WO2021208881A1 - 抗人白细胞介素-4受体α抗体及其制备方法和应用 - Google Patents

抗人白细胞介素-4受体α抗体及其制备方法和应用 Download PDF

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WO2021208881A1
WO2021208881A1 PCT/CN2021/086806 CN2021086806W WO2021208881A1 WO 2021208881 A1 WO2021208881 A1 WO 2021208881A1 CN 2021086806 W CN2021086806 W CN 2021086806W WO 2021208881 A1 WO2021208881 A1 WO 2021208881A1
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antibody
seq
amino acid
antigen
sequence
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PCT/CN2021/086806
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English (en)
French (fr)
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张成海
郭锦林
袁玉菁
陈林刚
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上海麦济生物技术有限公司
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Priority to IL297312A priority Critical patent/IL297312A/en
Application filed by 上海麦济生物技术有限公司 filed Critical 上海麦济生物技术有限公司
Priority to AU2021255445A priority patent/AU2021255445A1/en
Priority to MX2022013034A priority patent/MX2022013034A/es
Priority to CN202180028819.7A priority patent/CN115461368A/zh
Priority to BR112022021030A priority patent/BR112022021030A2/pt
Priority to CA3175786A priority patent/CA3175786A1/en
Priority to EP21788204.2A priority patent/EP4137517A1/en
Priority to JP2022563020A priority patent/JP7454882B2/ja
Priority to KR1020227039054A priority patent/KR20230004576A/ko
Publication of WO2021208881A1 publication Critical patent/WO2021208881A1/zh
Priority to US18/047,039 priority patent/US11939387B2/en
Priority to ZA2022/11889A priority patent/ZA202211889B/en
Priority to US18/583,713 priority patent/US20240190979A1/en

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Definitions

  • the present invention relates to the field of antibodies. More specifically, the present invention relates to an anti-interleukin receptor antibody and its preparation method and application.
  • T cells and B cells are the main effector cells.
  • T cells can directly kill target cells and secrete different types of cytokines to expand and enhance the immune response to exert immune effects.
  • Th2-type cytokines such as interlukin (IL)-4, IL-5, IL-9 and IL-13 mediate the main pathological development.
  • Asthma is a common respiratory disease, usually manifested as respiratory tract inflammation, bronchial hyperresponsiveness and structural changes in the bronchial wall (respiratory tract remodeling).
  • the genetic background and the stimulation of environmental factors including allergens and respiratory viruses have induced the occurrence of asthma.
  • the main pathological manifestations are repeated wheezing, shortness of breath, chest tightness and coughing.
  • wheezing wheezing
  • shortness of breath wheezing, shortness of breath, chest tightness and coughing.
  • asthma is treated gradually to reduce symptoms and control risks.
  • Inhaled corticosteroids are the standard treatment for moderate asthma, and long-acting beta antagonists are added to severe patients, and additional control medications may be needed for the most severe patients. These treatments can cause immune system disorders, as well as other serious side effects. Despite this, there are still 5%-10% of patients currently without medicine.
  • Th2 cytokines In allergic asthma, abnormally high expression of Th2 cytokines is found in the bronchi. At the same time, it has been confirmed that Th2 cytokines mediate the occurrence and development of inflammatory reactions and promote pathological changes in the respiratory tract. These cytokines promote the activation and chemotaxis of inflammatory cells including eosinophils and mast cells to the site of inflammation. IL-4 and IL-13 target B cells to transform their secreted antibodies from IgM to IgE. At the same time, they can induce the proliferation of goblet cells and transform bronchial fibroblasts into myofibroblasts, collagen deposition and airway smooth muscle cells. Proliferation and so on induce bronchial remodeling.
  • Both IL-4 and IL-13 can activate the corresponding signaling pathways by binding to interleukin-4 receptor ⁇ (IL-4R ⁇ ). Therefore, the development of IL-4R ⁇ targeting antibodies will block both IL-4 and IL- The pathological response of 13 is expected to be used to treat IL-4R ⁇ -related diseases including asthma.
  • IL-4R ⁇ interleukin-4 receptor ⁇
  • the inventors of the present invention conducted a large number of experiments and obtained a group that can block IL-4 and IL by specifically blocking the binding of IL-4 and IL-13 to the cell surface IL-4 receptor ⁇ (IL-4R ⁇ ).
  • -13 signal transduction monoclonal antibody which can block the biological activity mediated by IL-4/IL-13.
  • the present application provides an antibody or antigen-binding portion thereof that specifically binds IL-4 receptor ⁇ , which comprises a heavy chain variable region comprising HCDR3 sequence, and optionally Contains HCDR1 and/or HCDR2 sequences.
  • the aforementioned HCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs: 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197 and 203.
  • the aforementioned HCDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 105, 111, 117, 123, 129, 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195 and 201.
  • the aforementioned HCDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs: 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196 and 202.
  • the aforementioned antigen binding portion is selected from the group consisting of Fab fragments, Fab' fragments, F(ab')2 fragments, Fv fragments, scFv fragments, Fd fragments and single domain antibodies.
  • the aforementioned heavy chain variable region comprises SEQ ID NOs: 2,6,10,14,18,22,26,30,34,38,42,46,50,54,58, 62, 66, 70, 76, 82, 88, 94 and 100 have amino acid sequences with at least 80% homology, or the heavy chain variable region comprises SEQ ID NOs: 2, 6, 10, Amino acid sequences of 14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94 and 100.
  • the antibody or antigen binding portion thereof that specifically binds IL-4 receptor alpha further comprises a light chain variable region, wherein the light chain variable region comprises LCDR1, LCDR2, and/or LCDR3 sequences.
  • the LCDR1 sequence comprises an amino acid sequence selected from SEQ ID NOs: 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198 and 204.
  • the LCDR2 sequence comprises an amino acid sequence selected from SEQ ID NOs: 109, 115, 121, 127, 133, 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199 and 205.
  • the LCDR3 sequence comprises an amino acid sequence selected from SEQ ID NOs: 110, 116, 122, 128, 134, 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, and 206.
  • the above-mentioned light chain variable region comprises SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 73, 79, 85, 91, 97 and 103 have amino acid sequences with at least 80% homology; or the light chain variable region comprises SEQ ID NOs: 4, 8, 12, The amino acid sequences of 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 73, 79, 85, 91, 97 and 103.
  • the heavy chain of the antibody or antigen-binding portion thereof that specifically binds to IL-4 receptor ⁇ comprises an amino acid sequence selected from SEQ ID NOs: 71, 77, 83, 89, 95 and 101 or is a combination of the above sequence An amino acid sequence with at least 80% homology.
  • the light chain of the antibody or antigen-binding portion thereof comprises an amino acid sequence selected from SEQ ID NOs: 74, 80, 86, 92, 98 and 104 or an amino acid sequence having at least 80% homology with the above sequence .
  • the antibody that specifically binds to IL-4 receptor ⁇ described in the first aspect is a monoclonal antibody.
  • the antibody that specifically binds to IL-4 receptor ⁇ described in the first aspect is a humanized antibody.
  • the IL-4 receptor ⁇ antibody or antigen-binding portion thereof disclosed herein binds to the same epitope on IL-4 receptor ⁇ as the antibody 131-Hu, 136-Hu, or 236-Hu, or with 131-Hu, 136-Hu, or 236-Hu.
  • the heavy chain sequence of the antibody 131-Hu is shown in SEQ ID NO: 83, and the light chain sequence is shown in SEQ ID NO: 86
  • the heavy chain sequence of the antibody 136-Hu is shown in SEQ ID NO: 89, and the light chain sequence is shown in SEQ ID NO: 92; and the heavy chain sequence of the antibody 236-Hu is shown in SEQ ID NO: 101 As shown, the light chain sequence is shown in SEQ ID NO: 104.
  • the antibodies or antigen binding portions thereof disclosed herein are capable of inhibiting IgE secretion by B cells.
  • the antibody or antigen-binding portion thereof binds to IL-4R ⁇ with a KD of less than 600 pM, preferably less than 350 pM.
  • the antibody or antigen-binding portion thereof can inhibit IL-4 induced TF-1 cell proliferation.
  • the present application provides a nucleotide molecule that encodes the antibody or antigen-binding portion thereof that specifically binds to IL-4 receptor ⁇ as described above.
  • this application provides an expression vector containing the nucleotide molecule as described above.
  • the expression vector is pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF, or pCHO 1.0, etc.
  • the present application provides a host cell containing the expression vector as described above.
  • the host cell is HEK293, COS, CHO, NS0, sf9, sf21, DH5 ⁇ , BL21(DE3), TG1, or the like.
  • this application provides a method for preparing the antibody or antigen-binding portion thereof that specifically binds to IL-4 receptor ⁇ as described in the first aspect, the method comprising the following steps:
  • the present application provides a pharmaceutical composition comprising the anti-IL-4 receptor ⁇ antibody or its antigen binding portion as described in the first aspect and a pharmaceutically acceptable carrier.
  • the composition is used to treat IL-4R ⁇ -related diseases.
  • the present application provides the anti-IL-4 receptor ⁇ antibody or the antigen-binding portion thereof according to the first aspect, or the composition according to the sixth aspect in preparation for the prevention or treatment of IL-4R ⁇ -related diseases, For example, the application of immune-mediated inflammatory response or inflammatory disease drugs.
  • the immune-mediated inflammatory response or inflammatory disease includes: asthma, allergy, atopic dermatitis, chronic sinusitis, eosinophilic esophagitis, nasal polyps, psoriasis, rheumatoid Arthritis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, uveitis, Behçet’s uveitis, dry eye and chronic spontaneous urticaria.
  • the present application provides a method for preventing or treating IL-4R ⁇ -related diseases, including administering the antibody or antigen-binding portion thereof according to the first aspect or the pharmaceutical composition according to the sixth aspect to an individual in need.
  • the anti-IL-4R ⁇ antibody or its antigen binding portion of the present invention can specifically bind to IL-4R ⁇ , and has one or more of the following effects: blocking the binding of IL-4 or IL-13 to IL-4R ⁇ ; inhibiting IL -4 or IL-13 induced proliferation of cell lines such as TF-1 cells; and/or inhibit IgE secretion by B cells.
  • the in vivo pharmacodynamic test fully shows that the antibody of the present invention can antagonize the occurrence of downstream Th2-type responses by inhibiting the IL-4 and IL-13 signal pathways, has a strong asthma inhibitory function, and has a rapid onset of action.
  • the anti-IL-4R ⁇ antibody or antigen-binding portion thereof of the present invention can be used to prevent or treat IL-4R ⁇ -related diseases, such as immune-mediated inflammatory diseases.
  • Figure 1 shows the results of the determination of the affinity of the established mouse anti-human IL-4R ⁇ monoclonal antibody to human IL-4R ⁇ .
  • Figure 2 shows the results of the established strain of murine anti-human IL-4R ⁇ monoclonal antibody inhibiting the proliferation of TF-1 cells induced by human IL-4.
  • Figure 3 shows the experimental results of the humanized anti-human IL-4R ⁇ monoclonal antibody inhibiting the proliferation of TF-1 cells induced by human IL-4.
  • Figure 4 shows the experimental results of the humanized anti-human IL-4R ⁇ monoclonal antibody inhibiting the IgE secretion of fresh peripheral blood-derived B cells.
  • Figure 5 shows the pharmacokinetic results of humanized anti-human IL-4R ⁇ monoclonal antibody
  • Figure 7 The proportion of eosinophils in white blood cells in the alveolar lavage fluid of each model group
  • Figure 9 Mucus secretion in lung tissue of animals in each model group
  • This application provides a new anti-IL-4R ⁇ antibody or antigen-binding portion thereof that specifically binds to IL-4R ⁇ .
  • the antibody or antigen-binding portion thereof of the present application binds to human IL-4R ⁇ with high affinity and inhibits the activity of IL-4R ⁇ .
  • the application also provides a polynucleotide encoding the antibody or antigen-binding fragment thereof, a vector containing the polynucleotide, a host cell containing the polynucleotide or the vector, methods for preparing and purifying the antibody, and the antibody Medical and biological applications of the antigen-binding fragments thereof, such as prevention or treatment of IL-4R ⁇ -related diseases or disorders.
  • This application also covers methods for using the antibody or antigen-binding fragment thereof to detect IL-4R ⁇ and modulate the activity of IL-4R ⁇ .
  • antibody refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light chains (L) interconnected by disulfide bonds, and its multimers (such as IgM ).
  • Each heavy chain includes a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • the heavy chain constant region contains three domains, namely CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • CL light chain constant region contains one domain (CL1).
  • the VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDR), which are interspersed with conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • antigen-binding portion of an antibody refers to a part or segment of a complete antibody molecule that is responsible for binding an antigen.
  • the antigen binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both.
  • Antigen-binding fragments of antibodies can be prepared from intact antibody molecules using any suitable standard techniques, including proteolytic digestion or recombinant genetic engineering techniques.
  • Non-limiting examples of antigen-binding portions include: Fab fragments; F(ab')2 fragments; Fd fragments; Fv fragments; single-chain Fv (scFv) molecules; single domain antibodies; The smallest recognition unit consisting of residues (for example, isolated CDR).
  • the term "antigen-binding portion” also includes other engineered molecules, such as diabodies, tribodies, tetrabodies, and minibodies.
  • VH heavy chain variable region
  • VL light chain variable region
  • CDR complementarity determining region
  • the sequence of the CDR regions in the VH and VL sequences can be determined according to the Kabat definition or the Chothia definition.
  • Kabat is used to define CDR sequences.
  • the CDR1, CDR2, and CDR3 of the heavy chain variable region are referred to as HCDR1, HCDR2, and HCDR3, respectively;
  • the CDR1, CDR2, and CDR3 of the light chain variable region are referred to as LCDR1, LCDR2, and LCDR3, respectively.
  • variable region sequence of a given antibody the CDR region sequence in the variable region sequence can be analyzed in various ways, for example, it can be determined using the online software Abysis (http://www.abysis.org/).
  • the term "specific binding” refers to a non-random binding reaction between two molecules, such as the binding of an antibody to an antigen epitope, for example, an antibody having an affinity at least two times greater than its affinity for a non-specific antigen The ability to bind to a specific antigen with affinity. It should be understood, however, that an antibody can specifically bind to two or more sequence-related antigens.
  • the antibody of the present invention can specifically bind to human and non-human (e.g., mouse or non-human primate) IL-4R ⁇ .
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous antibody population, that is, each antibody constituting the population is the same except that there may be naturally occurring mutations in a small number of individuals.
  • the monoclonal antibodies described herein particularly include “chimeric" antibodies, in which a part of the heavy chain and/or light chain is the same or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and The remaining part of the chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, as long as they can exhibit the desired The biological activity (see, U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81: 6851-6855 (1984)).
  • homology is defined as the percentage of identical residues in amino acid or nucleotide sequence variants after sequence alignment and introduction of gaps, if necessary, to achieve the maximum percentage of homology. Methods and computer programs for comparison are well known in the art.
  • at least 80% homology means that the homology is any value from 80% to 100%, such as 85%, 90%, 95%, 99%, and so on.
  • IL-4R ⁇ -related diseases includes diseases and/or symptoms related to the activation of the IL-4R ⁇ signaling pathway.
  • exemplary IL-4R ⁇ -related diseases or disorders include immune-mediated inflammatory reactions, such as allergic diseases, asthma, and the like.
  • half-life and “serum half-life” refer to the time it takes for the serum concentration of the antigen binding protein according to the present disclosure to decrease by 50% in the body.
  • the application provides an antibody or antigen binding portion thereof that specifically binds IL-4R ⁇ , which comprises a heavy chain variable region and/or a light chain variable region.
  • IL-4R ⁇ which comprises a heavy chain variable region and/or a light chain variable region.
  • Table 1-5 exemplarily list the CDR, VH, VL, heavy chain and light chain amino acid sequences and corresponding nucleotide sequences suitable for the antibodies disclosed in the present application.
  • the anti-IL-4R ⁇ antibody or antigen binding portion thereof comprises HCDR3, HCDR2, and/or HCDR1 sequences, which are independently selected from any of the HCDR3, HCDR2, or HCDR1 sequences shown in Table 1.
  • the anti-IL-4R ⁇ antibody of the present application may further comprise a light chain CDR, which is independently selected from any of the light chain CDR1, CDR2, or CDR3 sequences shown in Table 2.
  • the anti-IL-4R ⁇ antibody of the present application may comprise any of the heavy chain variable domains shown in Tables 3 and 4, optionally with the light chain variable domains shown in Tables 3 and 4. Any one of them is paired.
  • Table 4 Nucleotide and amino acid sequences of the heavy chain variable region and light chain variable region of an exemplary humanized anti-IL-4R ⁇ antibody
  • the HCDR3 of the antibody or antigen-binding portion thereof disclosed herein is selected from the amino acid sequences shown in SEQ ID NOs: 107, 113, 119, 125, 131, 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197 and 203.
  • HCDR2 is selected from the amino acid sequence shown in SEQ ID NOs: 106, 112, 118, 124, 130, 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196 and 202; and/or the HCDR1 is selected from the amino acid sequence shown in SEQ ID NOs: 105, 111, 117, 123, 129, 135, 141, 147, 153, 201, 165, 171, 177, 183, 171, 177.
  • HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 107, 113, 119, 125 and 131. In another specific embodiment, HCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 137, 143, 149, 155, 161, 167, 173, 179, 185, 191, 197 and 203. In a preferred embodiment, HCDR3 is selected from the amino acid sequences shown in 125, 131 and 203.
  • HCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 106, 112, 118, 124 and 130. In another specific embodiment, HCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 136, 142, 148, 154, 160, 166, 172, 178, 184, 190, 196 and 202. In a preferred embodiment, HCDR2 is selected from the amino acid sequences shown in 124, 130 and 202.
  • HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 105, 111, 117, 123 and 129. In another specific embodiment, HCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 135, 141, 147, 153, 159, 165, 171, 177, 183, 189, 195 and 201. In a preferred embodiment, HCDR1 is selected from the amino acid sequences shown in 123, 129 and 201.
  • the antibody heavy chain variable region disclosed herein comprises SEQ ID NOs: 2,6,10,14,18,22,26,30,34,38,42,46,50,54, 58, 62, 66, 70, 76, 82, 88, 94 and 100 amino acid sequences.
  • the heavy chain variable region is selected from SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54, 58 , 62, 66, 70, 76, 82, 88, 94 and 100 amino acid sequences.
  • the amino acid sequence of the antibody heavy chain variable region disclosed herein is the same as SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30, 34, 38, 42, 46, 50, 54 ,58,62,66,70,76,82,88,94 or 100 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology.
  • the heavy chain variable region has more than 99% homology with the amino acid sequence shown in SEQ ID NOs: 82, 88 or 100.
  • the antibody or antigen-binding portion thereof disclosed herein may further include a light chain variable region in addition to the heavy chain variable region.
  • the CDR3 (LCDR3) of the light chain variable region is selected from the amino acid sequence shown in SEQ ID NOs: 110, 116, 122, 128, and 134, or selected from the amino acid sequence shown in SEQ ID NOs: 140, 146, 152, 158, 164, 170, 176, 182, 188, 194, 200, and 206.
  • LCDR3 is selected from the amino acid sequences shown in SEQ ID NOs: 128, 134 and 206.
  • LCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 109, 115, 121, 127, and 133, or selected from the amino acid sequences shown in SEQ ID NOs: 139, 145, 151, 157, 163, 169, 175, 181, 187, 193, 199, and 205. In a preferred embodiment, LCDR2 is selected from the amino acid sequences shown in SEQ ID NOs: 127, 133 and 205.
  • LCDR1 is selected from the amino acid sequence of SEQ ID NOs: 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, and 204, or selected from the amino acid sequence of SEQ ID NOs: 108, 114, 120, 126, 132, 138, 144, 150, 156, 162, 168, 174, 180, 186, 192, 198, and 204.
  • LCDR1 is selected from the amino acid sequences shown in SEQ ID NOs: 126, 132 and 204.
  • the antibody light chain variable region disclosed herein comprises SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 73, 79, 85, 91, 97 and 103 amino acid sequences.
  • the light chain variable region is selected from SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60 ,64,68,73,79,85,91,97 and 103 amino acid sequence composition.
  • the amino acid sequence of the variable region of the antibody light chain disclosed herein is the same as SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52, 56 ,60,64,68,73,79,85,91,97 or 103 has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homology.
  • the heavy chain variable region has more than 99% homology with the sequence shown in SEQ ID NOs: 85, 91 or 103.
  • the heavy chain of the antibody or antigen-binding portion thereof disclosed herein has at least 80% homology with an amino acid sequence selected from SEQ ID NOs: 71, 77, 83, 89, 95 and 101, for example 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
  • the aforementioned antibody heavy chain consists of an amino acid sequence selected from SEQ ID NOs: 71, 77, 83, 89, 95 and 101.
  • the amino acid sequence of the heavy chain of the aforementioned antibody is shown in SEQ ID NO: 83, 89 or 101.
  • the light chain of the antibody disclosed herein has at least 80% homology with a sequence selected from SEQ ID NOs: 74, 80, 86, 92, 98 and 104, such as 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology.
  • the antibody light chain consists of an amino acid sequence selected from SEQ ID NOs: 74, 80, 86, 92, 98, and 104.
  • the amino acid sequence of the above antibody light chain is shown in SEQ ID NO: 86, 92 or 104.
  • the heavy chain or heavy chain variable region, light chain or light chain variable region of the antibody disclosed herein can be substituted, deleted or added at least one on the basis of the respective corresponding specific amino acid sequences listed above. Amino acids, and the resulting variants still retain IL-4R ⁇ binding activity.
  • the number of the above-mentioned amino acid substitutions, deletions or additions is 1-30, preferably 1-20, and more preferably 1-10.
  • the sequence variant differs from the original amino acid sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions and/or additions.
  • the sequence variant differs from the original amino acid sequence by about 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions.
  • the amino acid substitutions are conservative substitutions.
  • the antibody disclosed herein is the antibody 131-Hu, 136-Hu or 236-Hu, wherein the heavy chain sequence of the antibody 131-Hu is shown in SEQ ID NO: 83, and the light chain sequence is shown in SEQ ID NO : 86, wherein the CDR sequence is the same as antibody 131; the heavy chain sequence of antibody 136-Hu is shown in SEQ ID NO: 89, and the light chain sequence is shown in SEQ ID NO: 92, wherein the CDR sequence is the same as antibody 136;
  • the heavy chain sequence of the antibody 236-Hu is shown in SEQ ID NO: 101, and the light chain sequence is shown in SEQ ID NO: 104, wherein the CDR sequence is the same as that of the antibody 236.
  • the antibody or antigen-binding portion thereof disclosed herein binds to the same epitope on the interleukin-4 receptor ⁇ as the antibody 131-Hu, 136-Hu, or 236-Hu, or binds to the same epitope on the interleukin-4 receptor ⁇ -Hu or 236-Hu compete for binding to interleukin-4 receptor alpha.
  • the antibodies disclosed herein are monoclonal antibodies. In specific embodiments, the antibodies disclosed herein are humanized antibodies.
  • the antibodies or antigen-binding portions thereof disclosed herein are capable of specifically binding IL-4R ⁇ .
  • the antibody or antigen binding portion thereof specifically binds human IL-4R ⁇ or murine IL-4R ⁇ .
  • the antibody or antigen binding portion thereof specifically binds to human IL-4R ⁇ .
  • the antibody or antigen binding portion thereof binds to IL-4R ⁇ with a KD of less than 600 pM. In a preferred embodiment, the antibody or antigen-binding portion thereof binds to IL-4R ⁇ , such as human IL-4R ⁇ , with a KD of less than 350 pM pM.
  • the antibodies or antigen binding portions thereof disclosed herein are capable of inhibiting IL-4 induced TF-1 cell proliferation. In some embodiments, the antibodies or antigen binding portions thereof disclosed herein are capable of inhibiting IgE secretion by B cells.
  • the inventors of the present application conducted in vitro and in vivo biological experiments on the anti-human IL-4R ⁇ monoclonal antibody disclosed herein, and the results showed that the antibody can bind to IL-4R ⁇ well.
  • the inventors of the present application conducted experiments such as affinity detection, experimental analysis for blocking the binding of IL-4/IL-13 to IL-4R ⁇ , and in vitro cell function detection for the anti-human IL-4R ⁇ monoclonal antibody.
  • Experimental results show that the anti-human IL-4R ⁇ monoclonal antibody disclosed herein can bind to IL-4R ⁇ on the cell surface, block the signal transduction between IL-4/IL-13 and IL-4R ⁇ , and inhibit the occurrence of inflammation.
  • the application also provides a nucleotide molecule encoding the antibody disclosed herein or an antigen-binding portion thereof, a vector containing the polynucleotide, a host cell containing the polynucleotide or the vector, and methods for preparing and purifying the antibody .
  • the nucleotide molecule encoding the antibody or antigen-binding portion thereof is operably linked to a regulatory sequence, which can be recognized by a host cell transformed with the vector.
  • any suitable expression vector can be used in this application.
  • the expression vector may be one of pTT5, pUC57, pDR1, pcDNA3.1(+), pDHFF, and pCHO 1.0.
  • the expression vector may include a fusion DNA sequence linked with appropriate transcription and translation regulatory sequences.
  • the usable host cell is a cell containing the above-mentioned expression vector, which may be a eukaryotic cell.
  • a eukaryotic cell for example, mammalian or insect host cell culture systems can be used for the expression of the antibody or antigen-binding portion thereof in the present application.
  • HEK293 cells, COS, CHO, NS0, sf9, and sf21 are all suitable for the present invention.
  • the host cell may also be a prokaryotic cell containing the above-mentioned expression vector, for example, it may be DH5 ⁇ , BL21(DE3) or TG1.
  • the preparation method of the anti-human IL-4R ⁇ monoclonal antibody disclosed herein includes: culturing host cells under expression conditions to express the anti-human IL-4R ⁇ monoclonal antibody; and isolating and purifying the expressed anti-human IL -4R ⁇ monoclonal antibody.
  • the recombinant protein can be purified into a substantially uniform substance, such as a single band on SDS-PAGE electrophoresis.
  • affinity chromatography can be used to separate and purify the anti-IL-4R ⁇ antibodies disclosed herein. According to the characteristics of the affinity column used, conventional methods such as high-salt buffers and pH changes can be used. Methods such as eluting the anti-IL-4R ⁇ antibody bound to the affinity column.
  • the humanized anti-human IL-4R ⁇ monoclonal antibody disclosed herein is obtained by the following method: immunizing Balb/c mice with IL-4R ⁇ antigen prepared in the laboratory, and immunizing mice multiple times After the titer is higher, the mouse spleen cells are fused with hybridoma cells and the hybridoma cell lines with the functional activity of inhibiting IL-4 are screened out. More specifically, the inventors of the present application, through a large number of experiments, first expressed IL-4R ⁇ extracellular segment antigen, IL-4 and IL-13 respectively, and on this basis used different adjuvants mixed with IL-4R ⁇ antigen to immunize small cells.
  • the spleen cells of the above-mentioned mice were further fused with the hybridoma cell line sp2/0.
  • the hybridoma used the IL-4R ⁇ extracellular segment antigen to screen out the positive cell line, and then verified its IL-4/IL- 13 Obtain the target cell line after blocking the binding of IL-4R ⁇ and indeed inhibiting the function of IL-4/IL-13.
  • the target molecule is humanized
  • the light chain and heavy chain genes are simultaneously cloned into the eukaryotic expression vector pCHO1.0.
  • the above expression vector was transfected into CHO cells by liposome method, and then positive cell clones were screened with puromycin and methotrexate.
  • the selected high expression clones were expanded and cultured in serum-free medium, and protein A affinity column was used. Isolation or purification of humanized anti-human IL-4R ⁇ monoclonal antibody.
  • conventional techniques in the art can be used to further alter the murine parent antibody to produce a chimeric or humanized form or other variant form of the antibody.
  • the parent antibody of the present application can be mutagenized in, for example, an antigen complementarity determining region (CDR) domain to produce a variant antibody, which can be screened for the existence of its intended properties, such as binding affinity (lower KD), IC50, specificity, preferential binding and many more.
  • the target property in the variant antibody is an improvement relative to the property in the parent antibody.
  • the amino acid substitution variant antibody and at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues of the parent antibody molecule are removed and different residues are inserted in its position.
  • the most interesting sites for replacement mutagenesis are one or more CDR regions, but framework region (FR) changes are also considered.
  • Conservative amino acid substitutions are preferred, and non-conservative amino acid changes can also be introduced and the obtained variant antibodies can be used to screen the properties of interest.
  • the Fc region of the antibody is modified to increase the serum half-life of the antibody.
  • the main mutation sites that have been identified that can improve the ability of human FcRn to bind to antibodies are T250Q, M252Y, S254T, T256E, V308P, M428L, N434A, and N434S.
  • amino acid mutations at these positions can achieve antibody serum half-life. extend.
  • the application provides a pharmaceutical composition comprising the antibody or antigen binding portion thereof disclosed herein and a pharmaceutically acceptable carrier.
  • the above-mentioned anti-IL-4R ⁇ antibodies disclosed herein such as anti-human IL-4R ⁇ monoclonal antibodies, can be formulated into pharmaceutical preparations together with pharmaceutically acceptable carriers, thereby exerting a more stable therapeutic effect.
  • these formulations can ensure the conformational integrity of the amino acid core sequence of the anti-IL-4R ⁇ antibody disclosed herein, such as the anti-human IL-4R ⁇ monoclonal antibody, while also protecting the protein's multifunctional groups from degradation (including but It is not limited to agglomeration, deamidation or oxidation).
  • for liquid formulations it can generally be stored at 2°C-8°C for at least one year. In some embodiments, for lyophilized formulations, it is stable at 30°C for at least six months.
  • the anti-IL-4R ⁇ antibody such as anti-human IL-4R ⁇ monoclonal antibody preparation
  • Disclosed anti-IL-4R ⁇ antibodies such as water injection or lyophilized preparations of anti-human IL-4R ⁇ monoclonal antibodies.
  • Pharmaceutically acceptable excipients include, but are not limited to: surfactants, solution stabilizers, isotonic regulators and buffers Or a combination.
  • surfactants include, but are not limited to: non-ionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80), poloxamers (such as poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, tetradecyl, linoleyl or octadecyl sarcosine, Pluronics, MONAQUATTM, etc.
  • non-ionic surfactants such as polyoxyethylene sorbitol fatty acid esters (Tween 20 or 80), poloxamers (such as poloxamer 188), Triton, sodium dodecyl sulfate (SDS), sodium lauryl sulfate, tetradecyl, linoleyl or octadecyl sarcosine, Pluronics, MONAQUATTM, etc.
  • the amount added
  • the solution stabilizer includes, but is not limited to, one or a combination of the following: sugars, such as reducing sugars and non-reducing sugars; amino acids, such as monosodium glutamate or histidine; alcohols Class, such as triol, higher sugar alcohol, propylene glycol, polyethylene glycol, etc.
  • sugars such as reducing sugars and non-reducing sugars
  • amino acids such as monosodium glutamate or histidine
  • alcohols Class such as triol, higher sugar alcohol, propylene glycol, polyethylene glycol, etc.
  • Isotonic adjusting agents include, but are not limited to, sodium chloride, mannitol, or a combination thereof.
  • the buffer includes but is not limited to: Tris, histidine buffer, phosphate buffer, or a combination thereof.
  • the present application also provides a method for preventing or treating IL-4R ⁇ -related diseases, which comprises administering to an individual an anti-IL-4R ⁇ antibody, or a composition comprising an anti-IL-4R ⁇ antibody, such as an anti-human IL-4R ⁇ monoclonal antibody.
  • a method for preventing or treating IL-4R ⁇ -related diseases which comprises administering to an individual an anti-IL-4R ⁇ antibody, or a composition comprising an anti-IL-4R ⁇ antibody, such as an anti-human IL-4R ⁇ monoclonal antibody.
  • an anti-IL-4R ⁇ antibody such as an anti-human IL-4R ⁇ monoclonal antibody.
  • the anti-IL-4R ⁇ antibody disclosed herein can effectively prevent and/or treat immune-mediated inflammation, and can be used as an anti-inflammatory drug.
  • the application also provides the use of an anti-IL-4R ⁇ antibody or a composition containing an anti-IL-4R ⁇ antibody in the preparation of a medicament for preventing or treating IL-4R ⁇ -related diseases or symptoms.
  • the IL-4R ⁇ -related disease or symptom is an immune-mediated inflammatory response or an immune-mediated inflammatory disease.
  • the above-mentioned immune-mediated inflammatory response or immune-mediated inflammatory diseases include, but are not limited to: asthma, allergy, atopic dermatitis, chronic sinusitis, eosinophilic esophagitis, nasal polyps , Psoriasis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, multiple sclerosis, uveitis, Behcet's uveitis, dry eye, chronic spontaneous urticaria, etc.
  • the anti-IL-4R ⁇ antibodies disclosed herein can also be used for the prevention or prevention of multiple sclerosis, Crohn’s disease, colitis, ulcerative colitis, systemic lupus erythematosus, graft-versus-host disease, etc. treatment.
  • the anti-IL-4R ⁇ antibody disclosed herein can be used as an anti-immune-mediated inflammatory response drug.
  • the anti-immune-mediated inflammatory response drugs referred to in this application refer to drugs that inhibit and/or treat immune-mediated inflammatory responses. For example, it can delay the development of immune-mediated inflammatory response-related symptoms and/or reduce these The severity of the symptoms.
  • the drug can reduce the symptoms associated with the existing inflammatory response and prevent the appearance of other symptoms.
  • the drug can also reduce or prevent the metastasis of the inflammatory response.
  • the dosage of administration varies depending on the age and weight of the patient, the characteristics and severity of the disease, and the route of administration. With reference to the results of animal experiments and the overall situation, the total dose should not exceed a certain range. In a specific embodiment, the dose for intravenous injection is 1-1800 mg/day.
  • the dosage and frequency of administration of the antibody or its composition may vary depending on the prevention or treatment of the disease.
  • a composition containing the antibody of the present application or a mixture thereof is administered to a patient who is not yet in a disease state to enhance the patient's resistance, and this amount is defined as a "prophylactic effective dose".
  • the specific dosage depends on the patient's health and systemic immunity. Generally, relatively low doses are administered at relatively infrequent intervals for longer periods of time. In therapeutic applications, it is sometimes necessary to administer relatively high doses at relatively short intervals until disease progression is slowed or terminated, and preferably until the patient shows partial or complete improvement of disease symptoms. Thereafter, a prophylactic regimen can be administered to the patient.
  • Those of ordinary skill in the art can easily grasp the specific dosage and frequency according to actual needs.
  • the human IL-4R ⁇ extracellular segment antigen sequence is from UniProt (UniProtKB-P24394). According to the codon usage preference of Cricetulus griseus, the codon is optimized and the gene synthesis of the N-terminal amino acid fragment 26-232 is performed, and the sequence is subcloned into In the pUC57 vector, pUC57-hIL4R ⁇ -ECD was obtained.
  • the constant region sequence of human IgG1 is synthesized according to the sequence of Secukinumab (see WHO Drug Information Vol. 23, No. 4, 2009. P342). The sequence was subcloned into the pUC57 vector to obtain pUC57-IgG1-CH.
  • Dupilumab The amino acid sequence of Dupilumab (IgG4, ⁇ ) comes from who.int (see WHO Drug Information Vol. 26, No. 4, 2012. P412). After codon optimization, the nucleotide sequence of the whole gene is synthesized and cloned into the pTT5 expression vector. Sequencing verification confirmed that the correct cloning vector labeled pTT5 (Dupilumab) was obtained.
  • the pTT5 (Dupilumab) vector was transiently transfected into the HEK293E cell line, cultured in Freestyle293 medium containing 3mM valproic acid for 5 days, and purified from the cell culture supernatant using a Protein A affinity chromatography column (purchased from Pharmacia) Dupilumab antibody protein.
  • the human IL-4 sequence was derived from UniProt (UniProtKB-P05112). According to the codon usage preference of Cricetulus griseus, codon optimization was performed and the N-terminal amino acid fragment 25-153 was synthesized, and the sequence was subcloned into the pUC57 vector.
  • the Flag tag (DYKDDDDK) was inserted into the C-terminus of the hIL4 fragment by PCR and constructed on the pTT5 expression vector to obtain pTT5 (hIL4-Flag), and then sequenced, and clones with completely correct sequences were selected for transfection.
  • the plasmid was transfected into the HEK 293E cell line (preserved in the laboratory) by the PEI method. After culturing for 5 days in Freestyle293 medium (purchased from Gibco) containing 3mM valproic acid, use Protein A affinity chromatography column (purchased from Pharmacia) or Flag affinity chromatography (purchased from Sigma) to culture cells Purify the target protein from the supernatant. The quantification of the protein was carried out by the Bicinchoninic acid (BCA) method, and the purified protein was used for the following further analysis and research. The purified protein was used for the following mouse immunization and further analysis and research.
  • BCA Bicinchoninic acid
  • mice Dilute 100 ⁇ g/mouse of hIL-4R ⁇ -ECD-Fc antigen into 75 ⁇ l with saline, mix it with an equal volume of Freund’s complete adjuvant, and treat 4-5 weeks old Balb/c mice after phacoemulsification. (Purchased from Shanghai Lingchang Biotechnology Co., Ltd., animal production license number: SCXK (Shanghai) 2013-0018) for subcutaneous multi-point injection. Three weeks later, the 50 ⁇ g/mouse protein was also diluted to 75 ⁇ l and mixed with an equal volume of Freund's incomplete adjuvant. After the phacoemulsification was completed, the mice were injected subcutaneously at multiple points, and the immunization was repeated again two weeks later.
  • mice After the third immunization, all mice were cut off their tails and blood was collected to separate serum, and the serum titer was detected by ELISA coated with hIL-4R ⁇ -Fc antigen.
  • serum antibody titer > 10000, impulse immunization was performed one week after blood collection: 10 ⁇ g antigen/100 ⁇ l normal saline/mouse was injected into the tail vein.
  • the titer was detected by ELISA method: the ELISA plate was coated with hIL-4R ⁇ ECD-Fc antigen at a coating concentration of 1 ⁇ g/ml, 100 ⁇ l per well, and coated overnight at 4°C. The plate was washed twice with PBST (PBS containing 0.5% Tween-20) and then patted dry. Add a coating solution containing 1% BSA to each well to seal 200 ⁇ l, seal it at room temperature for 4 hours and then pat dry, and store it in a refrigerator at -20°C until use. When testing, add 100 ⁇ l of mouse serum of different concentrations to each well of the ELISA plate, set up 2 multiple wells, and incubate at room temperature for 1.5 hours. Wash 3 times with PBST and pat dry.
  • PBST PBS containing 0.5% Tween-20
  • Hybridoma sp2/0 cells (from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, the deposit number is TCM-18) were cultured in a 37°C, 5% CO 2 incubator, and the medium was changed one day before fusion. Three days after the mice were immunized, the spleen cells of the mice were taken for fusion.
  • the method of fusion and screening is as follows: take the mouse spleen, grind and wash, and count the spleen cells. The splenocytes and sp2/0 cells were mixed at a ratio of 10:1, and centrifuged at 1500 rpm for 7 minutes. Wash off the supernatant.
  • the blocking of IL-4 binding to hIL-4R ⁇ -ECD-Fc by murine anti-human hIL-4R ⁇ monoclonal antibody was studied by ELISA method.
  • the hIL-4 antigen was coated with the ELISA plate, and after blocking, hIL-4R ⁇ -ECD-Fc and 300 ⁇ l of murine anti-human hIL-4R ⁇ monoclonal antibody in the subclones were added to the culture supernatant of hybridoma cells, and finally added to HRP-sheep Anti-human IgG antibody for color detection. Retain cell lines that can block IL-4 and IL-4R ⁇ -ECD-Fc binding to the next round of subcloning.
  • Example 5 EC50 of murine-derived anti-human hIL-4R ⁇ monoclonal antibody binding to human IL-4R ⁇
  • the preferred mouse-derived anti-human hIL-4R ⁇ monoclonal antibody is affinity purified by a Protein G affinity chromatography column, and then quantified by the BCA method.
  • the EC50 of the binding of the anti-human hIL-4R ⁇ monoclonal antibody to hIL-4R ⁇ was detected by ELISA.
  • the detection method is as in Example 3.
  • the 1 ⁇ g/ml hIL-4R ⁇ -ECD-Fc antigen was coated on the ELISA plate, and murine anti-human hIL-4R ⁇ monoclonal antibodies of different concentrations were added for detection.
  • Example 6 Mouse anti-human hIL-4R ⁇ embodiment of monoclonal antibody inhibition of hIL-4 or hIL-13-induced TF-1 cell proliferation of
  • the hybridoma cell line after the third round of subcloning was expanded and serum-free culture, and then the cell supernatant was collected and purified using a Protein G (purchased from GE) affinity column for antibody purification. Quantify the purified antibody and verify its functional activity.
  • TF-1 cells are cytokine-dependent cell lines isolated and cultured from the bone marrow of human erythroid leukemia patients. Studies have shown that TF-1 cells grow well under the stimulation of hIL-4 or hIL-13, so they can become a better model for functional verification of IL-4 signaling pathways.
  • TF-1 cells from ATCC, deposit number: CRL-2003
  • hIL-4 or hIL-13 purchased from R&D Systems
  • the medium is RPMI1640 medium (purchased from Gibco), which contains 10% fetal bovine serum (purchased from Sigma), 100U/ml penicillin (purchased from Gibco) and 100mg/ml streptomycin (purchased from Gibco) , Called RPMI-1640 complete medium.
  • mice-derived anti-human hIL-4R ⁇ monoclonal antibodies (20 ⁇ g/ml-3ng/ml, 3-fold dilution, 9 different concentrations) with medium solution, 100 ⁇ l per well, add to 96-well flat-bottomed cell culture plate Medium (purchased from Corning), and then add 100 ⁇ l of cell suspension to each well. Two replicate wells were set up in each group , incubated at 37°C and 5% CO 2 for 72 hours. Add 20 ⁇ l of CCK-8 solution (purchased from Dojindo Company) to each well, continue to incubate for 8 hours, mix well, measure the OD value of each well at 450nm wavelength with a microplate reader (Molecular Device), and calculate the cell proliferation ratio.
  • CCK-8 solution purchased from Dojindo Company
  • Table 7 Exemplary murine anti-human hIL-4R ⁇ monoclonal antibody inhibits IL-4 induced TF-1 proliferation
  • RNA of each hybridoma cell line was extracted using Trizol (purchased from Shanghai Shenggong Biotechnology), and the mRNA was reverse transcribed into cDNA using a reverse transcription kit (purchased from Takara).
  • the light chain variable region and heavy chain variable region genes of the mouse-derived anti-human hIL-4R ⁇ monoclonal antibody were amplified by PCR with primers reported in the literature, and then the PCR product was cloned into the pGEM-T vector, sequenced and analyzed. Region gene sequence. Based on the results of various functional experiments and early druggability analysis, we finally selected 17 antibodies listed in Table 2 as lead antibodies, and sequenced them to obtain the nucleotide sequences of their light and heavy chain variable regions.
  • the transformed amino acid sequences were compared and analyzed in GenBank, and all sequences were consistent with the characteristics of mouse IgG variable region genes. Further sequence analysis showed that the CDR sequences of the light and heavy chains of antibody Nos. 54, 55, 57, 64, 75, 81, 83, 84, 88, 100, 228, and 236 are all highly similar, with only a few amino acid differences between each other. The CDR sequences of the light and heavy chains of antibodies 29, 59, 120, 131, and 136 are highly similar. The nucleotide sequence of the heavy chain variable region of antibody No.
  • SEQ ID NO: 1 the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 2; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 3, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 4.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 59 is shown in SEQ ID NO: 5, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 7, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8.
  • SEQ ID NO: 8 the amino acid sequence of the heavy chain variable region of antibody No.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 131 is shown in SEQ ID NO: 13, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 15, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 131 is shown in SEQ ID NO: 13
  • the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14
  • the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 15
  • the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • SEQ ID NO: 17 the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 18; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 19, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 20.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 54 is shown in SEQ ID NO: 21, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 23, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 24.
  • SEQ ID NO: 25 is shown in SEQ ID NO: 25; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 26; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 27, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 28.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 57 is shown in SEQ ID NO: 29, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 30; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 31, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 32.
  • SEQ ID NO: 33 the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 34; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 35, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 36.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 75 is shown in SEQ ID NO: 37, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 38; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 39, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 40.
  • SEQ ID NO: 41 the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 42; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 43, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 44.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 83 is shown in SEQ ID NO: 45, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 46; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 47, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 48.
  • SEQ ID NO: 49 the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 50; the nucleotide sequence of the light chain variable region is shown in SEQ ID NO: 51, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 52.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 88 is shown in SEQ ID NO: 53, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 54; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 55, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 56.
  • SEQ ID NO: 57 the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 58; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 59, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 60.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 228 is shown in SEQ ID NO: 61, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 62; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 63, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 64.
  • the nucleotide sequence of the heavy chain variable region of antibody No. 236 is shown in SEQ ID NO: 65, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 66; the nucleotide sequence of the light chain variable region is shown in SEQ ID As shown in NO: 67, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 68.
  • antibodies Nos. 29, 59, 131, 136, 228 and 236 were selected for the construction of chimeric antibodies and humanized antibodies.
  • the chimeric antibody was constructed by intercepting the variable region of the heavy chain and the variable region of the light chain of the murine antibody, and connecting them to the constant region of the light and heavy chain of human IgG4 (from the Dupilumab antibody) by overlapping PCR.
  • variable region of the light chain and variable region of the heavy chain of the murine anti-human IL-4R ⁇ monoclonal antibody were analyzed according to Kabat's law, and 3 CDRs and 4 FRs were determined. Taking antibody No.
  • the amino acid sequence of its heavy chain complementarity determining region is HCDR1: DYGMH (SEQ ID NO: 129), HCDR2: YISSGSTTIYYADTVKG (SEQ ID NO: 130) and HCDR3: ISTVVAKRYAMDY (SEQ ID NO: 131),
  • the amino acid sequence of the light chain complementarity determining region is LCDR1: RASQDISNYLN (SEQ ID NO: 132), LCDR2: YTSRLHS (SEQ ID NO: 133) and LCDR3: QQINALPLT (SEQ ID NO: 134).
  • IGHV3-48*01 was selected as the heavy chain CDR grafting template, and the heavy chain CDR region of the mouse-derived anti-human IL-4R ⁇ monoclonal antibody 136 Transplanted into the framework region of IGHV3-48*01 to construct a heavy chain CDR grafted antibody.
  • IGKV1-33*01 was selected as the light chain CDR grafting template, and the light chain CDR region of the mouse-derived anti-human IL-4R ⁇ monoclonal antibody 136 was transplanted into IGKV1-
  • the framework region of 33*01 is constructed as a light chain CDR grafted antibody, and the resulting antibody is defined as 136-Gr (136-Grafting).
  • some amino acid positions in the framework regions were back-mutated. During the back mutation, the amino acid sequence was coded by Kabat, and the position of the site was indicated by the Kabat code.
  • the F at position 73 of the Kabat encoding is restored to the murine L, and at the same time, the L at position 96 in CDR3 is mutated to F.
  • the above-mentioned variable region gene sequence is codon optimized and synthesized by Biotech according to the codon usage preference of Cricetulus griseus.
  • the synthesized humanized variable region sequence is connected to the constant region of human IgG4, and this antibody is defined as the humanized antibody of antibody No. 136 (136-Humanization, 136-Hu).
  • the pTT5 vector (purchased from NRC Biotechnology Research Institute) was used to construct transient expression vectors of humanized heavy and light chains respectively, and the combination of the above light and heavy chains was used to transiently transfect and express antibodies using the HEK293 system (purchased from NRC biotechnology Research Institute).
  • HEK293 cells were cultured in Free Style 293 Expression Medium (purchased from Gibco), and the plasmid was transferred into the cells by PEI transfection for 5 days, and then the cell supernatant was collected. The antibody was purified by Protein A.
  • the humanized heavy chain variable region gene sequence of antibody No. 29 is 366 bp in length and encodes 122 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 69, and the amino acid sequence is shown in SEQ ID NO: 70.
  • the humanized light chain variable region gene sequence is 321 bp in length and encodes 107 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 72, and the amino acid sequence is shown in SEQ ID NO: 73.
  • the humanized heavy chain variable region gene sequence of antibody No. 59 is 354 bp in length and encodes 118 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 75, and the amino acid sequence is shown in SEQ ID NO: 76;
  • the humanized light chain variable region gene sequence is 318 bp in length and encodes 106 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 78, and the amino acid sequence is shown in SEQ ID NO: 79.
  • the humanized heavy chain variable region gene sequence of antibody No. 131 has a full length of 366 bp and encodes 122 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 81, and the amino acid sequence is shown in SEQ ID NO: 82;
  • the humanized light chain variable region gene sequence is 321 bp in length and encodes 107 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 84, and the amino acid sequence is shown in SEQ ID NO: 85.
  • the 448 amino acid 131-Hu humanized heavy chain (sequence is shown in SEQ ID NO: 83) and the 214 amino acid 131-Hu humanized light chain (sequence shown in SEQ ID NO: shown in 86).
  • the humanized heavy chain variable region gene sequence of antibody No. 136 has a full length of 366 bp and encodes 122 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 87, and the amino acid sequence is shown in SEQ ID NO: 88;
  • the humanized light chain variable region gene sequence is 321 bp in length and encodes 107 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 90, and the amino acid sequence is shown in SEQ ID NO: 91.
  • the 136-Hu humanized heavy chain of 448 amino acids (sequence shown in SEQ ID NO: 89) and the 136-Hu humanized light chain of 214 amino acids (sequence shown in SEQ ID NO: shown in 92).
  • the humanized heavy chain variable region gene sequence of antibody No. 228 has a full length of 360 bp and encodes 120 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 93, and the amino acid sequence is shown in SEQ ID NO: 94;
  • the humanized light chain variable region gene sequence is 318 bp in length and encodes 106 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 96, and the amino acid sequence is shown in SEQ ID NO: 97.
  • the humanized heavy chain variable region gene sequence of antibody No. 236 has a full length of 360 bp and encodes 120 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 99, and the amino acid sequence is shown in SEQ ID NO: 100;
  • the humanized light chain variable region gene sequence is 318 bp in length and encodes 106 amino acid residues.
  • the nucleotide sequence is shown in SEQ ID NO: 102, and the amino acid sequence is shown in SEQ ID NO: 103.
  • the 236-Hu humanized heavy chain of 446 amino acids (sequence is shown in SEQ ID NO: 101) and the 236-Hu humanized light chain of 213 amino acids (sequence shown in SEQ ID NO: shown in 104).
  • Example 9 Affinity of humanized anti-human IL-4R ⁇ monoclonal antibody to IL-4R ⁇
  • the affinity of the 6 humanized antibodies expressed and purified was tested by Biacore T200 (GE healthcare), and the reference antibody Dupilumab was used as a control.
  • the specific experimental method is: using Protein-A CM5 sensor chip (GE healthcare), FC1 (Flow cell 1) as the reference channel, and FC2 (Flow cell 2) as the sample channel. Capture human antibody or control antibody in FC2 channel, and then inject different concentrations of hIL-4R ⁇ -ECD-Flag.
  • the circulation conditions are: inject the analyte in all channels of FCs at 50 ⁇ l/min for 4min, dissociation time is 20min, and inject 6M guanidine hydrochloride (Sinopharm Chemical Reagent Co., Ltd.) at a rate of 10 ⁇ l/min for 30s for surface regeneration, and then use Biacore T200Evaluation Software Ver 1.0 calculates the difference between the signal of the capture antibody and the signal without the capture antibody and the affinity of the interaction. As shown in Table 8, the humanized antibodies 136-Hu and 236-Hu have significantly higher affinity for hIL-4R ⁇ -ECD-Flag than the reference antibody Dupilumab.
  • Antibody IC50(ng/ml) Dupilumab 137.9 29-hu 155.9 59-hu 295.5 131-hu 90.6 136-hu 84.3 236-hu 54.6
  • Example 11 Humanized anti-human IL-4R ⁇ inhibition of IgE secreting monoclonal antibody derived from peripheral blood B cells embodiment
  • IL-4 and IL-13 induce the conversion of IgM in B cells to IgE, and a large amount of IgE is secreted.
  • IgE is activated by binding to receptors on the surface of neutrophils, lymphocytes and mast cells. Immune response narrows the airways and causes inflammation, which makes asthma symptoms worse. Therefore, one of the functional activities of the anti-human IL-4R ⁇ monoclonal antibody is to inhibit the secretion of IgE from B cells. We verified this activity of the humanized anti-human IL-4R ⁇ monoclonal antibody.
  • Mononuclear cells were isolated from fresh human peripheral blood (provided by Changhai Hospital) using Histopaque-1077 (purchased from Sigma). Resuspend the cells into a 5x10 5 /ml cell suspension with RPMI-1640 complete medium, add 100 ⁇ l of cell suspension to a 96-well cell culture plate, and at the same time add 100 ⁇ l of 20ng/ml recombinant hIL-4 and different concentrations Anti-human hIL-4R ⁇ antibody (20 ⁇ g/ml-3ng/ml), incubated at 37°C, 5% CO 2 for 14 days. Then draw 100 ⁇ l of cell fluid supernatant, and detect the content of IgE by ELISA.
  • the ELISA analysis method is as follows: the mouse anti-human IgE is used to coat the ELISA plate (purchased from R&D Systems), the coating concentration is 2.5 ⁇ g/ml, 100 ⁇ l per well, and the coating is overnight at 4°C. Wash the plate twice with PBST and pat dry. Add a coating solution containing 1% BSA to each well to seal 200 ⁇ l, seal it at room temperature for 4 hours and then pat dry, and store it in a refrigerator at -20°C until use.
  • hIgE standard product purchased from R&D Systems
  • culture medium to make the final concentration of 100ng/ml, 33ng/ml, 11ng/ml, 3.7ng/ml, 1.2ng/ml, 410pg/ml, 130pg/ml, respectively.
  • ml and 46pg/ml 100 ⁇ l per well is added to the coated ELISA plate, each concentration is set to 2 replicate holes; 100 ⁇ l of cell supernatant is added to the coated ELISA plate, and incubated at room temperature for 1.5 hours. Wash 3 times with PBST and pat dry.
  • the Fc region of the antibody is modified on the basis of humanized antibodies.
  • the main mutation sites of the Fc region are T250Q, M252Y, S254T, T256E, V308P, M428L, N434A, and N434S.
  • the pharmacokinetic parameters of the Fc unmutated control antibody and the Fc region modified antibody after sc administration are shown in Table 10 and Table 11. There are two animals in each group, one male and one male, the route of administration is subcutaneous injection (sc), and the dosage is 5 mg/kg.
  • the Fc region modified antibody after sc administration has a smoother drug clearance, and the serum half-life of the antibody is significantly prolonged (Figure 5).
  • 305-A-sc is the control group
  • 305-B-sc is the control group.
  • Example 13 Establishment of an asthma-like pneumonia model in mice based on B-hIL-4/hIL-4RA two-person originated mice and drug efficacy experiment
  • the target molecule has undergone pilot production and formulation development, and the obtained 150mg/ml stable formulation is named 305-B.
  • IL-4/IL-4R ⁇ humanized mice B-hIL-4/hIL-4RA or B-hIL-4/IL4RA mice.
  • murine IL-4 and IL-4R ⁇ were knocked out and replaced by human IL-4 and IL-4R ⁇ genes.
  • the humanized mice behaved in the same way as normal mice, and there was no abnormality.
  • the construction of humanized mice and the pharmacodynamic study of 305-B on mouse asthma models were commissioned by Beijing Biocytogen Biotechnology Co., Ltd. to complete.
  • the experiment used a mouse model of asthma-like pneumonia sensitized by Ovalbumin (OVA). After the mice were sensitized by OVA, nebulized inhalation of OVA stimulated, triggering a Th2-type immune response, an increase in eosinophil infiltration can be observed in the lungs, and the concentration of IgE in the serum increased significantly.
  • OVA Ovalbumin
  • Dupilumab served as a positive control.
  • 305-B is the test product, and its main component is a humanized monoclonal antibody against human IL-4R ⁇ .
  • G1 was the PBS model group
  • G2 was the Dupilumab positive control (50mg/kg) group
  • G3 was the test product 305-B (0.5mg/kg) group
  • G4 is the test product 305-B (5mg/kg) group
  • G5 is the test product 305-B (25mg/kg) group
  • G6 is the test product 305-B (50mg/kg) group
  • G7 is no model Group.
  • mice were sensitized by intraperitoneal injection of OVA on days 0, 7, and 14, and they were stimulated by nebulized inhalation of OVA on days 21-25.
  • the animals in each group were administered intervention on the 20th day and the 23rd day.
  • the serum IgE in the PBS model group was significantly increased, and the proportion of eosinophils in the white blood cells in the alveolar lavage fluid was significantly increased. Histopathological results showed that mixed inflammatory cell infiltration and eosinophil infiltration in lung tissue were significantly enhanced, and mucus secretion was significantly increased, indicating that the model was successful.
  • the serum IgE of the test product 305-B (0.5mg/kg) group was significantly reduced (p ⁇ 0.05), the positive control Dupilumab (50mg/kg) group and the test product 305-B (5mg/kg) ), (25mg/kg), (50mg/kg) groups of serum IgE were significantly reduced (p ⁇ 0.0001), see Figure 6 for details; except for the 305-B (0.5mg/kg) group, each administration group is addicted to The proportion of acidic granulocytes in white blood cells (Eos/WBC%) was significantly reduced (p ⁇ 0.0001), as shown in Figure 7; except for the 305-B (0.5mg/kg) group, all the lung tissues of each administration group There was no obvious eosinophil infiltration, and the eosinophil score was significantly reduced (p ⁇ 0.05), see Figure 8 for details; 305-B (25mg/kg) and (50mg/kg) groups significantly improved lung mucus formation (p ⁇ 0.05), see Figure 8 for details;
  • 305-B in asthma-like pulmonary inflammation dominated by Th2-type immune response, two therapeutic administrations of 25mg/kg or 50mg/kg of 305-B can significantly reduce serum IgE levels and alveolar lavage. Eos/WBC% in the liquid reduces the degree of eosinophil infiltration in the lungs and reduces the secretion of mucus. It fully shows that 305-B antagonizes the occurrence of downstream Th2-type responses by inhibiting IL-4 and IL-13 signaling pathways, has a strong asthma inhibitory function, and has a rapid onset of action. This inhibitory effect is dose-dependent. There was no significant difference between 305-B and Dupilumab at the same dose in each evaluation index.
  • the antibody of the present invention shows consistent and stronger biological activity than the control antibody Dupilumab.
  • the results of in vivo pharmacodynamic studies show that there is no significant difference between the antibody of the present invention and the control antibody Dupilumab.

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Abstract

提供了能够结合人白细胞介素4受体α(hIL-4Rα)的抗体以及其制备方法和应用。该抗hIL-4Rα抗体能够与hIL-4Rα特异性结合，具有良好的抑制IL-4和IL-13诱导的细胞系增殖等效果，可应用于治疗IL-4Rα相关疾病，例如免疫介导的炎症性疾病。

Description

抗人白细胞介素-4受体α抗体及其制备方法和应用
相关申请的交叉引用
本申请要求于2020年04月17日提交的中国专利申请第202010309238.8号的优先权。
技术领域
本发明涉及抗体领域，更具体地，本发明涉及抗白细胞介素受体的抗体及其制备方法和应用。
背景技术
当机体受抗原刺激时，体内的抗原特异性淋巴细胞会识别抗原并发生活化、增殖、分化等应答，并最终清除入侵的抗原。T细胞和B细胞是主要的效应细胞。针对不同类型的抗原，T细胞可通过直接杀伤靶细胞和分泌不同类型的细胞因子，扩大和增强免疫应答来发挥免疫效应。研究表明，在过敏性哮喘等过敏性疾病中，白细胞介素(interlukin,IL)-4、IL-5、IL-9和IL-13等Th2型细胞因子介导了主要的病理发展。
哮喘是一种常见的呼吸道疾病，通常表现为呼吸道炎症、支气管高反应性和支气管壁的结构改变(呼吸道重构)。基因背景和包括过敏原及呼吸系统病毒等环境因素的刺激等诱发了哮喘的发生，主要的病理表现为反复喘息、气短、胸闷和咳嗽。哮喘在全球有230万患者，而且预期在今后的几十年中，患病率仍将升高。当前哮喘通过渐进式治疗来减轻症状并控制风险。吸入式皮质激素是中度哮喘的标准疗法，而严重患者会加上长效β拮抗剂，对于最严重的患者，可能还需要额外的控制药物。这些治疗会导致免疫系统紊乱，同时还会产生其他严重副作用。尽管如此，仍有5％-10％的患者目前无药可治。
在过敏性哮喘中，发现Th2型细胞因子在支气管中的异常高表达，同时已经证实Th2细胞因子介导了炎症反应的发生和发展并促进了呼吸道的病理改变等。这些细胞因子促进了包括嗜酸性粒细胞和肥大细胞等炎症细胞的活化和向炎症位点的趋化。IL-4和IL-13靶向B细胞，使其分泌的抗体由IgM向IgE转化， 同时通过诱导杯状细胞增生、将支气管成纤维细胞转化成肌成纤维细胞、胶原蛋白沉积和呼吸道平滑肌细胞增殖等诱导支气管重构。
IL-4和IL-13均可以通过结合白细胞介素-4受体α(IL-4Rα)而激活对应的信号通路，因此开发IL-4Rα靶向抗体将能同时阻断IL-4和IL-13的病理反应，有望用于治疗包括哮喘在内的IL-4Rα相关疾病。
发明内容
本发明的发明人进行了大量试验，得到了一组可以通过特异性阻断IL-4和IL-13与细胞表面IL-4受体α(IL-4Rα)结合从而阻断IL-4和IL-13信号传导的单克隆抗体，其能够阻断IL-4/IL-13介导的生物学活性。
第一方面，本申请提供了一种特异性结合IL-4受体α的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR3序列，任选地还包含HCDR1和/或HCDR2序列。在一些实施方案中，上述HCDR3序列包含选自SEQ ID NOs:107,113,119,125,131,137,143,149,155,161,167,173,179,185,191,197和203的氨基酸序列。在一些实施方案中，上述HCDR1序列包含选自SEQ ID NOs:105,111,117,123,129,135,141,147,153,159,165,171,177,183,189,195和201的氨基酸序列。在一些实施方案中，上述HCDR2序列包含选自SEQ ID NOs:106,112,118,124,130,136,142,148,154,160,166,172,178,184,190,196和202的氨基酸序列。在可选的实施方案中，上述抗原结合部分选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、scFv片段、Fd片段和单域抗体。
在一些实施方案中，上述重链可变区包含与选自SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94和100的氨基酸序列具有至少80％同源性的氨基酸序列，或者所述重链可变区包含选自SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94和100的氨基酸序列。
在一些实施方案中，特异性结合IL-4受体α的抗体或其抗原结合部分还包含轻链可变区，其中所述轻链可变区包含LCDR1、LCDR2和/或LCDR3序列。在某些实施方案中，所述LCDR1序列包含选自SEQ ID NOs:108,114,120,126,132,138,144,150,156,162,168,174,180,186,192,198和204的氨基酸序列。在某些实施方案中，所述LCDR2序列包含选自SEQ ID NOs:109,115,121,127,133, 139,145,151,157,163,169,175,181,187,193,199和205的氨基酸序列。在某些实施方案中，所述LCDR3序列包含选自SEQ ID NOs:110,116,122,128,134,140,146,152,158,164,170,176,182,188,194,200和206的氨基酸序列。
在一些实施方案中，上述轻链可变区包含与选自SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56,60,64,68,73,79,85,91,97和103的氨基酸序列具有至少80％同源性的氨基酸序列；或者所述轻链可变区包含选自SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56,60,64,68,73,79,85,91,97和103的氨基酸序列。
在一些实施方案中，特异性结合IL-4受体α的抗体或其抗原结合部分的重链包含选自SEQ ID NOs:71,77,83,89,95和101的氨基酸序列或者与上述序列具有至少80％同源性的氨基酸序列。任选地，所述抗体或其抗原结合部分的轻链包含选自SEQ ID NOs：74,80,86,92,98和104的氨基酸序列或者与上述序列具有至少80％同源性的氨基酸序列。
在一些实施方案中，第一方面所述的特异性结合IL-4受体α的抗体为单克隆抗体。
在一些实施方案中，第一方面所述的特异性结合IL-4受体α的抗体为人源化抗体。
在一些实施方案中，本文公开的IL-4受体α抗体或其抗原结合部分与抗体131-Hu、136-Hu或236-Hu结合IL-4受体α上的相同表位，或者与131-Hu、136-Hu或236-Hu竞争结合于IL-4受体α，其中所述抗体131-Hu的重链序列如SEQ ID NO：83所示，轻链序列如SEQ ID NO：86所示；所述抗体136-Hu的重链序列如SEQ ID NO：89所示，轻链序列如SEQ ID NO：92所示；以及所述抗体236-Hu的重链序列如SEQ ID NO：101所示，轻链序列如SEQ ID NO：104所示。
在一些实施方案中，本文公开的抗体或其抗原结合部分能够抑制B细胞的IgE分泌。在一些实施方案中，所述抗体或其抗原结合部分以小于600pM、优选为小于350pM的KD结合于IL-4Rα。在另一些实施方案中，所述抗体或其抗原结合部分能够抑制IL-4诱导的TF-1细胞增殖。
第二方面，本申请提供了一种核苷酸分子，所述核苷酸分子编码如上所述的特异性结合IL-4受体α的抗体或其抗原结合部分。
第三方面，本申请提供了一种表达载体，所述表达载体含有如上所述的核苷 酸分子。
在一些实施方案中，所述表达载体为pTT5、pUC57、pDR1、pcDNA3.1(+)、pDHFF或pCHO 1.0等。
第四方面，本申请提供了一种宿主细胞，所述宿主细胞含有如上所述的表达载体。在一些实施方案中，所述宿主细胞为HEK293、COS、CHO、NS0、sf9、sf21、DH5α、BL21(DE3)或TG1等。
第五方面，本申请提供了一种制备第一方面所述的特异性结合IL-4受体α的抗体或其抗原结合部分的方法，所述方法包括以下步骤：
a)在使得第四方面所述的宿主细胞能够产生所述抗体或其抗原结合部分的表达条件下，培养所述的宿主细胞，从而表达所述抗体或其抗原结合部分；以及
b)分离并纯化a)表达的所述抗体或其抗原结合部分。
第六方面，本申请提供了一种药物组合物，所述组合物包含第一方面所述的抗IL-4受体α抗体或其抗原结合部分以及药学上可接受的载体。
在一些实施方案中，所述组合物用于治疗IL-4Rα相关的疾病。
第七方面，本申请提供了第一方面所述的抗IL-4受体α抗体或其抗原结合部分、或第六方面所述的组合物在制备用于预防或治疗IL-4Rα相关疾病，例如免疫介导的炎症反应或炎症性疾病的药物中的应用。
在一些实施方案中，所述免疫介导的炎症反应或炎症性疾病包括：哮喘、过敏、特应性皮炎、慢性鼻窦炎、嗜酸性粒细胞性食道炎、鼻息肉、银屑病、类风湿性关节炎、银屑性关节炎、强直性脊柱炎、多发性硬化、葡萄膜炎、白塞氏葡萄膜炎、干眼症和慢性自发性荨麻疹等。
在其他方面，本申请提供了预防或治疗IL-4Rα相关疾病的方法，包括给予有需要的个体第一方面所述的抗体或其抗原结合部分、或第六方面所述的药物组合物。
本发明的抗IL-4Rα抗体或其抗原结合部分能够特异性与IL-4Rα结合，具有以下的一种或多种效应：阻断IL-4或IL-13与IL-4Rα的结合；抑制IL-4或IL-13诱导的细胞系如TF-1细胞增殖；和/或抑制B细胞的IgE分泌。体内药效试验充分显示本发明抗体能够通过抑制IL-4、IL-13信号通路拮抗下游Th2型应答的发生，具有很强的哮喘抑制功能，且起效迅速。本发明的抗IL-4Rα抗体或其抗原结合部分可以用于预防或治疗IL-4Rα相关疾病，例如免疫介导的炎症性疾病。
附图说明
图1为建株鼠源抗人IL-4Rα单克隆抗体对人IL-4Rα的亲和力测定结果。
图2为建株鼠源抗人IL-4Rα单克隆抗体抑制人IL-4诱导的TF-1细胞增殖的结果。
图3为人源化的抗人IL-4Rα单克隆抗体抑制人IL-4诱导的TF-1细胞增殖的实验结果。
图4为人源化的抗人IL-4Rα单克隆抗体抑制新鲜外周血来源B细胞IgE分泌的实验结果。
图5为人源化的抗人IL-4Rα单克隆抗体的药代动力学实验结果
图6 305-B对模型鼠血清IgE的抑制
图7各模型组动物的肺泡灌洗液中嗜酸性粒细胞在白细胞中的占比
图8各模型组动物的肺组织中的嗜酸性粒细胞浸润
图9各模型组动物的肺组织粘液分泌
具体实施方式
本申请提供了特异性结合于IL-4Rα的新的抗IL-4Rα抗体或其抗原结合部分。在优选实施方案中，本申请的抗体或其抗原结合部分以高亲和力结合于人IL-4Rα且抑制IL-4Rα的活性。本申请还提供了编码该抗体或其抗原结合片段的多核苷酸、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、制备和纯化该抗体的方法以及所述抗体或其抗原结合片段的医学和生物学应用，例如预防或治疗IL-4Rα相关疾病或病症。本申请还涵盖使用所述抗体或其抗原结合片段来检测IL-4Rα及调节IL-4Rα活性的方法。
为容易地理解本申请，首先定义本文中使用的某些术语。
本文所用术语“抗体”指包含四条多肽链，即通过双硫键互连的两条重链(H)链及两条轻链(L)的免疫球蛋白分子，以及其多聚体(例如IgM)。各重链包含重链可变区(缩写为VH)及重链恒定区(缩写为CH)。重链恒定区包含三个域，即CH1、CH2及CH3。各轻链包含轻链可变区(缩写为VL)及轻链恒定区(缩写为CL)。轻链恒定区包含一个域(CL1)。VH及VL区可进一步细分成称为互补决定区(CDR)的高变区，其中穿插有称为构架区(FR)的保守区。
如本文所用，术语抗体的“抗原结合部分”是指负责结合抗原的完整抗体分子的一部分或区段。抗原结合域可以包含重链可变区(VH)、轻链可变区(VL)或上述两者。抗体的抗原结合片段可使用任何适合的标准技术从完整抗体分子制备，所述标准技术包括蛋白水解消化或重组遗传工程化技术等。抗原结合部分的非限制性实例包括：Fab片段；F(ab')2片段；Fd片段；Fv片段；单链Fv(scFv)分子；单域抗体；dAb片段及由模拟抗体高变区的氨基酸残基组成的最小识别单元(例如分离的CDR)。术语“抗原结合部分”也包括其它工程化的分子，如双抗体、三抗体、四抗体及微型抗体等。
如本文所用，术语“重链可变区(VH)”及“轻链可变区(VL)”分别指单一抗体可变重链及轻链区，其包含FR1、2、3及4及CDR 1、2及3。
本领域技术人员公知，互补决定区(CDR，通常有CDR1、CDR2及CDR3)是可变区中对抗体的亲和力和特异性影响最大的区域。VH或VL的CDR序列有两种常见的定义方式，即kabat定义和Chothia定义，例如参见Kabat et al,“Sequences of Proteins of Immunological Interest”，National Institutes of Health,Bethesda，Md.(1991)；A1-Lazikani et al.，J.Mol.Biol.273:927-948(1997)；以及Martin et al.，Proc.Natl.Acad.Sci.USA86:9268-9272(1989)。对于给定抗体的可变区序列，可以根据Kabat定义或者Chothia定义来确定VH和VL序列中CDR区序列。在本申请的实施方案中，利用Kabat定义CDR序列。在本文中，重链可变区的CDR1、CDR2及CDR3分别简称为HCDR1、HCDR2及HCDR3；轻链可变区的CDR1、CDR2及CDR3分别简称为LCDR1、LCDR2及LCDR3。
对于给定抗体的可变区序列，可以通过多种方式分析可变区序列中CDR区序列，例如可以利用在线软件Abysis确定(http://www.abysis.org/)。
如本文所用术语“特异性结合”，是指两个分子之间的非随机结合反应，例如抗体至抗原表位的结合，例如抗体以比其对非特异性抗原的亲和性大至少两倍的亲和性结合于特异性抗原的能力。然而应了解，抗体能够特异性结合于两种或更多其序列相关的抗原。例如，本发明的抗体可特异性结合于人类与非人类(例如小鼠或非人类灵长动物)的IL-4Rα。
本文所用术语“单克隆抗体”指由基本同质的抗体群体获得的抗体，即，除了可能在少量个体中存在自然发生的突变以外，组成群体的各个抗体是相同的。本文所述单克隆抗体特别包括“嵌合”抗体，其中重链和/或轻链的一部分与来 源于具体物种或属于具体抗体类或亚类的抗体中的对应序列相同或同源，而重链和/或轻链的余下部分与来源于另一物种或属于另一抗体类或亚类的抗体中的对应序列相同或同源，并且还包括这样的抗体的片段，只要它们能表现出所期望的生物学活性(参见，美国专利号4,816,567；和Morrison et al,Proc.Natl.Acad.Sci.USA 81:6851-6855(1984))。
如本文所用，术语“同源性”被定义为经过序列比对和引入空位后，氨基酸或核苷酸序列变体中相同的残基的百分比，如果需要，达到最大百分比的同源性。用于比对的方法和计算机程序在本领域内是公知的。本文所述的“至少80％同源性”是指同源性为80％至100％中的任一值，例如85％、90％、95％、99％等。
如本文所用，术语“IL-4Rα相关疾病”包括与IL-4Rα信号通路激活相关的疾病和/或症状。示例性IL-4Rα相关疾病或病症包括免疫介导的炎症反应，例如过敏性疾病、哮喘等。
如本文所用，术语“半衰期”和“血清半衰期”是指根据本公开的抗原结合蛋白的血清浓度在体内减少50％而花费的时间。
一方面，本申请提供了特异性结合IL-4Rα的抗体或其抗原结合部分，其包含重链可变区和/或轻链可变区。下表1-5中示例性列出了适用于本申请公开的抗体的CDR、VH、VL、重链和轻链氨基酸序列及对应的核苷酸序列。在某些实施方案中，抗IL-4Rα抗体或其抗原结合部分包含HCDR3、HCDR2和/或HCDR1序列，其独立地选自表1中所示的HCDR3、HCDR2或HCDR1序列中任一者。在某些实施方案中，本申请的抗IL-4Rα抗体可进一步包含轻链CDR，其独立地选自表2中所示的轻链CDR1、CDR2或CDR3序列中任一者。举例而言，本申请的抗IL-4Rα抗体可包含表3和4中所示的重链可变域中的任一者，任选地与表3和4中所示的轻链可变域中的任一者配对。
表1：示例性抗IL-4Rα抗体的重链CDR氨基酸序列
Figure PCTCN2021086806-appb-000001
Figure PCTCN2021086806-appb-000002
表2：示例性抗IL-4Rα抗体的轻链CDR氨基酸序列
Figure PCTCN2021086806-appb-000003
表3：示例性抗IL-4Rα抗体重链可变区和轻链可变区的核苷酸序列和氨基酸序列
Figure PCTCN2021086806-appb-000004
Figure PCTCN2021086806-appb-000005
表4：示例性人源化抗IL-4Rα抗体重链可变区和轻链可变区的核苷酸序列和氨基酸序列
Figure PCTCN2021086806-appb-000006
表5：示例性抗IL-4Rα抗体的重链和轻链的氨基酸序列
Figure PCTCN2021086806-appb-000007
在一些实施方案中，本文公开的抗体或其抗原结合部分的HCDR3选自SEQ ID NOs:107,113,119,125,131,137,143,149,155,161,167,173,179,185,191,197和203所示的氨基酸序列。在一些实施方案中，HCDR2选自SEQ ID NOs:106,112,118,124,130,136,142,148,154,160,166,172,178,184,190,196和202所示的氨基酸序列；和/或所述HCDR1选自SEQ ID NOs:105,111,117,123,129,135, 141,147,153,159,165,171,177,183,189,195和201所示的氨基酸序列。
在具体的实施方案中，HCDR3选自SEQ ID NOs:107,113,119,125和131所示的氨基酸序列。在另一具体的实施方案中，HCDR3选自SEQ ID NOs:137,143,149,155,161,167,173,179,185,191,197和203所示的氨基酸序列。在优选的实施方案中，HCDR3选自125、131和203所示的氨基酸序列。
在具体的实施方案中，HCDR2选自SEQ ID NOs:106,112,118,124和130所示的氨基酸序列。在另一具体的实施方案中，HCDR2选自SEQ ID NOs:136,142,148,154,160,166,172,178,184,190,196和202所示的氨基酸序列。在优选的实施方案中，HCDR2选自124、130和202所示的氨基酸序列。
在具体的实施方案中，HCDR1选自SEQ ID NOs:105,111,117,123和129所示的氨基酸序列。在另一具体的实施方案中，，HCDR1选自SEQ ID NOs:135,141,147,153,159,165,171,177,183,189,195和201所示的氨基酸序列。在优选的实施方案中，HCDR1选自123、129和201所示的氨基酸序列。
在一些实施方案中，本文公开的抗体重链可变区包含选自SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94和100的氨基酸序列。在具体的实施方案中，所述重链可变区由选自SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94和100的氨基酸序列组成。
在一些实施方案中，本文公开的抗体重链可变区的氨基酸序列与SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94或100所示序列具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％以上的同源性。在优选的实施方案中，所述重链可变区与SEQ ID NOs:82,88或100所示的氨基酸序列具有99％以上的同源性。
本文公开的抗体或其抗原结合部分在包含重链可变区的基础上还可以进一步包含轻链可变区。
在一些实施方案中，所述轻链可变区的CDR3(LCDR3)选自SEQ ID NOs:110,116,122,128和134所示的氨基酸序列,或者选自SEQ ID NOs:140,146,152,158,164,170,176,182,188,194,200和206所示的氨基酸序列。在优选的实施方案中，LCDR3选自SEQ ID NOs:128,134和206所示的氨基酸序列。
在一些实施方案中，LCDR2选自SEQ ID NOs:109,115,121,127和133所 示的氨基酸序列，或者选自SEQ ID NOs:139,145,151,157,163,169,175,181,187,193,199和205所示的氨基酸序列。在优选的实施方案中，LCDR2选自SEQ ID NOs:127,133和205所示的氨基酸序列。
在一些实施方案中，LCDR1选自SEQ ID NOs:108,114,120,126,132,138,144,150,156,162,168,174,180,186,192,198和204的氨基酸序列，或者选自SEQ ID NOs:108,114,120,126,132,138,144,150,156,162,168,174,180,186,192,198和204的氨基酸序列。在优选的实施方案中，LCDR1选自SEQ ID NOs:126,132和204所示的氨基酸序列。
在一些实施方案中，本文公开的抗体轻链可变区包含选自SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56,60,64,68,73,79,85,91,97和103的氨基酸序列。在具体的实施方案中，所述轻链可变区由选自SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56,60,64,68,73,79,85,91,97和103的氨基酸序列组成。
在一些实施方案中，本文公开的抗体轻链可变区的氨基酸序列与SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56,60,64,68,73,79,85,91,97或103所示序列具有至少80％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％以上的同源性。在优选的实施方案中，所述重链可变区与SEQ ID NOs:85,91或103所示序列具有99％以上的同源性。
在具体的实施方案中，本文公开的抗体或其抗原结合部分的重链与选自SEQ ID NOs:71,77,83,89,95和101的氨基酸序列具有至少80％的同源性，例如90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同源性。在更具体的实施方案中，上述抗体重链由选自SEQ ID NOs:71,77,83,89,95和101的氨基酸序列组成。在优选的实施方案中，上述抗体重链的氨基酸序列如SEQ ID NO:83,89或101所示。
在具体的实施方案中，本文公开的抗体的轻链与选自SEQ ID NOs：74,80,86,92,98和104的序列具有至少80％的同源性，例如具有90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同源性。在更具体的实施方案中，所述抗体轻链由选自SEQ ID NOs：74,80,86,92,98和104的氨基酸序列组成。在优选的实施方案中，上述抗体轻链的氨基酸序列如SEQ ID NO:86,92或104所示。
在一些实施方案中，本文公开的抗体的重链或重链可变区、轻链或轻链可变 区可以在上述所列举的各自对应的具体氨基酸序列的基础上取代、缺失或添加至少一个氨基酸，且得到的变体仍保持结合IL-4Rα的活性。
在某些实施方案中，上述氨基酸取代、缺失或添加的数目为1-30个，优选为1-20个，更优选为1-10个。在优选的实施方案中，序列变体与原氨基酸序列相差约1、2、3、4、5、6、7、8、9、或10个氨基酸的取代、缺失和/或添加。在更优选的实施方案中，序列变体与原氨基酸序列相差约1、2、3、4或5个氨基酸的取代、缺失或添加。在具体的实施方案中，所述氨基酸取代为保守性取代。
在优选的实施方案中，本文公开的抗体为抗体131-Hu、136-Hu或236-Hu，其中抗体131-Hu的重链序列如SEQ ID NO：83所示，轻链序列如SEQ ID NO：86所示，其中CDR序列与抗体131相同；抗体136-Hu的重链序列如SEQ ID NO：89所示，轻链序列如SEQ ID NO：92所示，其中CDR序列与抗体136相同；所述抗体236-Hu的重链序列如SEQ ID NO：101所示，轻链序列如SEQ ID NO：104所示其中CDR序列与抗体236相同。
在一些实施方案中，本文公开的抗体或其抗原结合部分与抗体131-Hu、136-Hu或236-Hu结合白细胞介素-4受体α上的相同表位，或者与131-Hu、136-Hu或236-Hu竞争结合于白细胞介素-4受体α。
在一些的实施方案中，本文公开的抗体为单克隆抗体。在具体的实施方案中，本文公开的抗体为人源化的抗体。
本文公开的抗体或其抗原结合部分能够特异性结合IL-4Rα。在具体的实施方案中，所述抗体或其抗原结合部分特异性结合人IL-4Rα或鼠IL-4Rα。在优选的实施方案中，所述抗体或其抗原结合部分特异性结合人IL-4Rα。
在一些实施方案中，所述抗体或其抗原结合部分以小于600pM的KD结合于IL-4Rα。在优选实施方案中，所述抗体或其抗原结合部分以小于350pM pM的KD结合于IL-4Rα，例如人IL-4Rα。
在一些实施方案中，本文公开的抗体或其抗原结合部分能够抑制IL-4诱导的TF-1细胞增殖。在一些实施方案中，本文公开的抗体或其抗原结合部分能够抑制B细胞的IgE分泌。
例如，本申请的发明人对本文公开的抗人IL-4Rα单克隆抗体进行了体外、体内生物学实验，结果表明此抗体能够很好地与IL-4Rα进行结合。
具体地，本申请的发明人对抗人IL-4Rα单克隆抗体进行了亲和力检测、阻 断IL-4/IL-13与IL-4Rα结合的实验分析、体外细胞功能检测等实验。实验结果表明，本文公开的抗人IL-4Rα单克隆抗体可以结合细胞表面的IL-4Rα，阻断IL-4/IL-13与IL-4Rα之间的信号传导，抑制了炎症反应的发生。
本申请还提供了编码本文公开的抗体或其抗原结合部分的核苷酸分子、包含所述多核苷酸的载体、包含所述多核苷酸或载体的宿主细胞、以及制备和纯化该抗体的方法。
在一些实施方案中，编码所述抗体或其抗原结合部分的核苷酸分子可操作地连接到调控序列，调控序列可以被用所述载体转化过的宿主细胞识别。
在一些实施方案中，任何合适的表达载体都可以用于本申请。例如，所述表达载体可以为pTT5、pUC57、pDR1、pcDNA3.1(+)、pDHFF及pCHO 1.0中的一种。表达载体中可以包括连接有合适的转录和翻译调节序列的融合DNA序列。
在一些实施方案中，可用的宿主细胞为含有上述表达载体的细胞，可以是真核细胞，如哺乳动物或昆虫宿主细胞培养系统均可用于本申请的抗体或其抗原结合部分的表达。例如，HEK293细胞、COS、CHO、NS0、sf9及sf21等均可适用于本发明。所述宿主细胞也可以为含有上述表达载体的原核细胞，例如可以为DH5α、BL21(DE3)或TG1等。
在一些实施方案中，本文公开的抗人IL-4Rα单克隆抗体的制备方法包括：在表达条件下，培养宿主细胞，从而表达抗人IL-4Rα单克隆抗体；分离和纯化表达的抗人IL-4Rα单克隆抗体。利用上述方法，可以将重组蛋白纯化为基本均一的物质，例如在SDS-PAGE电泳上为单一条带。
在一些实施方案中，可以利用亲和层析的方法对本文公开的抗IL-4Rα抗体进行分离纯化，根据所利用的亲和柱的特性，可以使用常规的方法例如高盐缓冲液、改变PH等方法洗脱结合在亲和柱上的抗IL-4Rα抗体。
在一些实施方案中，本文公开的人源化的抗人IL-4Rα单克隆抗体是通过以下方法得到的：利用实验室制备的IL-4Rα抗原免疫Balb/c小鼠，在多次免疫小鼠滴度较高后取小鼠脾细胞与杂交瘤细胞融合并筛选出具有抑制IL-4功能活性的杂交瘤细胞株。更具体地，本申请的发明人通过大量实验，首先分别表达了IL-4Rα胞外段抗原、IL-4和IL-13，在此基础上利用不同的佐剂与IL-4Rα抗原混合免疫小鼠，然后进一步将上述小鼠的脾细胞与杂交瘤细胞株sp2/0融合，融合后的杂交瘤利用IL-4Rα胞外段抗原筛选出阳性细胞株，在验证其对IL-4/IL-13 与IL-4Rα结合的阻断并确实抑制IL-4/IL-13的功能后获得目标细胞株。将目标分子进行人源化改造后，将轻链和重链基因同时克隆到真核表达载体pCHO1.0中。将上述表达载体通过脂质体法转染CHO细胞，然后用嘌呤霉素和甲胺蝶呤筛选阳性细胞克隆，将筛选得到的高表达克隆用无血清培养基扩大培养，用Protein A亲和柱分离或纯化人源化的抗人IL-4Rα单克隆抗体。
在另外一些实施方案中，可以使用本领域的常规技术，例如PCR诱变进一步改变鼠源的亲本抗体来产生抗体的嵌合或人源化形式或其他变异形式。本申请的亲本抗体可以在例如抗原互补决定区(CDR)结构域内被诱变来产生变异抗体，可筛选其目的性质的存在，例如结合亲和力(更低的KD)、IC50、特异性、优先结合等等。优选地，变异抗体中目的性质是相对于亲本抗体中性质的改善。优选氨基酸替代变异抗体，并且亲本抗体分子的至少1、2、3、4、5、6、7、8、9或10个氨基酸残基被去除且在它的位置上插入不同的残基。用于替代诱变的最感兴趣的位点是一个或更多个CDR区，但是也考虑框架区(FR)改变。优选保守的氨基酸替代，也可引入非保守氨基酸改变并用获得的变异抗体筛选目的性质。
在一些实施方案中，通过在抗体的Fc区域进行改造以增加抗体的血清半衰期。已确定的可以提高人FcRn与抗体结合能力的突变位点主要有T250Q、M252Y、S254T、T256E、V308P、M428L、N434A、N434S，在本实施方案中通过这些位置氨基酸的突变可以实现抗体血清半衰期的延长。
本申请提供了药物组合物，其包含本文公开的抗体或其抗原结合部分以及药学上可接受的载体。本文公开的上述抗IL-4Rα抗体例如抗人IL-4Rα单克隆抗体，可以和药学上可接受的载体一起配制成药物制剂，从而更稳定地发挥疗效。在一些实施方案中，这些制剂可以保证本文公开的抗IL-4Rα抗体例如抗人IL-4Rα单克隆抗体的氨基酸核心序列的构像完整性，同时还保护蛋白质的多官能团防止其降解(包括但不限于凝聚、脱酰胺或氧化)。在一些实施方案中，对于液体制剂，通常可以在2℃-8℃条件下保存至少稳定一年。在一些实施方案中，对于冻干制剂，在30℃下至少六个月保持稳定。
在一些实施方案中，所述抗IL-4Rα抗体例如抗人IL-4Rα单克隆抗体制剂可为制药领域常用的混悬、水针、冻干等制剂，优选水针或冻干制剂，对于本文公开的抗IL-4Rα抗体例如抗人IL-4Rα单克隆抗体的水针或冻干制剂，药学上可以接受的辅料包括但不限于：表面活性剂、溶液稳定剂、等渗调节剂和缓冲液或其 组合。在一些实施方案中，表面活性剂包括但不限于：非离子型表面活性剂如聚氧乙烯山梨醇脂肪酸酯(吐温20或80)，泊洛沙姆(如泊洛沙姆188)，Triton，十二烷基硫酸钠(SDS)，月桂硫酸钠，十四烷基、亚油基或十八烷基肌氨酸，Pluronics，MONAQUATTM等，其加入量应使抗人IL-4Rα单克隆抗体的颗粒化趋势最小。在一些实施方案中，溶液稳定剂包括但不限于以下列举的一种或其组合：糖类，例如还原性糖和非还原性糖；氨基酸类，例如谷氨酸单钠或组氨酸；醇类，例如三元醇、高级糖醇、丙二醇、聚乙二醇等。溶液稳定剂的加入量应该使最后形成的制剂在本领域的技术人员认为达到稳定的时间内保持稳定状态。等渗调节剂包括但不限于氯化钠、甘露醇或其组合。缓冲液包括但不限于：Tris、组氨酸缓冲液、磷酸盐缓冲液或其组合。
本申请还提供了预防或治疗IL-4Rα相关疾病的方法，其包括给予个体抗IL-4Rα抗体、或者包含抗IL-4Rα抗体例如抗人IL-4Rα单克隆抗体的组合物。在一些实施方案中，在对包括人在内的动物给药后，抗免疫介导的炎症反应效果明显。具体地说，本文公开的抗IL-4Rα抗体能够有效地预防和/或治疗免疫介导的炎症反应，可以作为抗炎症药物使用。
本申请还提供了抗IL-4Rα抗体、或者包含抗IL-4Rα抗体的组合物在制备用于预防或治疗IL-4Rα相关疾病或症状的药物中的用途。在一些实施方案中，所述IL-4Rα相关疾病或症状为免疫介导的炎症反应或免疫介导的炎症性疾病。
在一些实施方案中，上述免疫介导的炎症反应或免疫介导的炎症性疾病，包括但不限于：哮喘、过敏，特应性皮炎、慢性鼻窦炎、嗜酸性粒细胞性食道炎、鼻息肉、银屑病、类风湿性关节炎、银屑性关节炎、强直性脊柱炎、多发性硬化、葡萄膜炎、白塞氏葡萄膜炎、干眼症、慢性自发性荨麻疹等。除了上述的炎症相关疾病外，本文公开的抗IL-4Rα抗体还可用于多发性硬化、克罗恩病、结肠炎、溃疡性结肠炎、系统性红斑狼疮、移植物抗宿主病等的预防或治疗。
在一些实施方案中，本文公开的抗IL-4Rα抗体可以作为抗免疫介导的炎症反应药物使用。本申请所称的抗免疫介导的炎症反应药物，指具有抑制和/或治疗免疫介导的炎症反应的药物，例如，它可以延迟免疫介导的炎症反应相关症状的发展和/或降低这些症状的严重程度。在一些实施方案中，所述药物可以减轻已存在的炎症反应伴随症状并防止其他症状的出现。在一些实施方案中，所述药物还可以减少或防止炎症反应的转移。
本文公开的抗人IL-4Rα单克隆抗体及其组合物在对包括人在内的动物给药时，给药剂量因病人的年龄和体重、疾病特性和严重性以及给药途径而异，可以参考动物实验的结果和综合情况，总给药量不能超过一定范围。在具体的实施方案中，静脉注射的剂量是1-1800mg/天。
抗体或其组合物的施用剂量和频率可根据对疾病进行预防或治疗而变化。在预防性应用中，向尚未处于疾病状态的患者施用含有本申请的抗体或其混合物的组合物以增强患者抵抗力，此量定义为“预防性有效剂量”。在此用途中，具体的剂量又视患者健康状况及全身免疫性而定。通常以相对不频繁的间隔施用相对较低剂量较长时间。在治疗性应用中，有时需要以相对较短间隔施用相对较高剂量直至疾病进展减缓或终止为止，且优选直至患者显示疾病症状部分或完全改善为止。此后，可向患者施用预防性方案。本领域普通技术人员可以容易地根据实际需要掌握具体的剂量和频率。
本说明书和权利要求书中，词语“包括”、“包含”和“含有”意指“包括但不限于”，且并非意图排除其他部分、添加物、组分、或步骤。
应该理解，在本申请的特定方面、实施方案或实施例中描述的特征、特性、组分或步骤，可适用于本文所描述的任何其他的方面、实施方案或实施例，除非与之矛盾。
上述公开内容总体上描述了本发明。以下具体的实施例是对本发明作进一步的说明，不应理解为对本发明的限制。实施例不包括对传统常规方法的详细描述，这样的方法对于本领域具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，例如分子克隆手册、冷泉港的抗体技术实验手册。未注明试剂来源的为常规试剂。
实施例
实施例1 可溶性IL-4Rα胞外段Fc标签和Flag标签抗原、参比抗体Dupilumab 及IL-4的制备
人IL-4Rα胞外段抗原序列来自于UniProt(UniProtKB-P24394)，按照Cricetulus griseus的密码子使用偏好进行密码子优化并进行N端第26-232位氨基酸片段的基因合成，将序列亚克隆到pUC57载体中，得到了pUC57-hIL4Rα-ECD。人IgG1的恒定区序列根据Secukinumab的序列(参见WHO Drug Information  Vol.23,No.4,2009.P342)合成。将序列亚克隆到pUC57载体中，得到了pUC57-IgG1-CH。通过PCR法将hFc片段和Flag标签(DYKDDDDK)插入到hIL4Rα-ECD片段的C端，并构建至pTT5表达载体(实验室保存)上，获得pTT5(hIL4Rα-ECD-hFc)和pTT5(hIL4Rα-ECD-Flag)，然后进行测序，选取序列完全正确的克隆进行转染。
Dupilumab(IgG4，κ)的氨基酸序列来自who.int(参见WHO Drug Information Vol.26,No.4,2012.P412)，经过密码子优化后全基因合成核苷酸序列，克隆到pTT5表达载体，测序验证确认获得了正确的克隆载体标记为pTT5(Dupilumab)。将pTT5(Dupilumab)载体瞬时转染至HEK293E细胞系，利用含3mM的丙戊酸的Freestyle293培养基培养5天后，利用Protein A亲和层析柱(购自Pharmacia公司)从细胞培养上清中纯化Dupilumab抗体蛋白。
人IL-4序列来自于UniProt(UniProtKB-P05112)，按照Cricetulus griseus的密码子使用偏好进行密码子优化并合成N端第25-153位氨基酸片段，将序列亚克隆到pUC57载体中。通过PCR法将Flag标签(DYKDDDDK)插入到hIL4片段的C端并构建至pTT5表达载体上，获得pTT5(hIL4-Flag)，然后进行测序，选取序列完全正确的克隆进行转染。
通过PEI法将质粒转染至HEK 293E细胞系(实验室保存)。利用含3mM的丙戊酸的Freestyle293培养基(购自Gibco公司)培养5天后，利用Protein A亲和层析柱(购自Pharmacia公司)或Flag亲和层析(购自Sigma公司)从细胞培养上清中纯化目的蛋白。蛋白的定量通过二喹啉甲酸(Bicinchoninic acid，BCA)方法进行，纯化得到的蛋白用于以下的进一步分析与研究。纯化得到的蛋白用于以下的小鼠免疫及进一步分析与研究。
实施例2  hIL-4Rα-ECD-Fc的免疫
将100μg/鼠的hIL-4Rα-ECD-Fc抗原用生理盐水稀释成75μl后，与等体积的弗氏完全佐剂混合，并经超声乳化完全后对4-5周龄的Balb/c小鼠(购自上海灵畅生物科技有限公司，动物生产许可证号：SCXK(沪)2013-0018)进行皮下多点注射。三周后，将50μg/鼠的蛋白同样稀释成75μl后与等体积弗氏不完全佐剂混合，超声乳化完全后对小鼠进行皮下多点注射，两周后再次重复此免疫。所有小鼠在第三次免疫后一周剪尾取血分离血清，利用包被hIL-4Rα-Fc抗原的ELISA进行 血清滴度的检测。对于血清抗体效价>10000的小鼠，在取血后一周进行冲击免疫：尾静脉注射10μg抗原/100μl生理盐水/鼠。
滴度的检测通过ELISA方法进行：利用hIL-4RαECD-Fc抗原包被ELISA板，包被浓度为1μg/ml，每孔100μl，4℃包被过夜。PBST(含0.5％Tween-20的PBS)洗板2次后拍干。每孔加入含1％BSA的包被液封闭200μl，常温下封闭4小时后拍干，至-20℃冰箱中保存待用。检测时在ELISA板中每孔加入不同浓度的小鼠血清100μl，设2个复孔，室温孵育1.5小时。PBST洗涤3次后拍干。加入用PBST1:10000倍稀释的HRP标记的兔抗鼠Ig抗体(购自Sigma公司)100μl，室温孵育1小时。PBST洗涤3次后拍干。每孔加100μl显色液(临用前将ELISA显色A液与显色B液按照1：1的体积比混匀)显色，随后每孔加入100μl 2M H 2SO4终止液终止反应。立即用酶标仪(Molecular Device)在450nm波长处测量各孔OD值。
实施例3 杂交瘤融合和筛选
杂交瘤sp2/0细胞(来自中国科学院典型培养物保藏委员会细胞库，保藏号为TCM-18)在37℃、5％CO 2培养箱中培养，融合前一天换液。小鼠冲击免疫三天后取小鼠脾细胞进行融合。融合与筛选方法如下：取小鼠脾脏，研磨洗涤后进行脾细胞计数。将脾细胞和sp2/0细胞以10:1的比例混合，1500rpm离心7分钟。洗去上清液。1分钟内加入1ml PEG(1450)，轻摇90秒，在2.5分钟内加入无血清DMEM培养液(购自Gibco公司)5ml，再一次性加5ml无血清培养液终止反应，静置5分钟，1280rpm离心8分钟。按照一块96孔板两百万个sp2/0细胞的数量，将细胞均匀接种入96孔板，每孔200μl。先用含次黄嘌呤(hypoxanthine，H)、甲胺蝶呤(aminopterin，A)和胸腺嘧啶核苷(thymidine，T)的HAT培养基筛选，每3～4天半量换液，第10天改用HT培养基。10天后，待杂交瘤细胞铺满96孔板底部大于10％时，取上清用hIL-4Rα-ECD-Fc抗原包被的酶标板进行ELISA检测。ELISA检测方法如实施例2中所述方法相同。挑选出阳性杂交瘤克隆于24孔板中扩大培养，有限稀释法进行亚克隆，获得稳定表达目的抗体的杂交瘤株后进行保种建库。
实施例4 鼠源的抗人hIL-4Rα单克隆抗体对IL-4结合hIL-4RαECD-Fc的阻断
用ELISA方法研究鼠源的抗人hIL-4Rα单克隆抗体对IL-4结合hIL-4Rα-ECD-Fc的阻断。hIL-4抗原包被酶标板，封闭后同时加入hIL-4Rα-ECD-Fc和300μl亚克隆中的鼠源的抗人hIL-4Rα单克隆抗体杂交瘤细胞培养上清，最终加入HRP-羊抗人IgG抗体进行显色检测。保留能阻断IL-4和与IL-4Rα-ECD-Fc结合的细胞株进入下一轮的亚克隆。
实施例5 鼠源的抗人hIL-4Rα单克隆抗体对人IL-4Rα结合的EC50
将优选的鼠源抗人hIL-4Rα单克隆抗体通过Protein G亲和层析柱亲和纯化后，用BCA法完成定量。抗人hIL-4Rα单克隆抗体与hIL-4Rα的结合的EC50利用ELISA进行检测。检测方法如实施例3。将1μg/ml的hIL-4Rα-ECD-Fc抗原包被ELISA板，加入不同浓度的鼠源抗人hIL-4Rα单克隆抗体进行检测。
我们对266个建株抗体进行了分析，图1为代表性实验结果。表6所列为部分优选抗体的EC50数据，这些抗体对人IL-4Rα的亲和力较高，EC50均在10ng/ml左右。
表6：示例性鼠源的抗人hIL-4Rα单克隆抗体对人IL-4Rα的亲和力
抗体编号 EC50(ng/ml)
29 7.6
54 9.9
55 8.9
57 7.9
59 11.7
64 10.6
75 12.9
81 9.6
83 12.3
84 12.2
88 9.1
100 12.0
120 10.0
131 13.8
136 11.3
228 13.1
236 11.5
实施例6 鼠源的抗人hIL-4Rα单克隆抗体对hIL-4或hIL-13诱导的TF-1细胞增 殖的抑制
将第三轮亚克隆后的杂交瘤细胞株进行扩大无血清培养，然后收集细胞上清利用Protein G(购自GE公司)亲和柱进行抗体纯化。将纯化后的抗体进行定量并验证其功能活性。
TF-1细胞是从人红系白血病病人骨髓中分离培养的细胞因子依赖细胞株。研究表明TF-1细胞在hIL-4或hIL-13的刺激下生长良好，因此可成为较好的IL-4信号通路功能验证模型。
取生长状态良好的TF-1细胞(来自ATCC，保藏号为CRL-2003)，计数后与终浓度为20ng/ml的重组hIL-4或hIL-13(购自R&D Systems)重悬成2x10 5/100μl的细胞悬液。培养基为RPMI1640培养基(购自Gibco公司)，其含10％胎牛血清(购自Sigma公司)、100U/ml青霉素(购自Gibco公司)和100mg/ml链霉素(购自Gibco公司)，称为RPMI-1640完全培养基。用培养基溶液稀释不同浓度的鼠源的抗人hIL-4Rα单克隆抗体(20μg/ml-3ng/ml,3倍稀释，9个不同浓度)，每孔100μl，加入至96孔平底细胞培养板中(购自Corning公司)，然后每孔加入100μl的细胞悬液。每组设立2个复孔，37℃，5％CO 2中孵育培养72小时。每孔加入20μl的CCK-8溶液(购自Dojindo公司)，继续培养8小时，混匀后利用酶标仪(Molecular Device)在450nm波长处测量各孔OD值，并计算细胞增殖比率。
我们对上述266个鼠源的抗人hIL-4Rα单克隆抗体进行了IL-4诱导的TF-1细胞增殖抑制的功能活性分析(图2)，优选17个抗体的数据见表7。
表7：示例性鼠源的抗人hIL-4Rα单克隆抗体对IL-4诱导的TF-1增殖的抑制
抗体编号 IC50(ng/ml)
29 65.3
54 57.0
55 19.0
57 23.6
59 43.7
64 43.7
75 29.0
81 24.6
83 36.6
84 31.3
88 67.2
100 53.7
120 59.7
131 47.1
136 49.6
228 34.2
236 22.4
实施例7 鼠源的抗人hIL-4Rα单克隆抗体序列的测定
使用Trizol(购自上海生工生物)提取各杂交瘤细胞株的总RNA，用逆转录试剂盒(购自Takara公司)将mRNA逆转录成cDNA。以文献报道的引物通过PCR扩增鼠源的抗人hIL-4Rα单克隆抗体的轻链可变区和重链可变区基因，然后将PCR产物克隆入pGEM-T载体，测序并分析可变区基因序列。根据多种功能实验和早期成药性分析结果，我们最终挑取了表2所列17个抗体作为先导抗体，测序获取了其轻重链可变区核苷酸序列。在GenBank中对转化而来的氨基酸序列进行比对分析，所有序列均符合小鼠IgG可变区基因的特征。进一步序列分析结果显示，54、55、57、64、75、81、83、84、88、100、228、236号抗体的轻重链CDR序列均高度相似，相互间只有几个氨基酸的差异，而29、59、120、131、136号抗体的轻重链CDR序列高度相似。29号抗体的重链可变区核苷酸序列如SEQ ID NO：1所示，重链可变区氨基酸序列如SEQ ID NO：2所示；轻链可变区核苷酸序列如SEQ ID NO：3所示，轻链可变区氨基酸序列如SEQ ID NO：4所示。59号抗体的重链可变区核苷酸序列如SEQ ID NO：5所示，重链可变区氨基酸序列如SEQ ID NO：6所示；轻链可变区核苷酸序列如SEQ ID NO：7所示，轻链可变区氨基酸序列如SEQ ID NO：8所示。120号抗体的重链可变区核苷酸序列如SEQ ID NO：9所示，重链可变区氨基酸序列如SEQ ID NO：10所示；轻链可变区核苷酸序列如SEQ ID NO：11所示，轻链可变区氨基酸序列如SEQ ID NO：12所示。131号抗体的重链可变区核苷酸序列如SEQ ID NO：13所示，重链可变区氨基酸序列如SEQ ID NO：14所示；轻链可变区核苷酸序列如SEQ ID NO：15所示，轻链可变区氨基酸序列如SEQ ID NO：16所示。136号抗体的重链可变区核苷酸序列如SEQ ID NO：17所示，重链可变区氨基酸序列如SEQ ID NO：18所示；轻链可变区核苷酸序列如SEQ ID NO：19所示，轻链可变区氨基酸序列如SEQ ID NO：20所示。54号抗体的重链可变区核苷酸序列如SEQ ID NO：21所示，重链可变区氨基酸序列如SEQ ID NO：22所示；轻链可变区核苷酸序列如SEQ ID NO：23所示，轻链可变区氨基酸序列如SEQ ID  NO：24所示。55号抗体的重链可变区核苷酸序列如SEQ ID NO：25所示，重链可变区氨基酸序列如SEQ ID NO：26所示；轻链可变区核苷酸序列如SEQ ID NO：27所示，轻链可变区氨基酸序列如SEQ ID NO：28所示。57号抗体的重链可变区核苷酸序列如SEQ ID NO：29所示，重链可变区氨基酸序列如SEQ ID NO：30所示；轻链可变区核苷酸序列如SEQ ID NO：31所示，轻链可变区氨基酸序列如SEQ ID NO：32所示。64号抗体的重链可变区核苷酸序列如SEQ ID NO：33所示，重链可变区氨基酸序列如SEQ ID NO：34所示；轻链可变区核苷酸序列如SEQ ID NO：35所示，轻链可变区氨基酸序列如SEQ ID NO：36所示。75号抗体的重链可变区核苷酸序列如SEQ ID NO：37所示，重链可变区氨基酸序列如SEQ ID NO：38所示；轻链可变区核苷酸序列如SEQ ID NO：39所示，轻链可变区氨基酸序列如SEQ ID NO：40所示。81号抗体的重链可变区核苷酸序列如SEQ ID NO：41所示，重链可变区氨基酸序列如SEQ ID NO：42所示；轻链可变区核苷酸序列如SEQ ID NO：43所示，轻链可变区氨基酸序列如SEQ ID NO：44所示。83号抗体的重链可变区核苷酸序列如SEQ ID NO：45所示，重链可变区氨基酸序列如SEQ ID NO：46所示；轻链可变区核苷酸序列如SEQ ID NO：47所示，轻链可变区氨基酸序列如SEQ ID NO：48所示。84号抗体的重链可变区核苷酸序列如SEQ ID NO：49所示，重链可变区氨基酸序列如SEQ ID NO：50所示；轻链可变区核苷酸序列如SEQ ID NO：51所示，轻链可变区氨基酸序列如SEQ ID NO：52所示。88号抗体的重链可变区核苷酸序列如SEQ ID NO：53所示，重链可变区氨基酸序列如SEQ ID NO：54所示；轻链可变区核苷酸序列如SEQ ID NO：55所示，轻链可变区氨基酸序列如SEQ ID NO：56所示。100号抗体的重链可变区核苷酸序列如SEQ ID NO：57所示，重链可变区氨基酸序列如SEQ ID NO：58所示；轻链可变区核苷酸序列如SEQ ID NO：59所示，轻链可变区氨基酸序列如SEQ ID NO：60所示。228号抗体的重链可变区核苷酸序列如SEQ ID NO：61所示，重链可变区氨基酸序列如SEQ ID NO：62所示；轻链可变区核苷酸序列如SEQ ID NO：63所示，轻链可变区氨基酸序列如SEQ ID NO：64所示。236号抗体的重链可变区核苷酸序列如SEQ ID NO：65所示，重链可变区氨基酸序列如SEQ ID NO：66所示；轻链可变区核苷酸序列如SEQ ID NO：67所示，轻链可变区氨基酸序列如SEQ ID NO：68所示。
实施例8 抗人hIL-4Rα单克隆抗体的人源化
根据序列分析结果，挑取29、59、131、136、228和236号抗体进行了嵌合抗体和人源化抗体的构建。嵌合抗体的构建通过截取鼠源抗体的重链可变区和轻链可变区，利用overlapping PCR分别与人IgG4的轻重链恒定区(来自Dupilumab抗体)连接而成。
根据Kabat法则对鼠源的抗人IL-4Rα单克隆抗体的轻链可变区和重链可变区的氨基酸序列进行分析并确定了3个CDR和4个FR。以136号抗体为例，其重链互补决定区的氨基酸序列为HCDR1：DYGMH(SEQ ID NO：129)，HCDR2：YISSGSTTIYYADTVKG(SEQ ID NO：130)和HCDR3：ISTVVAKRYAMDY(SEQ ID NO：131)，轻链互补决定区的氨基酸序列为LCDR1：RASQDISNYLN(SEQ ID NO：132)，LCDR2：YTSRLHS(SEQ ID NO：133)和LCDR3：QQINALPLT(SEQ ID NO：134)。
通过在NCBI IgBlast与人IgG胚系序列(Germline)进行同源性比较，选择IGHV3-48*01为重链CDR移植模板，将鼠源的抗人IL-4Rα单克隆抗体136的重链CDR区移植入IGHV3-48*01骨架区，构建成重链的CDR移植抗体。同样地，经过与人IgG胚系序列同源性比较，选择IGKV1-33*01为轻链CDR移植模板，将鼠源的抗人IL-4Rα单克隆抗体136的轻链CDR区移植入IGKV1-33*01的骨架区，构建成轻链的CDR移植抗体，所得的抗体定义为136-Gr(136-Grafting)。同时，在此基础上，对一些框架区的氨基酸位点进行了回复突变。在进行回复突变时，将氨基酸序列进行了Kabat编码，位点的位置由Kabat码指示。优选地，对于轻链可变区序列，将Kabat编码第73位的F回复为鼠源的L，同时，将CDR3中第96位的L突变成F。重链可变区没有回复突变。上述可变区基因序列由生工生物按照Cricetulus griseus的密码子使用偏好进行密码子优化并合成。将合成的人源化可变区序列与人IgG4恒定区相连，此抗体定义为136号抗体的人源化抗体(136-Humanization，136-Hu)。
利用上述相同原理，对其余5个抗体同样进行了人源化。利用pTT5载体(购自NRC biotechnology Research Institute)分别构建人源化重链、轻链的瞬时表达载体，将上述轻重链组合利用HEK293系统(购自NRC biotechnology Research Institute)进行瞬时转染并表达抗体。HEK293细胞在Free Style 293 Expression Medium(购自Gibco公司)培养基中培养，利用PEI转染法将质粒转入细胞5 天后收取细胞上清，利用Protein A纯化后获得抗体。
最终，29号抗体人源化后的重链可变区基因序列全长366bp，编码122个氨基酸残基，核苷酸序列如SEQ ID NO：69所示，氨基酸序列如SEQ ID NO：70所示；人源化后的轻链可变区基因序列全长321bp，编码107个氨基酸残基，核苷酸序列如SEQ ID NO：72所示，氨基酸序列如SEQ ID NO：73所示。与人IgG4恒定区相连后，最终得到448个氨基酸的29-Hu人源化重链(序列如SEQ ID NO：71所示)和214个氨基酸的29-Hu人源化轻链(序列如SEQ ID NO：74所示)。
59号抗体人源化后的重链可变区基因序列全长354bp，编码118个氨基酸残基，核苷酸序列如SEQ ID NO：75所示，氨基酸序列如SEQ ID NO：76所示；人源化后的轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：78所示，氨基酸序列如SEQ ID NO：79所示。与人IgG4恒定区相连后，最终得到444个氨基酸的59-Hu人源化重链(序列如SEQ ID NO：77所示)和213个氨基酸的59-Hu人源化轻链(序列如SEQ ID NO：80所示)。
131号抗体人源化后的重链可变区基因序列全长366bp，编码122个氨基酸残基，核苷酸序列如SEQ ID NO：81所示，氨基酸序列如SEQ ID NO：82所示；人源化后的轻链可变区基因序列全长321bp，编码107个氨基酸残基，核苷酸序列如SEQ ID NO：84所示，氨基酸序列如SEQ ID NO：85所示。与人IgG4恒定区相连后，最终得到448个氨基酸的131-Hu人源化重链(序列如SEQ ID NO：83所示)和214个氨基酸的131-Hu人源化轻链(序列如SEQ ID NO：86所示)。
136号抗体人源化后的重链可变区基因序列全长366bp，编码122个氨基酸残基，核苷酸序列如SEQ ID NO：87所示，氨基酸序列如SEQ ID NO：88所示；人源化后的轻链可变区基因序列全长321bp，编码107个氨基酸残基，核苷酸序列如SEQ ID NO：90所示，氨基酸序列如SEQ ID NO：91所示。与人IgG4恒定区相连后，最终得到448个氨基酸的136-Hu人源化重链(序列如SEQ ID NO：89所示)和214个氨基酸的136-Hu人源化轻链(序列如SEQ ID NO：92所示)。
228号抗体人源化后的重链可变区基因序列全长360bp，编码120个氨基酸残基，核苷酸序列如SEQ ID NO：93所示，氨基酸序列如SEQ ID NO：94所示；人源化后的轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：96所示，氨基酸序列如SEQ ID NO：97所示。与人IgG4恒定区相连后，最终得到446个氨基酸的228-Hu人源化重链(序列如SEQ ID NO： 95所示)和213个氨基酸的228-Hu人源化轻链(序列如SEQ ID NO：98所示)。
236号抗体人源化后的重链可变区基因序列全长360bp，编码120个氨基酸残基，核苷酸序列如SEQ ID NO：99所示，氨基酸序列如SEQ ID NO：100所示；人源化后的轻链可变区基因序列全长318bp，编码106个氨基酸残基，核苷酸序列如SEQ ID NO：102所示，氨基酸序列如SEQ ID NO：103所示。与人IgG4恒定区相连后，最终得到446个氨基酸的236-Hu人源化重链(序列如SEQ ID NO：101所示)和213个氨基酸的236-Hu人源化轻链(序列如SEQ ID NO：104所示)。
实施例9 人源化的抗人IL-4Rα单克隆抗体对IL-4Rα的亲和力
表达纯化的6个人源化抗体的亲和力通过Biacore T200(GE healthcare)检测，参比抗体Dupilumab作为对照。具体实验方法为：利用Protein-A CM5传感芯片(GE healthcare)，以FC1(Flow cell 1)为参照通道，FC2(Flow cell 2)为样品通道。在FC2通道分别捕获人源抗体或对照抗体，随后注射不同浓度的hIL-4Rα-ECD-Flag。循环条件为：在FCs所有通道中以50μl/min注射4min分析物，解离时间为20min，以10μl/min速率注射6M盐酸胍(国药集团化学试剂有限公司)30s进行表面再生，然后利用Biacore T200Evaluation Software Ver 1.0计算捕获抗体的信号和无捕获抗体的信号差值及相互作用的亲和力。如表8所显示，人源化后的抗体136-Hu和236-Hu对hIL-4Rα-ECD-Flag的亲和力显著高于参比抗体Dupilumab。
表8:不同人源化抗体对IL-4Rα的亲和力
抗体 Ka(1/Ms) Kd(1/s) KD(pM)
Dupilumab 4.456 x 10 5 1.797 x 10 -4 403.1
29-hu 3.982 x 10 5 2.096 x 10 -4 526.5
59-hu 1.136 x 10 6 2.84 x 10 -4 250
131-hu 3.35 x 10 5 1.107 x 10 -4 330
136-hu 6.354 x 10 5 9.801 x 10 -5 154.2
228-hu 7.211 x 10 5 6.578x 10 -4 912.1
236-hu 1.475 x 10 6 2.555 x 10 -4 173.2
实施例10 人源化的抗人hIL-4Rα单克隆抗体对TF-1细胞增殖的抑制
人源化抗人hIL-4Rα单克隆抗体对TF-1细胞增殖的抑制实验参照实施例6 进行。其结果见图3。如表9所显示，人源化抗体131-Hu、136-Hu和236-Hu对IL-4介导的TF-1细胞增殖的抑制能力显著高于参比抗体Dupilumab。
表9:不同人源化抗体对IL-4诱导的TF-1细胞增殖的抑制
抗体 IC50(ng/ml)
Dupilumab 137.9
29-hu 155.9
59-hu 295.5
131-hu 90.6
136-hu 84.3
236-hu 54.6
实施例11 人源化的抗人IL-4Rα单克隆抗体对外周血来源B细胞的IgE分泌的 抑制
在哮喘发病过程中，IL-4和IL-13通过诱导B细胞的IgM向IgE转化，大量分泌IgE，而IgE在与嗜中性粒细胞、淋巴细胞和肥大细胞表面的受体结合后，激活免疫应答，使气道变窄并发生炎症反应，从而使哮喘症状加重。因此，抗人IL-4Rα单克隆抗体的功能活性之一是抑制B细胞的IgE分泌。我们验证了人源化抗人IL-4Rα单克隆抗体的这一活性。
利用Histopaque-1077(购自Sigma公司)从新鲜人外周血(由长海医院提供)中分离获得单个核细胞。用RPMI-1640完全培养基将细胞重悬成5x10 5/ml的细胞悬液，在96孔细胞培养板中加入100μl细胞悬液，同时加入100μl的20ng/ml的重组hIL-4及不同浓度的抗人hIL-4Rα抗体(20μg/ml-3ng/ml)，在37℃，5％CO 2条件下孵育培养14天。然后吸取100μl细胞液上清，用ELISA检测其中IgE的含量。
ELISA分析方法如下：采用小鼠抗人IgE包被ELISA板(购自R&D Systems)，包被浓度为2.5μg/ml，每孔100μl，4℃包被过夜。PBST洗板2次后拍干。每孔加入含1％BSA的包被液封闭200μl，常温下封闭4小时后拍干，置于-20℃冰箱中保存待用。检测时用培养基配制hIgE标准品(购自R&D Systems)，使其终浓度分别为100ng/ml、33ng/ml、11ng/ml、3.7ng/ml、1.2ng/ml、410pg/ml、130pg/ml及46pg/ml，每孔100μl加入包被好的ELISA板中，每个浓度设2个复孔；细胞上清100μl加入至包被好的ELISA板中，室温孵育1.5小时。PBST洗涤3次后 拍干。每孔加生物素化的大鼠抗人IgE(购自R&D Systems，用含1％BSA的PBST稀释1000倍)100μl，室温孵育1小时。PBST洗涤3次后拍干。每孔加Streptavidin HRP(购自BD Pharmingen，用含1％BSA的PBST稀释1000倍)100μl，室温孵育1小时。每孔加100μl显色液(临用前将ELISA显色A液与显色B液按照1：1的体积比混匀)显色，随后每孔加入100μl 2M H 2SO 4终止液终止反应。立即用酶标仪在450nm波长处测量各孔OD值。结果显示，相比于Dupilumab，131-Hu、136-Hu和236-Hu对外周血B细胞IgE分泌的抑制作用更强，表明它们具有优于Dupilumab的功能活性(图4)。
实施例12人源化的抗人IL-4Rα单克隆抗体在食蟹猴体内的药代动力学研究
在人源化抗体的基础上对抗体的Fc区域进行改造，Fc区域突变的位点主要为T250Q、M252Y、S254T、T256E、V308P、M428L、N434A、N434S。通过Fc的变化用以改变抗体的半衰期，更长的半衰期可以减少给药频率。
在食蟹猴单次给药后0h、1h、4h、10h、24h、48h、72h、120h、168h、240h、336h、408h、504h、672h、840h、1008h、1176h、1344h、1512h、1680h和1848h采集血样，进行了药代动力学分析。采用经验证的ELISA方法检测抗体的血药浓度。本研究中分析方法的定量范围为15.63-1,000.00ng/mL，所有浓度点的标准样品均由100％食蟹猴血清配制。Fc未突变对照组抗体和Fc区域改造后的抗体在sc给药后的药代动力学参数见表10、表11。每组动物数为两只，雌雄各一只，给药途径为皮下注射(sc)，给药剂量为5mg/kg。
表10：对照组抗体的药代动力学参数
Figure PCTCN2021086806-appb-000008
Figure PCTCN2021086806-appb-000009
表11：Fc区域改造后的抗体药代动力学参数
Figure PCTCN2021086806-appb-000010
相较于对照组，Fc区域改造后的抗体在sc给药后，药物清除比较平缓，抗体的血清半衰期显著延长(图5)，其中305-A-sc为对照组，305-B-sc为Fc区域改造后的抗体。本实验委托“军科正源(北京)药物研究有限责任公司”完成。
实施例13基于B-hIL-4/hIL-4RA双人源化小鼠类哮喘肺炎模型建立及药物药效 实验
将目标分子进行了中试生产和制剂开发，所获得的150mg/ml稳定制剂命名为305-B。对于体内药效研究，我们采用了IL-4/IL-4Rα双人源化小鼠(B-hIL-4/hIL-4RA或B-hIL-4/IL4RA小鼠)。在这些C57BL/6背景小鼠中，其鼠源IL-4和IL-4Rα被敲除并由人源的IL-4和IL-4Rα基因所取代。人源化小鼠与正常小鼠表现一致，未见异常。人源化小鼠构建和305-B在小鼠哮喘模型上的药效研究均委托北京百奥赛图基因生物技术有限公司完成。
试验采用鸡卵清白蛋白(Ovalbumin,OVA)致敏的小鼠类哮喘肺炎模型。小鼠经OVA致敏后雾化吸入OVA激发，引发Th2型免疫应答，在肺中可观察到嗜酸性粒细胞浸润的增加，同时血清中IgE浓度显著升高。利用此模型评价了抗体的抗过敏性哮喘功能。Dupilumab作为阳性对照。305-B为受试品，其主要成分为抗人IL-4Rα人源化单克隆抗体。
49只动物按照体重随机分为7组，每组7只，G1为PBS模型组，G2为Dupilumab阳性对照(50mg/kg)组，G3为受试品305-B(0.5mg/kg)组，G4为受试品305-B(5mg/kg)组，G5为受试品305-B(25mg/kg)组，G6为受试品305-B(50mg/kg)组，G7为不造模
Figure PCTCN2021086806-appb-000011
组。G1-G6组的动物都进行OVA致敏：小鼠在第0、7、14天腹腔注射OVA致敏，在第21-25天雾化吸入OVA激发。各组动物在第20天和第23天给药干预。
与
Figure PCTCN2021086806-appb-000012
组相比，PBS模型组血清中IgE显著升高，肺泡灌洗液中嗜酸性粒细胞在白细胞中的占比显著提高。组织病理学结果显示，肺组织中混合炎细胞浸润及嗜酸性粒细胞浸润显著增强，且粘液分泌显著增多，提示造模成功。
与PBS模型组相比，受试品305-B(0.5mg/kg)组血清IgE显著降低(p<0.05)，阳性对照Dupilumab(50mg/kg)组以及受试品305-B(5mg/kg)、(25mg/kg)、(50mg/kg)组的血清IgE均显著降低(p<0.0001)，详见图6；除305-B(0.5mg/kg)组之外，各给药组嗜酸性粒细胞在白细胞的占比(Eos/WBC％)均显著降低(p<0.0001)，详见图7；除305-B(0.5mg/kg)组外，各给药组的肺组织中均无明显的嗜酸性粒细胞浸润，嗜酸性粒细胞评分显著降低(p<0.05)，详见图8；305-B(25mg/kg)、(50mg/kg)组对肺部粘液形成有显著改善(p<0.05)，详见图9。上述各指标，相同剂量(50mg/kg)的305-B与Dupilumab没有显著差异。
综上所述，在以Th2型免疫应答为主的类哮喘肺部炎症中，25mg/kg或50mg/kg的305-B两次治疗性给药即能显著降低血清中IgE水平及肺泡灌洗液中Eos/WBC％，减轻肺部嗜酸性粒细胞浸润程度，减少粘液的分泌。充分显示305-B通过抑制IL-4、IL-13信号通路拮抗下游Th2型应答的发生，具有很强的哮喘抑制功能，且起效迅速。此抑制作用具有一定的剂量相关性。同等剂量的305-B与Dupilumab在各评价指标中未见显著差异。
结论：在亲和力测定和多种体外功能活性分析中，本发明的抗体均显示了一致的强于对照抗体Dupilumab的生物活性。体内药效研究结果表明本发明所示抗体与对照抗体Dupilumab未见显著差异。
可以理解，尽管本申请以某种形式被说明，但本申请并不局限于本说明书中所显示和描述的内容。对本领域的技术人员显而易见的是，在不偏离本申请的范围的前提下还可对所述实施方式和/或某一特征或参数做出各种变化。这些变 化都在本申请要求保护的范围内。

Claims (15)

  1. 特异性结合白细胞介素-4受体α的抗体或其抗原结合部分，其包含重链可变区，所述重链可变区包含HCDR3序列，任选地还包含HCDR1和/或HCDR2序列，其中所述HCDR3序列包含选自SEQ ID NOs:107,113,119,125,131,137,143,149,155,161,167,173,179,185,191,197和203的氨基酸序列；和/或所述HCDR1序列包含选自SEQ ID NOs:105,111,117,123,129,135,141,147,153,159,165,171,177,183,189,195和201的氨基酸序列；和/或所述HCDR2序列包含选自SEQ ID NOs:106,112,118,124,130,136,142,148,154,160,166,172,178,184,190,196和202的氨基酸序列；优选地，所述抗原结合部分选自Fab片段、Fab’片段、F(ab’)2片段、Fv片段、scFv片段、Fd片段和单域抗体。
  2. 如权利要求1所述的抗体或其抗原结合部分，其中所述重链可变区包含与选自SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94和100的氨基酸序列具有至少80％同源性的氨基酸序列，或者所述所述重链可变区包含选自SEQ ID NOs:2,6,10,14,18,22,26,30,34,38,42,46,50,54,58,62,66,70,76,82,88,94和100的氨基酸序列。
  3. 如权利要求1或2所述的抗体或其抗原结合部分，其还包含轻链可变区，所述轻链可变区包含LCDR1、LCDR2和/或LCDR3序列，其中所述LCDR1序列包含选自SEQ ID NOs:108,114,120,126,132,138,144,150,156,162,168,174,180,186,192,198和204的氨基酸序列；和/或所述LCDR2序列包含选自SEQ ID NOs:109,115,121,127,133,139,145,151,157,163,169,175,181,187,193,199和205的氨基酸序列；和/或所述LCDR3序列包含选自SEQ ID NOs:110,116,122,128,134,140,146,152,158,164,170,176,182,188,194,200和206的氨基酸序列。
  4. 如权利要求3所述的抗体或其抗原结合部分，其中所述轻链可变区包含与选自SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56,60,64,68,73,79,85,91,97和103的氨基酸序列具有至少80％同源性的氨基酸序列；或者所述轻链可变区包含选自SEQ ID NOs:4,8,12,16,20,24,28,32,36,40,44,48,52,56, 60,64,68,73,79,85,91,97和103的氨基酸序列。
  5. 如前述权利要求任一项所述的抗体或其抗原结合部分，其中所述重链包含选自SEQ ID NOs:71,77,83,89,95和101的氨基酸序列或者与上述序列具有至少80％同源性的氨基酸序列；和/或所述轻链包含选自SEQ ID NOs：74,80,86,92,98和104的氨基酸序列或者与上述序列具有至少80％同源性的氨基酸序列。
  6. 如前述权利要求任一项所述的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分与抗体131-Hu、136-Hu或236-Hu结合白细胞介素-4受体α上的相同表位，或者与抗体131-Hu、136-Hu或236-Hu竞争结合于白细胞介素-4受体α，其中所述抗体131-Hu的重链序列如SEQ ID NO：83所示，轻链序列如SEQ ID NO：86所示；所述抗体136-Hu的重链序列如SEQ ID NO：89所示，轻链序列如SEQ ID NO：92所示；以及所述抗体236-Hu的重链序列如SEQ ID NO：101所示，轻链序列如SEQ ID NO：104所示。
  7. 如前述权利要求任一项所述的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分以小于600pM、优选以小于350pM的KD结合于白细胞介素-4受体α。
  8. 如前述权利要求任一项所述的抗体或其抗原结合部分，其中所述抗体或其抗原结合部分特异性结合于人白细胞介素-4受体α或鼠白细胞介素-4受体α，其中优选地，所述抗体或其抗原结合部分能够抑制IL-4诱导的TF-1细胞增殖和/或抑制B细胞的IgE分泌。
  9. 药物组合物，其包含权利要求1-8中任一项所述的抗体或其抗原结合部分以及药学上可接受的载体。
  10. 核苷酸分子，其编码权利要求1-8中任一项所述的抗体或其抗原结合部分。
  11. 表达载体，其包含权利要求10所述的核苷酸分子。
  12. 宿主细胞，其包含权利要求10所述的核苷酸分子或权利要求11所述的表达载体。
  13. 产生权利要求1-8中任一项所述的抗体或其抗原结合部分的方法，其包括：
    a)在使得权利要求12所述的宿主细胞能够产生所述抗体或其抗原结合部分的表达条件下，培养所述宿主细胞，从而表达抗体或其抗原结合部分；以及
    b)分离并纯化步骤a)中表达的所述抗体或其抗原结合部分。
  14. 权利要求1-8中任一项所述的抗体或其抗原结合部分、或者权利要求9所述的组合物在制备用于预防或治疗IL-4Rα相关疾病的药物中的用途，优选地，所述IL-4Rα相关疾病为免疫介导的炎症性疾病。
  15. 如权利要求14所述的用途，其中所述免疫介导的炎症性疾病选自哮喘、过敏、特应性皮炎、慢性鼻窦炎、嗜酸性粒细胞性食道炎、鼻息肉、银屑病、类风湿性关节炎、银屑性关节炎、强直性脊柱炎、多发性硬化、葡萄膜炎、白塞氏葡萄膜炎、干眼症和慢性自发性荨麻疹。
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