来将抗体和细胞在冰上共孵育40分钟。后续实验步骤同实施例3，分析结果如表4和图5A至图5C所示。
实验结果显示，25G7-IT与参照样品MDNA55均能在IL-4R过表达GBM细胞中有效内吞，在4小时时间点于三种细胞中内吞效率达60％至80％，而且25G7-IT在LN229-、U251-IL4R稳转细胞株中的内吞效率高于MDNA55。
表4.GBM靶细胞中的内吞效率测定结果
Figure PCTCN2021140310-appb-000043
实施例5.GBM靶细胞中的体外杀伤效率测定
为测定25G7-IT与参照样品MDNA55在GBM靶细胞中的体外杀伤效率，通过CTG(CELL TITER-GLO)法比较了两者在上述三株稳转人IL-4R脑瘤细胞中的体外药效。
DMEM培养基中加入10％FBS和1％青霉素/链霉素制备成完全培养基。在37℃含5％CO 2的培养箱中，培养U87-IL4R、U251-IL4R、LN229-IL4R稳转细胞株和U87、U251、LN229对照细胞。
在96孔板中按照每孔4000个细胞以及130μl培养基的密度进行铺板。过夜后，待细胞完全贴壁，加入20μl分别含25G7-IT或MDNA55的梯度浓度药物(终浓度：100nM、20nM、4nM、0.8nM、0.16nM、0.032nM、0.0064nM、0.00128nM、0.000256nM、0nM)，在培养箱中培养3天。三天后，将96孔板拿出培养箱并平衡到室温，在细胞悬液中加入50μl的CTG试剂(CellTiter-Glo Luminescent，Promega，#G7573)，用摇床混匀2分钟后，室温静置10分钟。用EnVision2105 Multimode Plate Reader(PerkinElmer)中内置的Luminescence模式检测细胞活率。将吸光度结果换算成百分比后用Prism 8分析结果。
实验结果(图6A至图6F)显示，25G7-IT与参照样品MDNA55均能在IL-4R过表达GBM细胞中高效杀伤靶细胞，特别是对LN229-IL4R有较高的药效，于0.1nM浓度达到80％的体外杀伤效果。总体来看，25G7-IT在LN229、U251、U87三株IL-4R稳转脑胶质瘤细胞株中的体外杀伤效率高于MDNA55。
此外，还比较了没有链间二硫键形式的药物25G7-scFv-PE38KDEL与25G7-scdsFv-PE38KDEL(25G7-IT)、MDNA55在LN229和U251IL-4R稳转脑胶质瘤细胞株中的体外杀伤效率。图7A至图7D显示25G7-scFv-PE38KDEL与25G7-IT、MDNA55在脑胶质瘤细胞中的体外杀伤效率相当。
实施例6.靶向IL-4R的药物在GBM肿瘤模型中的体内药效
为测定25G7-IT与参照样品MDNA55在GBM肿瘤模型中的体内药效，首先构建了IL-4R表达为1+(免疫组织化学评分)的U87-MG皮下接种的模型。
在实验小鼠于右侧背部皮下接种2×10 6U87-MG细胞，细胞重悬在1：1的PBS与基质胶中(0.1ml/只)，定期观察肿瘤生长情况。当皮下接种肿瘤的平均大小达到约100-150mm 3时，进行分组，每组8只小鼠。多位点瘤内给药，药物25G7-scdsFv-PE38KDEL(25G7-IT)和MDNA55的给药剂量均为0.5mg/kg(mpk)，每周给药一次，总共给药四次。开始给药后，每周测量两次小鼠的体重和肿瘤的大小，肿瘤体积计算公式：肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。
实验结果如图8A至图8C和表5显示，在IL-4R低表达的U87-MG肿瘤模型中(免疫组织化学评分为1+)，0.5mpk每周给药一次，给药4次后，25G7-IT的TGI(肿瘤生长抑制率)为80％，而参照样品MDNA55的TGI为51％。该结果说明在IL-4R低表达的神经胶质瘤模型中，25G7-IT的肿瘤生长抑制效果显著优于MDNA55。
表5.靶向IL-4R的药物在小鼠胶质瘤模型中的体内药效总结
Figure PCTCN2021140310-appb-000044
注：s.c.：皮下；mpk：mg/kg(毫克每千克体重)；i.t.：瘤内注射；i.v.：静脉注射；QW：每周一次。**表示p<0.01,***表示p<0.001。
实施例7.靶向IL-4R的药物在GBM肿瘤模型(IL-4R 3+)的体内药效
为进一步测定25G7-IT与参照样品MDNA55在GBM肿瘤模型中的体内药效，利用IL-4R过表达的LN229细胞系(标注为LN229-IL4R，免疫组织化学评分为3+)建立了皮下接种的模型。
在实验小鼠于右侧背部皮下接种1×10 7LN229-IL4R细胞，细胞重悬在1：1 的PBS(0.1ml/只)，定期观察肿瘤生长情况。当皮下接种肿瘤的平均大小达到约100-150mm 3时，进行分组，每组6-7只小鼠。MDNA55给药剂量为0.25mpk(i.t.)，25G7-scdsFv-PE38KDEL(25G7-IT)剂量为0.25mpk(i.t.或i.v.)，每周给药一次，总共给药四次，肿瘤生长监测和计算同实施例6。
实验结果如图9A至图9C和表6显示，在IL-4R高表达的LN229-IL4R肿瘤模型中(免疫组织化学评分为3+)，0.25mpk(i.t.)的25G7-IT、MDNA55的TGI(肿瘤生长抑制率)均达到99％(完全缓解)，而同剂量0.25mpk的25G7-IT(i.v.)的TGI为68％，瘤内给药效果优于静脉给药。
此外，在IL-4R高表达的LN229-IL4R肿瘤模型中(免疫组织化学评分为3+)，进一步降低了免疫毒素的剂量，25G7-scdsFv-PE38KDEL(25G7-IT)给药剂量为0.1(第二次及以后降为0.05)、0.3(第二次及以后降为0.15)mpk，MDNA55给药剂量为0.1(第二次及以后降为0.05)mpk，瘤内给药。
实验结果如图10A至图10C和表6显示，在LN229-IL4R肿瘤模型中(免疫组织化学评分为3+)，0.1(第一次给药)或0.05(第二至第四次给药)mpk每周给药一次，给药4次后，25G7-IT的TGI(肿瘤生长抑制率)为77％，而参照样品MDNA55的TGI为59％；而且25G7-IT药物在0.3(第一次给药)或0.15(第二至第四次给药)mpk的剂量下，荷瘤小鼠的肿瘤完全缓解(TGI＝99％)。该结果说明在IL-4R高表达的神经胶质瘤模型中，低浓度的25G7-IT的肿瘤生长抑制效果优于等剂量的MDNA55，而高剂量的25G7-IT药物可以让荷瘤小鼠完全缓解，呈现明显的剂量效应。
表6.靶向IL-4R的药物在小鼠胶质瘤模型中的体内药效总结
Figure PCTCN2021140310-appb-000045
注：s.c.：皮下；mpk：mg/kg(毫克每千克)；i.t.：瘤内注射；i.v.：静脉注射；QW：每周一次。**表示p<0.01,***表示p<0.001。
本公开中实验动物的使用及福利遵照国际实验动物评估和认可委员会(AAALAC)的规定。每天监测动物的健康状况及死亡情况，例行检查包括观察受试物和药物对动物日常行为表现的影响如行为活动，体重变化，外观体征等。

Claims (22)

  1. 一种抗IL-4R抗体或其抗原结合片段的复合物，其包含：
    -抗IL-4R抗体或其抗原结合片段、以及
    -一个或多个毒素分子；
    所述抗IL-4R抗体或其抗原结合片段与所述毒素分子共价或非共价连接；
    所述抗IL-4R抗体或其抗原结合片段包含选自以下(I)至(IV)中的任一项：
    (I)
    重链可变区，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含氨基酸序列分别如SEQ ID NO：38、SEQ ID NO：7和SEQ ID NO：40所示的LCDR1、LCDR2和LCDR3；
    (II)
    重链可变区，其包含氨基酸序列分别如SEQ ID NO：11、SEQ ID NO：12和SEQ ID NO：13所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含氨基酸序列分别如SEQ ID NO：14、SEQ ID NO：15和SEQ ID NO：16所示的LCDR1、LCDR2和LCDR3；
    (III)
    重链可变区，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含氨基酸序列分别如SEQ ID NO：6、SEQ ID NO：7和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3；
    (IV)
    重链可变区，其包含氨基酸序列分别如SEQ ID NO：3、SEQ ID NO：4和SEQ ID NO：5所示的HCDR1、HCDR2和HCDR3；和
    轻链可变区，其包含氨基酸序列分别如SEQ ID NO：42、SEQ ID NO：39和SEQ ID NO：8所示的LCDR1、LCDR2和LCDR3。
  2. 如权利要求1所述的抗IL-4R抗体或其抗原结合片段的复合物，所述抗IL-4R抗体选自以下的任一项：鼠源抗体、嵌合抗体、全人抗体、人源化抗体。
  3. 如权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其中：
    所述抗IL-4R抗体或其抗原结合片段包含来源于人种系轻链IGKV3-11*01的FR或与其有至少95％序列同一性FR；优选地，所述FR包含回复突变，所述回复突变选自46P、47W、71Y中的一个或多个；和/或
    所述抗IL-4R抗体或其抗原结合片段包含来源于人种系重链IGHV3-48*01的FR或与其有至少95％序列同一性的FR；优选地，所述FR包含回复突变，所述回复突变选自49A、67S、93T中的一个或多个。
  4. 如权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其中：
    所述抗IL-4R抗体或其抗原结合片段包含来源于人种系轻链IGKV2D-29*01的FR或与其有至少95％序列同一性的FR，优选地，所述FR包含回复突变，所述回复突变选自4L和/或58I；和/或
    所述抗IL-4R抗体或其抗原结合片段包含来源于人种系重链IGHV1-2*02的FR或与其有至少95％序列同一性的FR，优选地，所述FR包含回复突变，所述回复突变选自69L、71I、73K、94K中的一个或多个。
  5. 如权利要求1-2任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其中：
    所述抗IL-4R抗体或其抗原结合片段包含重链恒定区，所述重链恒定区为人源IgG1、IgG2、IgG3或IgG4的重链恒定区、或其变体；和/或
    所述抗原结合片段为Fab、Fv、scFv、F(ab’)2、dsfv或ScdsFv，优选为scFv。
  6. 如权利要求5所述的抗IL-4R抗体或其抗原结合片段的复合物，其中：
    所述scFv自N端至C端依次包含重链可变区、连接肽和轻链可变区，或者
    所述scFv自N端至C端依次包含轻链可变区、连接肽和重链可变区；
    所述连接肽选自以下任一项或其组合：(GxS) n、(SxG) n、(GGGGS) n、(G) n，其中x是1-6的任一整数，且n是1-30的任一整数；或
    所述连接肽选自以下任一项或其组合：GKSSGSGSESKS、EGKSSGSGSESKEF、GSTSGSGKSSEGKG、GSTSGSGKSSEGSGSTKG、GSTSGSGKPGSGEGSTKG、SRSSG和SGSSC，优选(GGGGS) 3。
  7. 如权利要求1-6任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其包含选自以下(I)至(IV)中的任一项：
    (I)
    重链可变区，其包含如SEQ ID NO：43所示的氨基酸序列或与SEQ ID NO：43具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：37所示的氨基酸序列或与SEQ ID NO：37具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (II)
    重链可变区，其包含如SEQ ID NO：9所示的氨基酸序列或与SEQ ID NO：9 具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：10所示的氨基酸序列或与SEQ ID NO：10具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (III)
    重链可变区，其包含如SEQ ID NO：1所示的氨基酸序列或与SEQ ID NO：1具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：2所示的氨基酸序列或与SEQ ID NO：2具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (IV)
    重链可变区，其包含如SEQ ID NO：43所示的氨基酸序列或与SEQ ID NO：43具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：41所示的氨基酸序列或与SEQ ID NO：41具有至少90％、95％、98％、99％同一性的氨基酸序列；
    (V)
    重链可变区，其包含如SEQ ID NO：47所示的氨基酸序列或与SEQ ID NO：47具有至少90％、95％、98％、99％同一性的氨基酸序列；和
    轻链可变区，其包含如SEQ ID NO：48所示的氨基酸序列或与SEQ ID NO：48具有至少90％、95％、98％、99％同一性的氨基酸序列；
    优选地，
    重链可变区序列如SEQ ID NO：43所示，且轻链可变区序列如SEQ ID NO：37所示；或
    重链可变区序列如SEQ ID NO：9所示，且轻链可变区序列如SEQ ID NO：10所示；或
    重链可变区序列如SEQ ID NO：1所示，且轻链可变区序列如SEQ ID NO：2所示；或
    重链可变区序列如SEQ ID NO：43所示，且轻链可变区序列如SEQ ID NO：41所示；或
    重链可变区序列如SEQ ID NO：47所示，且轻链可变区序列如SEQ ID NO：48所示。
  8. 如权利要求1-6任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其中：
    重链可变区包含如SEQ ID NO：25-27之一所示的氨基酸序列或与SEQ ID NO：25-27之一具有至少90％、95％、98％或99％同一性的氨基酸序列；
    轻链可变区包含如SEQ ID NO：28-30之一所示的氨基酸序列或与SEQ ID NO：28-30之一具有至少90％、95％、98％或99％同一性的氨基酸序列；
    或者
    重链可变区包含如SEQ ID NO：31-33之一所示的氨基酸序列或与SEQ ID NO：31-33之一具有至少90％、95％、98％、或99％同一性的氨基酸序列；
    轻链可变区包含如SEQ ID NO：34-36之一所示的氨基酸序列或与SEQ ID NO：34-36之一具有至少90％、95％、98％或99％同一性的氨基酸序列；
    优选地，
    重链可变区序列如SEQ ID NO：25-27之一所示，且轻链可变区序列如SEQ ID NO：28-30之一所示；或
    重链可变区序列如SEQ ID NO：31-33之一所示，且轻链可变区序列如SEQ ID NO：34-36之一所示；
    更优选地，重链可变区和/或轻链可变区包含用于稳定结构的突变；
    进一步优选地，重链可变区第44位的氨基酸残基突变成半胱氨酸残基，
    进一步优选地，轻链可变区第100位的氨基酸残基突变成半胱氨酸残基。
  9. 如权利要求1-6任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，所述抗IL-4R抗体或其抗原结合片段包含选自(I)至(IV)中的任一项：
    (I)
    重链，其包含如SEQ ID NO：44所示的氨基酸序列或与SEQ ID NO：44具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：45所示的氨基酸序列或与SEQ ID NO：45具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (II)
    重链，其包含如SEQ ID NO：19所示的氨基酸序列或与SEQ ID NO：19具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：20所示的氨基酸序列或与SEQ ID NO：20具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (III)
    重链，其包含如SEQ ID NO：17所示的氨基酸序列或与SEQ ID NO：17具有至少90％、95％、98％或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：18所示的氨基酸序列或与SEQ ID NO：18具有至少90％、95％、98％或99％同一性的氨基酸序列；
    (IV)
    重链，其包含如SEQ ID NO：44所示的氨基酸序列或与SEQ ID NO：44具有至少90％、95％、98％、或99％同一性的氨基酸序列；和
    轻链，其包含如SEQ ID NO：46所示的氨基酸序列或与SEQ ID NO：46具有至少90％、95％、98％或99％同一性的氨基酸序列；
    优选地，
    重链序列如SEQ ID NO：44所示，且轻链序列如SEQ ID NO：45所示；或
    重链序列如SEQ ID NO：19所示，且轻链序列如SEQ ID NO：20所示；或
    重链序列如SEQ ID NO：17所示，且轻链序列如SEQ ID NO：18所示；或
    重链序列如SEQ ID NO：44所示，且轻链序列如SEQ ID NO：46所示。
  10. 如权利要求1-9任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其中：
    所述抗IL-4R抗体或其抗原结合片段通过接头连接至所述毒素分子；
    所述接头为蛋白性或非蛋白性的接头；
    优选地，所述复合物为融合蛋白。
  11. 如权利要求10所述的抗IL-4R抗体或其抗原结合片段的复合物，
    所述接头选自以下任一项或其组合：ASGGPE、VM、AM、AM(G 2-4S) pAM、ASGCGPE、ASGCCGPE、ASGCGSCPE、ASCGTTGCPE、KASGKKYGCKKGPE、KGGGCAGGPE；优选ASGCGPE；
    其中p是1-10的任一整数。
  12. 如权利要求1-11任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，所述毒素分子选自以下任一项或其组合：成孔毒素、嗜水气单胞菌气溶素、气单胞菌溶素原、布加宁、蓖麻毒素、假单胞菌外毒素、霍乱毒素或白喉毒素。
  13. 如权利要求12所述的抗IL-4R抗体或其抗原结合片段的复合物，所述毒素选自以下任一项或其组合：PE-LR、PE-LO10R456A、PE-T20、PE-T20-KDEL、PE4E、PE40、PE38、PE24、PE25、PE38QQR、PE35、PE38KDEL、PE38DKEL、PE38RDEL、PE38KNEL，优选如SEQ ID NO：49所示的PE38KDEL。
  14. 如权利要求1-13任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，其包含如SEQ ID NO：52或SEQ ID NO：53所示的序列。
  15. 一种抗IL-4R抗体或其抗原结合片段的复合物，其包含：
    -抗IL-4R抗体或其抗原结合片段、以及
    -一个或多个毒素分子；
    所述抗IL-4R抗体或其抗原结合片段与所述毒素分子共价或非共价连接；
    其中，
    所述毒素分子选自以下任一项或其组合：成孔毒素、嗜水气单胞菌气溶素、气单胞菌溶素原、布加宁、蓖麻毒素、假单胞菌外毒素、霍乱毒素或白喉毒素；
    优选地，所述毒素分子选自以下任一项或其组合：PE-LR、PE-LO10R456A、PE-T20、PE-T20-KDEL、PE4E、PE40、PE38、PE24、PE25、PE38QQR、PE35、PE38KDEL、PE38DKEL、PE38RDEL、PE38KNEL；
    更优选地，所述毒素分子是如SEQ ID NO：49所示的PE38KDEL。
  16. 一种多核苷酸，其编码权利要求1至15任一项所述的抗IL-4R抗体或其抗原结合片段的复合物。
  17. 一种载体，其含有权利要求16所述的多核苷酸，其为真核表达载体、原核表达载体或病毒载体。
  18. 一种宿主细胞，其包含权利要求17所述的载体，
    优选地，所述宿主细胞为细菌、酵母或哺乳动物细胞，
    更优选地，所述宿主细胞为大肠杆菌、毕赤酵母、中国仓鼠卵巢细胞或人胚肾293细胞。
  19. 一种药物组合物，其含有：
    权利要求1至15任一项所述的抗IL-4R抗体或其抗原结合片段的复合物，以及
    任选地，可药用的赋形剂、稀释剂或载体。
  20. 一种治疗和/或预防癌症或肿瘤的方法，包括：
    向受试者施用治疗有效量或预防有效量的权利要求1至15任一项所述的抗IL-4R抗体或其抗原结合片段的复合物、权利要求16所述的多核苷酸、或权利要求19所述的药物组合物，
    优选地，所述癌症或肿瘤选自以下任一项或其组合：***癌、卵巢癌、乳腺癌、子宫内膜癌、多发性骨髓瘤、黑素瘤、淋巴瘤、肺癌、肾癌、肝癌、大肠癌(例如结肠癌)、胰腺癌、胃癌、白血病和中枢神经***肿瘤；
    更优选地，中枢神经***肿瘤选自以下任一项或其组合：神经胶质瘤、神经胶质母细胞瘤、神经母细胞瘤、星形细胞瘤、髓母细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤、听神经瘤、少突胶质瘤、血管瘤、脑膜瘤、视网膜母细胞瘤。
  21. 根据权利要求20所述的方法，所述中枢神经***肿瘤为神经胶质母细胞瘤；
    优选地，所述中枢神经***肿瘤为复发性或难治性胶质母细胞瘤、或O6-甲基鸟嘌呤-DNA甲基转移酶表达阳性或阴性的神经胶质母细胞瘤。
  22. 一种用于制备抗IL-4R抗体或其抗原结合片段的复合物的方法，包括如下步骤：
    在权利要求18所述的宿主细胞中表达抗IL-4R抗体或其抗原结合片段的复合物，以及
    从所述宿主细胞中分离所述抗IL-4R抗体或其抗原结合片段的复合物。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11939387B2 (en) 2020-04-17 2024-03-26 Shanghai Mabgeek Biotech. Co., Ltd Anti-human interleukin-4 receptor alpha antibody and preparation method and application thereof

Citations (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4892827A (en) 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects
WO1995027732A2 (en) 1994-04-08 1995-10-19 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Circularly permuted ligands and circularly permuted chimeric molecules
US5512658A (en) 1990-05-11 1996-04-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pseudomonas exotoxins (PE) and conjugates thereof having lower animal toxicity with high cytocidal activity through substitution of positively charged amino acids
US5602095A (en) 1992-06-18 1997-02-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Recombinant pseudomonas exotoxin with increased activity
US5608039A (en) 1990-10-12 1997-03-04 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Single chain B3 antibody fusion proteins and their uses
WO2001092340A2 (en) 2000-05-26 2001-12-06 Immunex Corporation Use of interleukin-4 antagonists and compositions thereof
WO2008054606A2 (en) 2006-10-02 2008-05-08 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
WO2010053751A1 (en) 2008-10-29 2010-05-14 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
CN102153655A (zh) * 2010-12-29 2011-08-17 吉林大学 一种靶向抗肿瘤重组蛋白质及其制备方法
WO2014031610A1 (en) 2012-08-21 2014-02-27 Sanofi Methods for treating or preventing asthma by administering an il-4r antagonist
WO2014124227A1 (en) 2013-02-07 2014-08-14 Immunomedics, Inc. Pro-drug form (p2pdox) of the highly potent 2-pyrrolinodoxorubicin conjugated to antibodies for targeted therapy of cancer
US8871906B2 (en) 2007-09-04 2014-10-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Deletions in domain II of pseudomonas exotoxin a that remove immunogenic epitopes
US8907060B2 (en) 2005-07-29 2014-12-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Mutated Pseudomonas exotoxins with reduced antigenicity
US8936792B2 (en) 2009-09-11 2015-01-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Pseudomonas exotoxin a with reduced immunogenicity
US9206240B2 (en) 2011-09-16 2015-12-08 The United States of America, as represented by the Secretary, Department of Helath and Human Services Pseudomonas exotoxin A with less immunogenic B cell epitopes
EP2954933A1 (en) * 2014-06-10 2015-12-16 3B Pharmaceuticals GmbH Conjugate comprising a neurotensin receptor ligand
WO2015188934A1 (en) 2014-06-10 2015-12-17 3B Pharmaceuticals Gmbh Conjugate comprising a neurotensin receptor ligand and use thereof
US9346859B2 (en) 2011-06-09 2016-05-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Pseudomonas exotoxin A with less immunogenic T cell and/or B cell epitopes
US9388222B2 (en) 2013-10-06 2016-07-12 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Modified Pseudomonas exotoxin A
WO2018217227A1 (en) 2017-05-24 2018-11-29 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1
WO2020038454A1 (zh) 2018-08-24 2020-02-27 江苏恒瑞医药股份有限公司 结合人il-4r的抗体、其抗原结合片段及其医药用途

Patent Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4892827A (en) 1986-09-24 1990-01-09 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxins: construction of an active immunotoxin with low side effects
US5512658A (en) 1990-05-11 1996-04-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Pseudomonas exotoxins (PE) and conjugates thereof having lower animal toxicity with high cytocidal activity through substitution of positively charged amino acids
US5608039A (en) 1990-10-12 1997-03-04 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Single chain B3 antibody fusion proteins and their uses
US5602095A (en) 1992-06-18 1997-02-11 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Recombinant pseudomonas exotoxin with increased activity
US5821238A (en) 1992-06-18 1998-10-13 The United States Of America As Represented By The Department Of Health And Human Services Recombinant pseudomonas exotoxin with increased activity
US5854044A (en) 1992-06-18 1998-12-29 National Institutes Of Health Recombinant pseudomonas exotoxin with increased activity
WO1995027732A2 (en) 1994-04-08 1995-10-19 The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services Circularly permuted ligands and circularly permuted chimeric molecules
US5635599A (en) * 1994-04-08 1997-06-03 The United States Of America As Represented By The Department Of Health And Human Services Fusion proteins comprising circularly permuted ligands
WO2001092340A2 (en) 2000-05-26 2001-12-06 Immunex Corporation Use of interleukin-4 antagonists and compositions thereof
US8907060B2 (en) 2005-07-29 2014-12-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Mutated Pseudomonas exotoxins with reduced antigenicity
WO2008054606A2 (en) 2006-10-02 2008-05-08 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
US8871906B2 (en) 2007-09-04 2014-10-28 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Deletions in domain II of pseudomonas exotoxin a that remove immunogenic epitopes
CN102197052A (zh) * 2008-10-29 2011-09-21 瑞泽恩制药公司 抗人il-4受体的高亲和性人抗体
WO2010053751A1 (en) 2008-10-29 2010-05-14 Regeneron Pharmaceuticals, Inc. High affinity human antibodies to human il-4 receptor
CN103739711A (zh) 2008-10-29 2014-04-23 瑞泽恩制药公司 抗人il-4受体的高亲和性人抗体
US8936792B2 (en) 2009-09-11 2015-01-20 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Pseudomonas exotoxin a with reduced immunogenicity
CN102153655A (zh) * 2010-12-29 2011-08-17 吉林大学 一种靶向抗肿瘤重组蛋白质及其制备方法
US9346859B2 (en) 2011-06-09 2016-05-24 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Pseudomonas exotoxin A with less immunogenic T cell and/or B cell epitopes
US9206240B2 (en) 2011-09-16 2015-12-08 The United States of America, as represented by the Secretary, Department of Helath and Human Services Pseudomonas exotoxin A with less immunogenic B cell epitopes
WO2014031610A1 (en) 2012-08-21 2014-02-27 Sanofi Methods for treating or preventing asthma by administering an il-4r antagonist
WO2014124227A1 (en) 2013-02-07 2014-08-14 Immunomedics, Inc. Pro-drug form (p2pdox) of the highly potent 2-pyrrolinodoxorubicin conjugated to antibodies for targeted therapy of cancer
US9388222B2 (en) 2013-10-06 2016-07-12 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Modified Pseudomonas exotoxin A
EP2954933A1 (en) * 2014-06-10 2015-12-16 3B Pharmaceuticals GmbH Conjugate comprising a neurotensin receptor ligand
WO2015188934A1 (en) 2014-06-10 2015-12-17 3B Pharmaceuticals Gmbh Conjugate comprising a neurotensin receptor ligand and use thereof
WO2018217227A1 (en) 2017-05-24 2018-11-29 Immunomedics, Inc. Novel anti-pd-1 checkpoint inhibitor antibodies that block binding of pd-l1 to pd-1
WO2020038454A1 (zh) 2018-08-24 2020-02-27 江苏恒瑞医药股份有限公司 结合人il-4r的抗体、其抗原结合片段及其医药用途

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
"Biotechnological Pharmaceutics", January 2016, CHINA MEDICAL SCIENCE PRESS, pages: 209 - 211
"China Tumor Clinical Yearbook", July 2010, PEKING UNION MEDICAL COLLEGE PRESS, pages: 61
"The three-letter and single-letter codes for amino acids used in the present disclosure are described", J. BIOL. CHEM, vol. 243, 1968, pages 3558
ALFTHAN ET AL., PROTEIN ENG., vol. 8, 1995, pages 725 - 731
ALLAHYARI HOSSEIN, ET AL.: "Immunotoxin: A New Tool for Cancer Therapy", TUMOR BIOLOGY, vol. 39, no. 2, 28 February 2017 (2017-02-28), pages 1 - 11, XP055944527, DOI: 10.1177/1010428317692226 *
BIOCHEM. J., vol. 307, 1995, pages 29 - 37
CANCER RESEARCH, vol. 55, 1 August 1995 (1995-08-01), pages 3357 - 3363
CHOI ET AL., EUR. J. IMMUNOL., vol. 31, 2001, pages 94 - 106
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 6444 - 6448
HU ET AL., CANCER RES., vol. 56, 1996, pages 3055 - 3061
JOHANNES, L. ET AL.: "Protein Toxins: Intracellular Trafficking for Targeted Therapy", GENE THERAPY, vol. 12, 19 May 2005 (2005-05-19), pages 1360 - 1368, XP037772410, DOI: 10.1038/sj.gt.3302557 *
KIPRIYANOV ET AL., J. MOL. BIOL., vol. 293, 1999, pages 41 - 56
LI, MENG ET AL.: "Clinical Targeting Recombinant Immunotoxins for Cancer Therapy", ONCOTARGETS AND THERAPY, vol. 10, 20 July 2017 (2017-07-20), pages 3645 - 3665, XP055526991, DOI: 10.2147/OTT.S134584 *
ZHANG YUJIAN, ET AL.: "Fusion Protein of Interleukin4and Pseudomonas Exotoxin with High Cytotoxicity to Cancer Cells ", CHINESE JOURNAL OF BIOTECHNOLOGY, vol. 20, no. 6, 30 November 2004 (2004-11-30), pages 862 - 867, XP055944532, DOI: 10.13345/j.cjb.2004.06.012 *

Cited By (1)

* Cited by examiner, † Cited by third party
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US11939387B2 (en) 2020-04-17 2024-03-26 Shanghai Mabgeek Biotech. Co., Ltd Anti-human interleukin-4 receptor alpha antibody and preparation method and application thereof

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