Pharmaceutical composition containing an antibody against IL-5 and use thereof

Abstract

This application discloses pharmaceutical compositions containing antibodies against IL-5 and uses thereof. In particular, this application discloses pharmaceutical compositions containing an IL-5 antibody or an antigen-binding fragment thereof in a buffer solution. Furthermore, the pharmaceutical composition further comprises a sugar and a non-ionic surfactant.

Description

[Technical field]

[0001] The present disclosure is in the field of pharmaceutical formulations, and in particular relates to pharmaceutical compositions comprising anti-IL-5 antibodies or antigen-binding fragments thereof, and the use of said pharmaceutical compositions as diagnostic and therapeutic agents for IL-5-related diseases. [Background technology]

[0002] The statements made in this specification are merely intended to provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Interleukin-5 (IL-5), an important member of the interleukin family, is also known as T cell replacing factor (TRF), B cell growth factor-II (BCGF-II), IgA enhancing factor (IgA-EF) or eosinophil differentiation factor (EDF), a homodimeric glycoprotein secreted primarily by helper T cells type 2 (Th2). Human IL-5 consists of 134 amino acid residues, including a signal peptide of 22 amino acids and two glycosylation sites. Active IL-5 is an oligodimer of two peptide chains in an antiparallel configuration linked by disulfide bonds; however, IL-5 monomers have no biological activity (Adv Immunol. 1994; 57: 145-90).

[0004] Eosinophils (EOS) have been implicated in various pulmonary inflammatory diseases, including allergic diseases associated with allergic reactions. Asthma is a chronic inflammatory respiratory disease. There are approximately 300 million patients worldwide, with an incidence rate of 10%. The pathogenesis of asthma is associated with various cytokines. IL-5 and its receptor IL-5R play an important role in the pathogenesis of asthma. Currently, the most effective method of treating asthma is to administer sterols intranasally or orally to inhibit the expression of several important mediators involved in asthma (including IL-5), thereby reducing lung inflammation. However, the long-term application of steroids has numerous side effects. Therefore, it is necessary to find new drug targets for the treatment of asthma. Studies have shown that administration of anti-IL-5 antibodies inhibits the binding of IL-5 to the IL-5 receptor; significantly reduces eosinophil accumulation in the lungs and lowers the levels of eosinophils in blood, tissues and sputum; reduces inflammatory responses that can be mediated by eosinophils; improves lung function; and has a favorable effect on eosinophilic asthma and recurrent asthma (Drugs. May 2017; 77(7):777-784).

[0005] Antibody drugs have high molecular weight and complex structure. In the process of production, transportation and storage, antibodies often encounter problems due to denaturation, aggregation, contamination and formation of particles. To keep antibodies effective, the biological activity of antibodies must be maintained during production, purification, transportation and storage. Currently, new technologies for production and purification are being developed to produce a large number of highly purified monoclonal antibodies. However, how to stabilize these antibodies during transportation and storage, and how to prepare antibodies in a suitable dosage form for administration have always been problems.

[0006] As for anti-IL-5 antibodies, only GSK's mepolizumab and Teva Pharma's reslizumab are currently approved for marketing. Related patents include WO 2018 / 119016, WO 2017 / 033121, WO 2014 / 141149, WO 2016 / 040007, WO 2015 / 095539, WO 2012 / 138958, and WO 95 / 35375, etc. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2018 / 119016 [Patent Document 2] International Publication No. 2017 / 033121 [Patent Document 3] International Publication No. 2014 / 141149 [Patent Document 4] International Publication No. 2016 / 040007 [Patent Document 5] International Publication No. 2015 / 095539 [Patent Document 6] International Publication No. 2012 / 138958 [Patent Document 7] WO 95 / 35375 [Non-patent literature]

[0008] [Non-Patent Document 1] Adv Immunol.1994;57:145-90 [Non-Patent Document 2] Drugs.2017 May;77(7):777~784 Summary of the Invention

[0009] The present disclosure relates to a pharmaceutical composition comprising an IL-5 antibody or an antigen-binding fragment thereof, a buffer, and a surfactant, wherein the buffer is any one selected from the group consisting of acetic acid-sodium acetate buffer, succinic acid-sodium succinate buffer, histidine-hydrochloride buffer, and citric acid-sodium citrate buffer, and is preferably acetic acid-sodium acetate buffer or succinic acid-sodium succinate buffer; the anti-IL-5 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (i) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in amino acid SEQ ID NOs: 16, 17 and 18, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in amino acid SEQ ID NOs:19, 20 and 21, respectively; (ii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in amino acid SEQ ID NOs: 22, 23 and 24, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in amino acid SEQ ID NOs:25, 26 and 27, respectively; (iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as set forth in amino acid SEQ ID NOs: 28, 29 and 30, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in amino acid SEQ ID NOs:31, 32 and 33, respectively; (iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in amino acid SEQ ID NOs: 34, 35 and 36, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in amino acid SEQ ID NOs:37, 38 and 39, respectively; (v) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in amino acid SEQ ID NOs: 40, 41 and 42, respectively; the light chain variable region comprises an LCDR1, an LCDR2 and an LCDR3 set forth in amino acid SEQ ID NOs: 43, 44 and 45, respectively; or (vi) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 set forth in amino acid SEQ ID NOs: 34, 82 and 36, respectively; the light chain variable region comprises LCDR1, LCDR2 and LCDR3 set forth in amino acid SEQ ID NOs: 37, 38 and 39, respectively; Pharmaceutical compositions are provided.

[0010] In an alternative embodiment, the pH of the buffer in the pharmaceutical composition is about 5.0 to about 6.5, preferably about 5.5 to about 6.5, preferably about 6.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.0, preferably about 5.0 to about 5.5, preferably about 5.0 to about 5.8, preferably about 5.2 to about 5.8; non-limiting examples of the pH of the buffer include about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, with about 5.5 being most preferred.

[0011] In an alternative embodiment, the concentration of the buffer in the pharmaceutical composition is about 10 mM to about 40 mM, about 10 mM to about 30 mM, preferably about 15 mM to about 30 mM, preferably about 20 mM to about 30 mM, preferably about 25 mM to about 30 mM, preferably about 10 mM to about 25 mM, preferably about 15 mM to about 25 mM, preferably about 20 mM to about 25 mM, preferably about 10 mM to about 15 mM; non-limiting examples of the buffer concentration include about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, about 32 mM, about 34 mM, with about 30 mM being most preferred.

[0012] In an alternative embodiment, the concentration of the anti-IL-5 antibody or antigen-binding fragment thereof in the pharmaceutical composition is about 1 mg / ml to about 120 mg / ml, preferably about 1 mg / ml to about 100 mg / ml, preferably about 10 mg / ml to about 120 mg / ml, preferably about 20 mg / ml to about 120 mg / ml, preferably about 30 mg / ml to about 120 mg / ml, preferably about 40 mg / ml to about 120 mg / ml, preferably about 50 mg / ml to about 120 mg / ml, Preferably about 60 mg / ml to about 120 mg / ml, preferably about 70 mg / ml to about 120 mg / ml, preferably about 80 mg / ml to about 120 mg / ml, preferably about 90 mg / ml to about 120 mg / ml, preferably about 100 mg / ml to about 120 mg / ml, preferably about 110 mg / ml to about 120 mg / ml, preferably about 20 mg / ml to about 100 mg / ml, preferably about 30 mg / ml to about 100 mg / ml, preferably about 40 mg / ml to about 100 mg / ml, preferably about 50 mg / ml to about 100 mg / ml, preferably about 60 mg / ml to about 100 mg / ml, preferably about 70 mg / ml to about 100 mg / ml, preferably about 80 mg / ml to about 100 mg / ml, preferably about 90 mg / ml to about 100 mg / ml; non-limiting examples of the concentration of the anti-IL-5 antibody or antigen-binding fragment thereof are about 80 mg / ml, about 85 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, about 94 mg / ml, about 95 mg / ml, about 96 mg / ml, about 97 mg / ml, about 98 mg / ml, about 99 mg / ml, about 100 mg / ml, about 101 mg / ml, about 102 mg / ml, about 103 mg / ml, about 104 mg / ml, about 105 mg / ml, about 106 mg / ml, about 107 mg / ml, about 108 mg / ml, about 109 mg / ml, about 110 mg / ml, about 115 mg / ml, about 120 mg / ml, and most preferably about 100 mg / ml.

[0013] In an alternative embodiment, the surfactant included in the pharmaceutical composition is selected from the group consisting of polysorbate 20, polysorbate 80, polyhydroxyalkylene, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauramidopropyl betaine, cocamidopropyl betaine, linoleyl propyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isosteamidopropyl betaine, myristamidopropyl dimethylamine, palmamidopropyl dimethylamine, isostearamidopropyl dimethylamine, sodium methyl cocoyl The surfactant may be selected from the group consisting of polysorbate 80, polysorbate 20, polysorbate 80, etc. The surfactant may be selected from the group consisting of sodium methylcocoyl, sodium methyloleoyl taurate, polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, etc. The preferred surfactant is polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0014] In an alternative embodiment, the concentration of polysorbate 80 in the pharmaceutical composition is about 0.05 mg / ml to about 0.6 mg / ml, preferably about 0.1 mg / ml to about 0.6 mg / ml, preferably about 0.2 mg / ml to about 0.6 mg / ml, preferably about 0.3 mg / ml to about 0.6 mg / ml, preferably about 0.4 mg / ml to about 0.6 mg / ml, preferably about 0.5 mg / ml to about 0.6 mg / ml, preferably about 0.2 mg / ml to about 0.5 mg / ml. / ml, preferably about 0.3 mg / ml to about 0.5 mg / ml, preferably about 0.4 mg / ml to about 0.5 mg / ml, preferably about 0.3 mg / ml to about 0.4 mg / ml, and non-limiting examples of the concentration of the surfactant in the pharmaceutical composition are about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, and most preferably about 0.4 mg / ml.

[0015] Moreover, in an alternative embodiment, the pharmaceutical composition further comprises an excipient, wherein the excipient is selected from a stabilizer.

[0016] In an alternative embodiment, the stabilizer is selected from a sugar or an amino acid; the sugar may be selected from the group consisting of sucrose, trehalose, mannitol and sorbitol, preferably sucrose. The amino acid is selected from the group consisting of glycine, methionine and proline.

[0017] In alternative embodiments, the concentration of the sugar is from about 50 mg / ml to about 80 mg / ml, preferably from about 60 mg / ml to about 80 mg / ml, preferably from about 70 mg / ml to about 80 mg / ml, preferably from about 75 mg / ml to about 80 mg / ml, preferably from about 70 mg / ml to about 75 mg / ml; as non-limiting examples, the concentration of the stabilizer in the pharmaceutical composition includes about 70 mg / ml, about 71 mg / ml, about 72 mg / ml, about 73 mg / ml, about 74 mg / ml, about 75 mg / ml, about 76 mg / ml, about 77 mg / ml, about 78 mg / ml, about 79 mg / ml, about 80 mg / ml, more preferably about 72 mg / ml.

[0018] In an alternative embodiment, the concentration of the amino acid is about 8 mg / ml.

[0019] In an alternative embodiment, the pharmaceutical composition comprises: (a) about 1 mg / ml to about 120 mg / ml of an anti-IL-5 antibody or an antigen-binding fragment thereof; (b) about 10 mM to about 30 mM acetic acid-sodium acetate buffer having a pH of about 5.0 to 6.5; and (c) about 0.1 mg / ml to about 0.6 mg / ml of polysorbate 80. Includes.

[0020] In an alternative embodiment, the pharmaceutical composition comprises: (a) about 80 mg / ml to about 100 mg / ml of an anti-IL-5 antibody or an antigen-binding fragment thereof; (b) about 10 mM to about 30 mM acetic acid-sodium acetate buffer having a pH of about 5.0 to about 6.0; and (c) about 0.1 mg / ml to about 0.4 mg / ml of polysorbate 80. Includes.

[0021] In an alternative embodiment, the pharmaceutical composition comprises: (d) about 80 mg / ml to about 120 mg / ml of an IL-5 antibody or an antigen-binding fragment thereof; (e) about 10 mM to about 30 mM acetic acid-sodium acetate buffer at a pH of about 5.0 to about 5.8; (f) about 0.2 mg / ml to about 0.6 mg / ml of polysorbate 80; and (g) about 70 mg / ml to about 75 mg / ml of sucrose. Preferably, the pharmaceutical composition comprises (h) about 100 mg / ml of an IL-5 antibody or antigen-binding fragment thereof, (i) about 30 mM acetic acid-sodium acetate buffer at a pH of about 5.5, (j) about 0.4 mg / ml of polysorbate 80, and (k) about 72 mg / ml of sucrose. Includes.

[0022] According to some preferred embodiments, the anti-IL-5 antibody or antigen-binding fragment thereof in the pharmaceutical composition of the present disclosure is a murine antibody, a chimeric antibody or a humanized antibody.

[0023] In an alternative embodiment, the humanized anti-IL-5 antibody in the pharmaceutical composition comprises a heavy chain variable region set forth in SEQ ID NO:49, 57, 63, 69 or 75, or a variant thereof; the variant comprises 1 to 10 amino acid backmutations in the heavy chain variable region sequence set forth in SEQ ID NO:49, 57, 63, 69 or 75, respectively.

[0024] In an alternative embodiment, the variant is (i) a variant comprising one or more amino acid back mutations selected from the group consisting of S49T, V93T, and K98S in the heavy chain variable region set forth in SEQ ID NO: 49; (ii) a variant comprising one or more amino acid back mutations selected from the group consisting of S49T, V93T, and K98T in the heavy chain variable region set forth in SEQ ID NO: 57; (iii) a variant comprising one or more amino acid back-mutations selected from the group consisting of R38K, M48I, R67K, V68A, M70L, R72V, T74K, and L83F in the heavy chain variable region set forth in SEQ ID NO: 63; (iv) a variant comprising one or more amino acid back mutations selected from the group consisting of F29I, R38K, V48I, R72A and T97F in the heavy chain variable region as set forth in SEQ ID NO: 69 and / or an N55V mutation in the CDR; or (v) A variant comprising one or more amino acid back-mutations selected from the group consisting of R38K, M48I, R67K, V68A, R72A, T74K, M81L, L83F, and D89E in the heavy chain variable region shown in SEQ ID NO: 75. The variant is any one of the variants shown in the above.

[0025] In an alternative embodiment, the humanized anti-IL-5 antibody in the pharmaceutical composition comprises: A heavy chain variable region as set forth in SEQ ID NO: 50 or 51; or A heavy chain variable region as set forth in SEQ ID NO: 58 or 59; or A heavy chain variable region as set forth in any one of the group consisting of SEQ ID NOs: 64, 65, and 66; or A heavy chain variable region as set forth in SEQ ID NO: 70 or 71; or A heavy chain variable region shown in any one of the following SEQ ID NOs: 76, 77, 78, and 79. Includes.

[0026] In an alternative embodiment, the humanized anti-IL-5 antibody in the pharmaceutical composition comprises a light chain variable region set forth in SEQ ID NO:46, 54, 60, 67 or 72, or a variant thereof; the variant comprises 1 to 10 amino acid backmutations in the light chain variable region set forth in SEQ ID NO:46, 54, 60, 67 or 72.

[0027] In an alternative embodiment, the variant is (i) a variant comprising one or more amino acid back-mutations selected from the group consisting of A43S, L47V, G66R, T69S, F71Y and Y87F in the light chain variable region set forth in SEQ ID NO: 46; (ii) a variant comprising one or more amino acid back-mutations selected from the group consisting of A43S, L47M, F71Y and Y87F in the light chain variable region set forth in SEQ ID NO: 54; (iii) a variant comprising an amino acid back mutation selected from the group consisting of E1D, I2T, I57V, V84T, and Y86F, or a combination thereof, in the light chain variable region set forth in SEQ ID NO:60; (iv) a variant comprising one or more amino acid back mutations selected from the group consisting of M4L, A42S, L45P and L46W in the light chain variable region set forth in SEQ ID NO: 67; and (v) a variant comprising one or more amino acid back-mutations selected from the group consisting of A43S, I48V, and F71Y in the light chain variable region set forth in SEQ ID NO: 72; The variant is any one of the variants shown in the above.

[0028] In an alternative embodiment, the humanized anti-IL-5 antibody in the pharmaceutical composition comprises: A light chain variable region as set forth in SEQ ID NO: 47 or 48; or A light chain variable region as set forth in SEQ ID NO: 55 or 56; or A light chain variable region as set forth in SEQ ID NO: 61 or 62; or A light chain variable region set forth in SEQ ID NO:68; or Light chain variable region shown in SEQ ID NO: 73 or 74 Includes.

[0029] In an alternative embodiment, the humanized anti-IL-5 antibody in the pharmaceutical composition comprises: (i) a heavy chain variable region as set forth in any one of SEQ ID NOs: 49, 50, and 51, or having 95% sequence identity with any one of SEQ ID NOs: 49, 50, and 51; and a light chain variable region as set forth in any one of SEQ ID NOs: 46, 47, and 48, or having 95% sequence identity between any one of SEQ ID NOs: 46, 47, and 48; (ii) a heavy chain variable region as set forth in any one of SEQ ID NOs:57, 58, and 59, or having 95% sequence identity with any one of SEQ ID NOs:57, 58, and 59; and a light chain variable region as set forth in any one of SEQ ID NOs: 54, 55, and 56, or having 95% sequence identity between any one of SEQ ID NOs: 54, 55, and 56; (iii) a heavy chain variable region as set forth in any one of SEQ ID NOs: 63, 64, 65, and 66, or having 95% sequence identity with any one of SEQ ID NOs: 63, 64, 65, and 66; and a light chain variable region as set forth in any one of SEQ ID NOs:60, 61, and 62, or having 95% sequence identity between any one of SEQ ID NOs:60, 61, and 62; (iv) a heavy chain variable region as set forth in any one of SEQ ID NOs:69, 70, and 71, or having 95% sequence identity with any one of SEQ ID NOs:69, 70, and 71; and A light chain variable region as set forth in any one of SEQ ID NOs:67 and 68, or having 95% sequence identity between any one of SEQ ID NOs:67 and 68; or (v) a heavy chain variable region set forth in any one of SEQ ID NOs:75, 76, 77, 78, and 79, or having 95% sequence identity between any one of SEQ ID NOs:75, 76, 77, 78, and 79; and Preferably, the humanized anti-IL-5 antibody comprises a light chain variable region as set forth in any one of SEQ ID NOs: 72, 73, and 74, or having 95% sequence identity between any one of SEQ ID NOs: 72, 73, and 74; (a) a heavy chain variable region set forth in SEQ ID NO:51 and a light chain variable region set forth in SEQ ID NO:47; (b) a heavy chain variable region set forth in SEQ ID NO:65 and a light chain variable region set forth in SEQ ID NO:62; (c) a heavy chain variable region set forth in SEQ ID NO:58 and a light chain variable region set forth in SEQ ID NO:56; (d) a heavy chain variable region set forth in SEQ ID NO: 71 and a light chain variable region set forth in SEQ ID NO: 68; or (e) a heavy chain variable region shown in SEQ ID NO: 79 and a light chain variable region shown in SEQ ID NO: 73 Includes.

[0030] The amino acid sequence having at least 95% sequence identity preferably has at least 95%, 96%, 97%, 98% or 99% sequence identity, more preferably has 97%, 98% or 99% or more sequence identity, and most preferably has at least 99% or more sequence identity, and the amino acid sequence having at least 95% sequence identity includes one or more amino acid deletions, insertions or substitutions obtained by mutation.

[0031] In an alternative embodiment, the anti-IL-5 antibody in the pharmaceutical composition comprises a human antibody constant region, preferably the human antibody heavy chain constant region set forth in SEQ ID NO:52 and the human antibody light chain constant region set forth in SEQ ID NO:53.

[0032] In an alternative embodiment, the anti-IL-5 antibody in the pharmaceutical composition is (i) a heavy chain set forth in SEQ ID NO:83 and a light chain set forth in SEQ ID NO:84; (ii) a heavy chain set forth in SEQ ID NO:85 and a light chain set forth in SEQ ID NO:86; (iii) a heavy chain set forth in SEQ ID NO:87 and a light chain set forth in SEQ ID NO:88; (iv) a heavy chain set forth in SEQ ID NO:89 and a light chain set forth in SEQ ID NO:90; or (v) a heavy chain as set forth in SEQ ID NO: 91 and a light chain as set forth in SEQ ID NO: 92 Includes.

[0033] In an alternative embodiment, the anti-IL-5 antibody in the pharmaceutical composition is a monoclonal antibody or an antigen-binding fragment thereof that competes with the above-mentioned anti-IL-5 antibody or an antigen-binding fragment thereof for binding to IL-5.

[0034] In a preferred embodiment, the antigen-binding fragment in the pharmaceutical composition of the present disclosure is selected from the group consisting of Fab, Fab', F(ab')2, single chain antibody (scFv), dimerized V region (diabody) and disulfide bond stabilized V region (dsFv).

[0035] The present disclosure further provides a method for preparing the pharmaceutical composition, comprising replacing a stock solution of anti-IL-5 antibody with a buffer. In an alternative embodiment, the buffer is preferably an acetic acid-sodium acetate buffer. The pH of the buffer is about 5.0 to about 6.5, preferably about 5.5 to about 6.5, preferably about 6.0 to about 6.5, preferably about 5.0 to about 6.0, preferably about 5.5 to about 6.0, preferably about 5.0 to about 5.5, preferably about 5.2 to about 5.8, and non-limiting examples of the pH value of the buffer include about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, with about 5.5 being the most preferred. In an alternative embodiment, the concentration of the buffer in the pharmaceutical composition is about 10 mM to about 30 mM, preferably about 15 mM to about 30 mM, preferably about 20 mM to about 30 mM, preferably about 25 mM to about 30 mM, preferably about 5 mM to about 25 mM, preferably about 10 mM to about 25 mM, preferably about 15 mM to about 25 mM, preferably about 20 mM to about 25 mM, preferably about 5 mM to about 20 mM, preferably about 10 mM to about 15 mM; non-limiting examples of the buffer concentration include about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30 mM, with about 30 mM being the most preferred.

[0036] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.5, and 0.2 mg / mL polysorbate 80.

[0037] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.5, and 0.05 mg / mL polysorbate 80.

[0038] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, and 0.2 mg / mL polysorbate 80.

[0039] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.5, and 0.2 mg / mL polysorbate 80.

[0040] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 6.0, and 0.2 mg / mL polysorbate 80.

[0041] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, and 0.05 mg / mL polysorbate 80.

[0042] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.5, and 0.05 mg / mL polysorbate 80.

[0043] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 6.0, and 0.05 mg / mL polysorbate 80.

[0044] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 6.5, and 0.2 mg / mL polysorbate 80.

[0045] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 5.5, and 0.2 mg / mL polysorbate 80.

[0046] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 6.0, and 0.2 mg / mL polysorbate 80.

[0047] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 6.5, and 0.05 mg / mL polysorbate 80.

[0048] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 5.5, and 0.05 mg / mL polysorbate 80.

[0049] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 6.0, and 0.05 mg / mL polysorbate 80.

[0050] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 6.5, and 0.2 mg / mL polysorbate 80.

[0051] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM citric acid-sodium citrate at pH 5.5, and 0.2 mg / mL polysorbate 80.

[0052] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM histidine-HCl at pH 6.0, and 0.2 mg / mL polysorbate 80.

[0053] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM histidine-HCl at pH 6.5, and 0.05 mg / mL polysorbate 80.

[0054] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM histidine-HCl at pH 5.5, and 0.05 mg / mL polysorbate 80.

[0055] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM histidine-HCl at pH 6.0, and 0.05 mg / mL polysorbate 80.

[0056] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, and 0.1 mg / mL polysorbate 20.

[0057] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, and 0.1 mg / mL polysorbate 80.

[0058] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 50 mg / mL sucrose, and 0.1 mg / mL polysorbate 80.

[0059] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 50 mg / mL trehalose and 0.1 mg / mL polysorbate 80.

[0060] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 50 mg / mL mannitol and 0.1 mg / mL polysorbate 80.

[0061] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 50 mg / mL sorbitol and 0.1 mg / mL polysorbate 80.

[0062] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 8 mg / mL glycine, and 0.1 mg / mL polysorbate 80.

[0063] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 8 mg / mL methionine and 0.1 mg / mL polysorbate 80.

[0064] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.0, 8 mg / mL proline and 0.1 mg / mL polysorbate 80.

[0065] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM succinic acid-sodium succinate at pH 5.5, 70 mg / mL sucrose, and 0.4 mg / mL polysorbate 80.

[0066] In some embodiments, the pharmaceutical composition comprises 80 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.8, and 0.2 mg / mL polysorbate 80.

[0067] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.4, and 0.6 mg / mL polysorbate 80.

[0068] In some embodiments, the pharmaceutical composition comprises 80 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.4, and 0.4 mg / mL polysorbate 80.

[0069] In some embodiments, the pharmaceutical composition comprises 80 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.0, and 0.2 mg / mL polysorbate 80.

[0070] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.4, and 0.2 mg / mL polysorbate 80.

[0071] In some embodiments, the pharmaceutical composition comprises 80 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.8, and 0.6 mg / mL polysorbate 80.

[0072] In some embodiments, the pharmaceutical composition comprises 120 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.0, and 0.2 mg / mL polysorbate 80.

[0073] In some embodiments, the pharmaceutical composition comprises 80 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.0, and 0.6 mg / mL polysorbate 80.

[0074] In some embodiments, the pharmaceutical composition comprises 120 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.8, and 0.6 mg / mL polysorbate 80.

[0075] In some embodiments, the pharmaceutical composition comprises 120 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.4, and 0.4 mg / mL polysorbate 80.

[0076] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.4, and 0.4 mg / mL polysorbate 80.

[0077] In some embodiments, the pharmaceutical composition comprises 120 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.0, and 0.6 mg / mL polysorbate 80.

[0078] In some embodiments, the pharmaceutical composition comprises 120 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.8, and 0.2 mg / mL polysorbate 80.

[0079] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.0, and 0.4 mg / mL polysorbate 80.

[0080] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.8, and 0.4 mg / mL polysorbate 80.

[0081] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 10 mM acetic acid-sodium acetate at pH 5.5, 70 mg / ml sucrose, and 0.4 mg / mL polysorbate 80.

[0082] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 20 mM acetic acid-sodium acetate at pH 5.5, 70 mg / ml sucrose, and 0.4 mg / mL polysorbate 80.

[0083] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 30 mM acetic acid-sodium acetate at pH 5.5, 70 mg / ml sucrose, and 0.4 mg / mL polysorbate 80.

[0084] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 30 mM acetic acid-sodium acetate at pH 5.5, 73 mg / ml sucrose, and 0.4 mg / mL polysorbate 80.

[0085] In some embodiments, the pharmaceutical composition comprises 100 mg / ml of anti-IL-5 antibody h1705-008, 30 mM acetic acid-sodium acetate at pH 5.5, 75 mg / ml sucrose, and 0.4 mg / mL polysorbate 80.

[0086] In some embodiments, the pharmaceutical compositions of the present disclosure are stable for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months at 2-8° C. The pharmaceutical compositions may be stable at 25° C. for at least 3 months, or at least 6 months.

[0087] The present disclosure further provides a method for preparing a lyophilized formulation comprising an anti-IL-5 antibody, comprising the step of lyophilizing the pharmaceutical composition.

[0088] In an alternative embodiment, the lyophilization in the method for preparing a lyophilized formulation comprising an anti-IL-5 antibody includes a pre-freezing step, a primary drying step, and a secondary drying step in sequence. Lyophilization is performed by freezing the formulation and then sublimating water at a temperature suitable for primary drying. Under such conditions, the temperature of the product is lower than the eutectic point or decomposition temperature of the formulation. Under suitable pressures, which are generally in the range of about 50 to 250 mTorr, the storage temperature for primary drying is usually about -30 to 25°C (assuming that the product remains frozen during primary drying). The formulation, the size and type of sample container (e.g., glass vial) and the volume of liquid determine the period required for drying, which may range from a few hours to several days (e.g., 40 to 60 hours). Secondary drying can be performed at about 0 to 40°C, which depends mainly on the type and size of the container and the type of protein used. The period of secondary drying is determined by the desired residual moisture level of the product, but usually requires at least about 5 hours. Generally, the moisture content of the lyophilized formulation prepared under low pressure is less than about 5%, preferably less than about 3%. The pressure of the secondary drying may be the same as that applied in the primary drying step; preferably, the pressure used in the secondary drying is lower than that used in the primary drying. The conditions for lyophilization may vary depending on the formulation and the size of the vial.

[0089] The present disclosure further provides a lyophilized formulation comprising an IL-5 antibody, prepared by the above-mentioned method for preparing a lyophilized formulation comprising an anti-IL-5 antibody.

[0090] In some embodiments, the lyophilized formulation is stable for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months at 2-8° C. In some embodiments, the lyophilized formulation is stable at 40° C. for at least 7 days, at least 14 days, or at least 28 days.

[0091] The present disclosure further provides a method for preparing a reconstituted solution of a lyophilized formulation comprising an anti-IL-5 antibody, comprising the step of reconstituting a lyophilized formulation comprising an anti-IL-5 antibody, wherein the solution used for reconstitution includes, but is not limited to, water for injection, saline, or glucose solution.

[0092] The present disclosure further provides a reconstituted solution of a lyophilized formulation comprising an IL-5 antibody, prepared by the above-mentioned method for preparing a reconstituted solution of a lyophilized formulation comprising an anti-IL-5 antibody.

[0093] The disclosure further provides articles or kits comprising a container comprising any of the stable pharmaceutical compositions herein, hi some embodiments, the container is a neutral borosilicate glass injection vial.

[0094] The present disclosure further provides an article comprising a container containing the pharmaceutical composition or lyophilized formulation or a reconstitution solution of the lyophilized formulation.

[0095] The present disclosure further provides a method for treating an IL-5 mediated disease comprising administering a therapeutically effective amount of said pharmaceutical composition or said lyophilized formulation or said reconstituted solution or said product to a subject in need thereof, wherein the IL-5 mediated disease is preferably selected from the group consisting of asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, parasitic infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, hypereosinophilic bronchitis, arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis and steroid-dependent eosinophilic bronchitis.

[0096] The present disclosure further provides the use of said pharmaceutical composition or said lyophilized formulation or said reconstituted solution of said lyophilized formulation or said product in the preparation of a medicament for treating an IL-5 mediated disease, wherein the IL-5 mediated disease is preferably selected from the group consisting of asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, parasitic infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, hypereosinophilic bronchitis, arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis and steroid-dependent eosinophilic bronchitis.

[0097] The present disclosure relates to a pharmaceutical composition or a lyophilized formulation or a reconstituted solution of a lyophilized formulation or a product as described above for use as a therapeutic medicament for the treatment of an IL-5 mediated disease, the IL-5 mediated disease being preferably selected from the group consisting of asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myopathy, pulmonary edema ... The present invention further provides the pharmaceutical composition, the freeze-dried preparation, or the reconstituted solution of the freeze-dried preparation, or the product, which is selected from the group consisting of chronic eosinophilic bronchitis, chronic pain syndrome, eosinophilic gastroenteritis, parasitic infection, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, hypereosinophilic bronchitis, arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis, and steroid-dependent eosinophilic bronchitis.

[0098] As will be appreciated by those skilled in the art, one, some or all of the features of each embodiment in the present disclosure may be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and further embodiments obtained by combination are further described in the following detailed description. [Brief description of the drawings]

[0099] [Figure 1] FIG. 1 shows the results of a FACS experiment in which anti-IL-5 antibodies block IL-5 binding to the IL-5 receptor. [Diagram 2] FIG. 1 shows the results of detecting the binding specificity of anti-IL-5 antibodies to Th2 cytokines. [Diagram 3] This figure shows that anti-IL-5 antibody improves the level of intermittent respiration (Penh). G1: normal control group (PBS); G2: model group (IgG); G3: h1705-008 antibody 10mpk group; G4: h1705-008 antibody 2mpk group; G5: h1706-009 antibody 10mpk group; G6: h1706-009 antibody 2mpk group; G7: Hu39D10 10mpk group; *p<0.05, **<0.01 (compared with G2 group by ANOVA / Bonferroni). [Figure 4A] FIG. 1 shows the levels of BALF eosinophils in the lungs of asthmatic mice. [Figure 4B] FIG. 1 shows the scores of tracheal mucosa thickness in asthmatic mice. G1: normal control group; G2: model group; G3: h1705-008 antibody 10mpk group; G4: h1705-008 antibody 2mpk group; G5: h1706-009 antibody 10mpk group; G6: h1706-009 antibody 2mpk group; G7: Hu39D10 10mpk group. [Figure 4C] FIG. 1 shows the percentage of BALF eosinophils in the lungs of asthmatic mice. [Figure 5A] FIG. 1 shows the ability of IL5 monoclonal antibody to reduce the levels of eosinophils in BALF. [Figure 5B] FIG. 1 shows the ability of IL5 monoclonal antibody to reduce the levels of eosinophils in BALF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0100] term In order to make this disclosure more readily understandable, certain technical and scientific terms are defined below: All other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs, unless expressly defined otherwise.

[0101] "Buffer" refers to a buffer that is resistant to changes in pH due to the action of acid-base conjugate components. Examples of buffers that control pH in the appropriate range include acetate buffers, succinate buffers, gluconate buffers, histidine salt buffers, oxalate buffers, lactate buffers, phosphate buffers, citrate buffers, tartrate buffers, fumarate buffers, glycyl-glycine buffers and other organic acid buffers.

[0102] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine-hydrochloride buffer, histidine-acetate buffer, histidine-phosphate buffer, and histidine-sulfate buffer; histidine-acetate buffer or histidine-hydrochloride buffer is preferred; histidine-acetate buffer is prepared by histidine and acetic acid, and histidine salt buffer is prepared by histidine and HCl.

[0103] A "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include citrate-sodium citrate buffer, citrate-potassium citrate buffer, citrate-calcium citrate buffer, and citrate-magnesium citrate buffer. A preferred citrate buffer is citrate-sodium citrate buffer.

[0104] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinic acid-sodium succinate buffer, succinic acid-potassium succinate buffer, and succinic acid-calcium succinate buffer. A preferred succinate buffer is succinic acid-sodium succinate buffer.

[0105] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, and disodium hydrogen phosphate-citrate buffer. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0106] An "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetate-sodium acetate buffer, histidine acetate buffer, acetate-potassium acetate buffer, acetate-calcium acetate buffer, and acetate-magnesium acetate buffer. A preferred acetate buffer is acetate-sodium acetate buffer.

[0107] "Pharmaceutical composition" refers to a mixture containing one or more of the compounds described herein (or physiologically / pharmaceutical acceptable salts or prodrugs thereof) and other chemical components, such as physiologically / pharmaceutical acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the antibody active ingredient and to facilitate administration to an organism, so as to enhance absorption and biological activity of the active ingredient.

[0108] As used herein, "pharmaceutical composition" and "formulation" are used interchangeably.

[0109] In the present disclosure, "sugar" refers to the conventional (CH 2 O) n and monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. (CH 2and derivatives of saccharides such as saccharides of formula (I) and (II). The sugar may be selected from the group consisting of glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, melitriose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose, etc. The preferred sugar is a non-reducing disaccharide, more preferably sucrose.

[0110] According to the present disclosure, the solvent contained in the solution form of the pharmaceutical composition is water, unless otherwise specified.

[0111] "Lyophilized formulation" means a formulation or pharmaceutical composition obtained by vacuum freeze-drying a liquid or solution form of the pharmaceutical composition or formulation.

[0112] Freeze-drying in the present disclosure includes pre-freezing, primary drying, and secondary drying. The purpose of pre-freezing is to freeze the product to obtain a crystalline solid. Pre-freezing temperature and pre-freezing rate are two important process parameters. In the present disclosure, the pre-freezing temperature is set at -45°C, and the pre-freezing rate is 1°C / min. Primary drying, also called drying, is the main stage for freeze-drying a sample. The purpose of primary drying is to remove ice from the product while maintaining the shape of the product so as to limit damage to the product to a minimum level. If the primary drying temperature and vacuum are not properly selected, the product will collapse. Increasing the temperature and vacuum will accelerate the efficiency of freeze-drying, but also increase the risk of product collapse. The temperature of primary drying in the present invention may be a conventional temperature in the art, such as -30°C to 0°C. Secondary drying, also called vacuum drying, is the main step of removing bound water from the product by drawing an ultimate vacuum (0.01 mbar) and increasing the temperature (20 to 40°C). Since most biological products are temperature sensitive, the selected secondary drying temperature should be at the lower end of the temperature range, 25° C. The duration of the lyophilization is related to the freezer, the volume of the formulation to be lyophilized, and the container of the formulation to be lyophilized. A person skilled in the art knows well how to adjust such duration.

[0113] As used herein, the terms "about" and "approximately" mean that a value is within an acceptable error range of a particular value as measured by one of ordinary skill in the art. The aforementioned particular value depends in part on how the value is measured or determined (i.e., the limitations of the measurement system). For example, in any practice in the art, "about" means within or more than 1 standard deviation. As an alternative, "about" or "substantially including" means within a range of up to ±20%, e.g., a pH of about 5.5 means pH 5.5±1.1. Furthermore, particularly with respect to biological systems or processes, the term "about" means up to 10-fold or up to 5-fold of a value. Unless otherwise specified, when a particular value is presented in the present application and claims, the meaning of "about" or "substantially including" should be within an acceptable error range of the particular value.

[0114] The pharmaceutical composition of the present disclosure can provide a stable effect, i.e., the antibody contained in the pharmaceutical composition substantially retains its physical and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and biological activity after storage. Generally, the storage period is determined based on the predetermined shelf life of the pharmaceutical composition. Currently, there are several assays for measuring protein stability, which can be used to measure stability after storage at a selected temperature for a selected period of time.

[0115] A stable pharmaceutical formulation of an antibody is one in which no significant physical and / or chemical and / or biological changes are observed under conditions of storage at low temperature (2-8° C.) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Furthermore, stable liquid formulations include liquid formulations that exhibit desirable properties after storage at a temperature of 25° C. for 1 month, 3 months, or 6 months. In general, the criteria for a stable formulation are as follows: typically no more than about 10%, preferably no more than about 5%, of the antibody monomer is degraded as measured by SEC-HPLC; upon visual inspection, the pharmaceutical formulation of the antibody is a light yellow, almost colorless, transparent liquid, or colorless, or transparent to slightly tinted; the concentration, pH, and osmolality of the formulation vary by ±10% or less; typically no more than about 10% or less, preferably no more than about 5% is observed to be lost; typically no more than about 10% or less, preferably no more than about 5% is formed of aggregates.

[0116] An antibody is considered to "retain physical stability" in a pharmaceutical formulation if it does not exhibit significantly increased aggregation, precipitation and / or denaturation as measured by visual inspection for color and / or clarity, or by ultraviolet light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines the tertiary structure of proteins, and FTIR spectroscopy, which determines the secondary structure of proteins.

[0117] An antibody is considered to "retain chemical stability" in a pharmaceutical formulation if it does not exhibit significant chemical modification. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and SDS-PAGE), oxidation (assessed by methods such as peptide spectroscopy combined with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement), and isomerization (assessed by methods such as measurement of isoaspartic acid content, peptide spectroscopy).

[0118] An antibody is considered to "retain biological activity" in a pharmaceutical formulation if the biological activity of the antibody after a given time period is still within a predetermined range relative to the biological activity exhibited at the time the pharmaceutical formulation was originally prepared. Antibody biological activity can be measured, for example, by antigen binding assays.

[0119] The three letter and one letter codes for amino acids used in this disclosure are as set forth in J. Biol. Chem. 243, p3558 (1968).

[0120] "Antibody" as used in this disclosure refers to an immunoglobulin; an intact antibody is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains joined by interchain disulfide bonds.

[0121] In the present disclosure, the antibody light chain of the present disclosure further comprises a light chain constant region, and the light chain constant region comprises a human or mouse κ chain, λ chain, or a variant thereof.

[0122] In the present disclosure, the antibody heavy chain of the present disclosure further comprises a heavy chain constant region, and the heavy chain constant region comprises human or mouse IgG1, IgG2, IgG3, IgG4, or a variant thereof.

[0123] Approximately 110 amino acids adjacent to the N-terminus of antibody heavy and light chains are highly variable and are known as the variable region (Fv region); the remainder of the amino acid sequence near the C-terminus is relatively stable and is known as the constant region. The variable region contains three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions that determine the specificity of the antibody are also known as complementarity determining regions (CDR). Each of the light chain variable region (LCVR, VL) and heavy chain variable region (HCVR, VH) consists of three CDR regions and four FR regions, in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2 and LCDR3, and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3. The numbers and positions of CDR amino acid residues belonging to the LCVR and HCVR regions in the antibodies or antigen-binding fragments thereof described in the present disclosure are according to the known Kabat numbering system (LCDR1-3, HCDR1-3).

[0124] Antibodies of the present disclosure include murine antibodies, chimeric antibodies, and humanized antibodies, preferably humanized antibodies.

[0125] In the present disclosure, an "antigen-binding fragment of an antibody" or a "functional fragment" refers to a Fab fragment, a Fab' fragment, and an F(ab') fragment that have antigen-binding activity. 2The term "scFv" refers to a fragment that binds to an antibody, and an Fv fragment and an scFv fragment that bind to an antibody. An Fv fragment contains the heavy and light chain variable regions of an antibody but does not have a constant region, and is the smallest antibody fragment that contains all antigen-binding sites. In general, an Fv antibody further comprises a polypeptide linker between the VH and VL domains to form a structure required for antigen binding. Different linkers can also be used to link two antibody variable regions to form a polypeptide chain called a single chain antibody and a single chain Fv (sFv).

[0126] The term "antigen-binding site" of the present disclosure refers to the contiguous or discontinuous three-dimensional spatial site on an antigen that is recognized by an antibody, or antigen-binding fragment thereof, of the present disclosure.

[0127] The term "mouse antibody" in this disclosure refers to a monoclonal antibody against human IL-5, prepared according to the knowledge and skill in the art, by injecting a test subject with an IL-5 antigen and then isolating a hybridoma expressing an antibody having the desired sequence or functional characteristics.

[0128] The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody that reduces the immune response induced by the mouse antibody. To establish a chimeric antibody, a hybridoma secreting a mouse-specific monoclonal antibody is newly established; then, the variable region genes are cloned from the mouse hybridoma cells; then, if necessary, the constant region genes of a human antibody are cloned; the mouse variable region genes are combined with the human constant region genes to form a chimeric gene, and this chimeric gene is inserted into a human vector; and finally, the chimeric antibody molecule is expressed in an industrial eukaryotic system or an industrial prokaryotic system. In a preferred embodiment of the present disclosure, the light chain of the IL-5 chimeric antibody further comprises a light chain constant region of a human kappa chain, a lambda chain, or a variant thereof. The heavy chain of the IL-5 chimeric antibody further comprises a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4, or a variant thereof. The constant region of the human antibody can be selected from the group consisting of the heavy chain constant regions of human IgG1, IgG2, IgG3, or IgG4, or variants thereof, and preferably includes a human IgG2 or IgG4 heavy chain constant region, or an IgG4 that has no ADCC toxicity (antibody-dependent cell-mediated cytotoxicity) after amino acid mutation.

[0129] The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody generated by grafting mouse CDR sequences onto a human antibody variable region framework, i.e., an antibody generated in a different type of human germline antibody framework sequence. Humanized antibodies avoid the strong heterologous reaction induced by chimeric antibodies that carry multiple mouse protein components. Such framework sequences can be obtained from public DNA databases or published references that list germline antibody gene sequences. For example, germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available at www.mrccpe.com.ac.uk / vbase) or in Kabat, EA et al. 1991 Sequences of Proteins of Immunological Interest, 5th Edition. To avoid reduced activity due to reduced immunogenicity, the framework sequences in the human antibody variable regions can be subjected to minimal reverse-mutations or back mutations to maintain activity. The humanized antibodies of the present disclosure also refer to humanized antibodies in which CDR affinity maturation has been performed by phage display.

[0130] In the present disclosure, "ADCC" (i.e., antibody-dependent cell-mediated cytotoxicity) refers to the direct killing of antibody-coated target cells by cells expressing Fc receptors, by recognizing the Fc segment of the antibody. The ADCC effector function of an antibody can be reduced or eliminated by modification of the Fc segment of IgG. The modification refers to the occurrence of mutations in the heavy chain constant region of the antibody, such as those selected from the group consisting of N297A, L234A, L235A in IgG1; IgG2 / 4 chimera, F234A / L235A mutations in IgG4.

[0131] The "mutation" in the mutant sequence in this disclosure includes, but is not limited to, a "back mutation," a "conservative modification," or a "conservative replacement or substitution." As used in this disclosure, a "conservative modification" or a "conservative replacement or substitution" refers to the replacement of an amino acid in a protein with another amino acid having similar properties (such as charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity) so that the replacement can be performed frequently without changing the biological activity of the protein. Those skilled in the art know that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). Moreover, the replacement of an amino acid with a similar structure or function is unlikely to disrupt biological activity.

[0132] In the present disclosure, a "mutated sequence" refers to a nucleotide sequence and / or amino acid sequence of the present disclosure that has been appropriately modified by mutation (such as substitution, insertion or deletion) to obtain a nucleotide sequence and / or amino acid sequence that has a different percentage of sequence homology from the nucleotide sequence and / or amino acid sequence of the present disclosure. In the present disclosure, the sequence homology can be at least 85%, 90% or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. Sequence alignment and determination of the percentage of identity between two sequences can be performed by the default settings of the BLASTN / BLASTP algorithm available on the National Center For Biotechnology Institute website.

[0133] The term "binding to IL-5" refers to interaction with IL-5, preferably human IL-5.

[0134] The terms "anti-IL-5 antibody" and "IL-5 antibody" are used interchangeably and both refer to an antibody that binds to IL-5.

[0135] In the present disclosure, a fusion protein is a protein product obtained by co-expression of two genes by DNA recombination. A recombinant IL-5 extracellular domain Fc fusion protein is a fusion protein obtained by co-expression of an IL-5 extracellular domain and a human antibody Fc fragment by DNA recombination. The IL-5 extracellular domain refers to the IL-5 protein expressed outside the cell membrane, and the sequence of the IL-5 extracellular domain is as presented in SEQ ID NO:1.

[0136] Methods for producing and purifying antibodies and antigen-binding fragments are well known and are provided in the prior art, such as Antibodies: A Laboratory Manual, Cold Spring Harbor press, Chapters 5-8 and 15. For example, mice can be immunized with human IL-5 or a fragment thereof, and the resulting antibodies can be renatured and purified, and amino acid sequencing can be performed by using conventional methods. Antigen-binding fragments can also be prepared by using conventional methods. The antibodies or antigen-binding fragments according to the present disclosure are genetically engineered to graft one or more human FR regions onto non-human CDR regions. Human FR germline sequences can be obtained from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from The Immunoglobulin Journal, 2001 ISBN012441351.

[0137] The engineered antibody or its antigen-binding fragment can be prepared and purified by conventional methods. For example, the cDNA sequences encoding the heavy and light chains can be cloned and recombined into a GS expression vector. The expression vector of the recombinant immunoglobulin can be stably transfected into CHO cells. In one preferred conventional technique, the antibody can be glycosylated by a mammalian expression system, in particular at the highly conserved N-terminal position belonging to the Fc region. Stable clones are obtained by expressing an antibody that specifically binds to human IL-5. Positive clones are grown in serum-free medium in a bioreactor to produce the antibody. The medium into which the antibody is secreted can be purified by conventional techniques. For example, a Protein A or Protein G Sepharose FF column with conditioned buffer may be used for purification. Non-specifically bound components are washed away. The bound antibody is then eluted by a pH gradient and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by conventional methods. Soluble mixtures and multimers can also be removed by conventional methods, such as molecular sieving and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0138] "Optionally" or "optionally" means that the events or circumstances following the term may or may not occur, but the description includes cases where the events or circumstances occur or do not occur. For example, "optionally includes one to three heavy chain CDR regions of an antibody" means that a heavy chain CDR region of an antibody having a specific sequence may be present, but is not necessarily present.

[0139] When applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, "administering" and "treating" refer to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering" and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell includes contact of a reagent with the cell and contact of a reagent with a fluid, where the fluid is in contact with the cell. "Administering" and "treating" also refer to, for example, the treatment of a cell with a reagent, diagnostic, binding composition, or another cell, in vitro and ex vivo. When applied to a human, veterinary subject, or research subject, "treating" refers to therapeutic treatment, preventative or prophylactic measures, research, and diagnostic applications.

[0140] "Treatment" refers to the application of an internal or external therapeutic agent, such as a composition containing any one of the binding compounds of the present disclosure, to a patient with one or more disease symptoms for which the therapeutic agent is known to have a therapeutic effect. In general, the therapeutic agent is given in an amount effective to alleviate one or more disease symptoms in the treated patient or population, thereby inducing regression of such symptoms to any clinically detectable extent or inhibiting the onset of such symptoms. The amount of therapeutic agent effective to alleviate any particular disease symptom (also called a "therapeutically effective amount") may vary depending on various factors, such as the disease state, age, and weight of the patient, and the ability of the therapeutic agent to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been alleviated can be evaluated by any clinical test method commonly used by physicians or other medical professionals to evaluate the severity or progression of the condition. An embodiment of the present disclosure (e.g., a method of treatment or article of manufacture) may not be effective in alleviating each targeted disease symptom, but should alleviate the targeted disease symptom in a statistically significant number of patients as determined by any statistical testing method known in the art, such as Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonkheel-Tapstra test, and Wilcoxon test.

[0141] There is no limitation on the disease associated with IL-5, so long as the disease is an IL-5 associated disease. For example, the therapeutic response induced by the molecules of the present disclosure can be generated by blocking or inhibiting the stimulatory effect of eosinophils after binding to human IL-5. Thus, when applied in suitable preparations and formulations for therapeutic use, the pharmaceutical compositions of the present disclosure are highly useful for subjects suffering from allergic and / or atopic reactions or reactions associated with eosinophils such as, but not limited to, asthma, asthma exacerbations, malignant asthma episodes, chronic pneumonia, allergic rhinitis, perennial allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, parasitic infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, hypereosinophilic bronchitis, arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis or steroid-dependent eosinophilic bronchitis. In a preferred embodiment, the treatment can inhibit or alleviate eosinophil infiltration into lung tissue. The frequency of administration of the pharmaceutical composition can be from 3 times per day to once every 6 months. The route of administration can be intravenous, subcutaneous, intramuscular, parenteral or topical.

[0142] The formulations of the present disclosure can be used to treat IL-5 mediated diseases.For example, the formulations can be used, but are not limited to, to inhibit or alleviate inflammatory responses and eosinophil maturation, activation, degranulation or tissue infiltration mediated by IL-5 in vivo and in vitro; suppress excessive stress of smooth muscle caused by IL-5; and reduce the level of IL-5 in lungs, airways or blood. Preferably, the formulations of the present disclosure can be used to treat an IL-5 mediated disease, preferably the IL-5 mediated disease is selected from the group consisting of asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, parasitic infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, hypereosinophilic bronchitis, arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis, and steroid-dependent eosinophilic bronchitis.

[0143] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also refers to an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as: the condition being treated, the general condition of the patient, the route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or administration schedule that avoids significant side effects or toxic effects.

[0144] "Exchange" refers to the replacement of the solvent system used to dissolve the antibody protein. For example, a high salt or hypertonic solvent system containing the antibody protein is replaced by the buffer system of the stable formulation by physical manipulation, such that the antibody protein is present in the stable formulation. Physical manipulation includes, but is not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation. EXAMPLES

[0145] (Examples and Test Examples) The present disclosure will be further described below in conjunction with examples, but these examples do not limit the scope of the present disclosure. Experimental methods that do not adopt specific conditions in the examples of the present disclosure are usually based on conventional conditions, such as those in the publication Antibodies: A Laboratory Manual, Molecular Cloning produced by Cold Spring Harbor Laboratory; or based on the conditions recommended by the manufacturers of materials or products. Reagents that do not indicate specific sources are conventional reagents purchased from the market.

[0146] Example 1 Proteins used in the preparation and detection of IL-5 antigen 1.1 Design and expression of IL-5 antigen The sequences encoding His-tagged human IL-5, His-tagged rhesus IL-5, His-tagged mouse IL-5, His-tagged rat IL-5, and human IL-5Rα receptor extracellular domain fusion protein containing human IgG1-Fc fragment were cloned into cphr vector to construct expression plasmids, and then transfected into HEK293. On the sixth day after transfection, samples were harvested and the cell supernatants were collected by centrifugation at 4500 rpm for 10 minutes. The supernatants containing recombinant IL-5 and IL-5α receptor proteins were purified by using a nickel column, and the recombinant human IL-5-Fc fusion protein was purified by using a protein A affinity chromatography column. The purified proteins can be used for subsequent experiments. The sequences of the specific protein antigens are as follows:

[0147] Amino acid sequence of human IL-5 with his tag (rhIL-5-his) [ka]

[0148] Amino acid sequence of his-tagged cynomolgus IL-5 [ka] Note: The italicized parts represent the His6 tag.

[0149] Amino acid sequence of his-tagged mouse IL-5 [ka] Note: The italicized parts represent the His6 tag.

[0150] Amino acid sequence of his-tagged rat IL-5 [ka] Note: The italicized parts represent the His6 tag.

[0151] Amino acid sequence of human IL-5α receptor fused to human Fc fragment [ka] Note: The italicized part represents the human IgG1-Fc tag.

[0152] Example 2 Construction and identification of recombinant IL-5α receptor and IL-5α / β receptor cell lines To screen for functional antibodies, the present disclosure constructed a CHO-S / IL-5α cell line expressing IL-5α and a CHO-S / IL-5α / IL-5β cell line expressing both IL-5α and IL-5β.

[0153] Specifically, human IL-5α full-length gene (Q01344) was cloned into mammalian cell expression vector pTargeT, and the linearized plasmid was electrotransfected into CHO-S cells and screened in the presence of G418 for 2 weeks, followed by two rounds of limiting dilution. The IL-5α gene on the cell surface was detected by FACS, and CHO-S / IL-5α cell lines with high IL-5α expression levels were selected. Based on this, linearized pcDNA3.1-IL-5β was electrotransfected, and screened in the presence of G418 and zeocin for 2 weeks, followed by two rounds of limiting dilution. The IL-5α and IL-5β genes on the cell surface were detected by FACS, and CHO-S / IL-5α / IL-5β cell lines with high IL-5α and IL-5β expression levels were selected.

[0154] Example 3 Preparation of anti-human IL-5 mouse monoclonal antibody Two doses of recombinant protein rhIL-5-his, Freund's adjuvant CFA (Sigma, Lot No. SLBQ1109V) and IFA (Sigma, Lot No. SLBJ2845V) (100g / 50g / 50g (high dose) and 25g / 12.5g / 12.5g (low dose)) were used to immunize two groups of Balb / c mice (5 mice / group) and four groups of SJL mice (5 mice / group), respectively. Specific immune responses against IL-5 were measured by detecting serum titers by ELISA, ligand-receptor blocking assay and TF-1 proliferation inhibition assay. Mice with good specific immune responses were selected and sacrificed; spleen cells were collected and fused with myeloma cells.

[0155] Primary screening was performed by ELISA binding assay for human IL-5. Once hybridoma cells were transferred to 24-well plates, the supernatants were screened again by using ELISA binding assays for human, cynomolgus and mouse IL-5, ELISA-based receptor blocking assay for IL-5 and inhibition assay of TF-1 proliferation. After subcloning the positive clones twice, hybridoma clones were obtained, which were used for antibody production; the obtained antibodies were purified by affinity chromatography.

[0156] The purified antibodies were subjected to SEC-HPLC, detection of endotoxin content, Biacore affinity assay for various species IL-5, FACS-based receptor blocking assay for IL-5, inhibition of TF-1 proliferation assay, eosinophil adhesion assay, and efficacy evaluation in mouse and guinea pig neutralization models of asthma in vivo; monoclonal hybridoma cell lines mAb1705, mAb1706, mAb1780, mAb1773 and mAb1779 were selected which showed favorable activity in vivo and in vitro.

[0157] The process for cloning sequences from positive hybridomas was as follows: logarithmic growth phase hybridoma cells were harvested, and RNA was extracted using Trizol (Invitrogen, Cat. No. 15596-018) according to the kit's instructions, and reverse transcribed using PrimeScript™ Reverse Transcriptase Kit (Takara, Cat. No. 2680A). The cDNA obtained by reverse transcription was amplified by PCR using Mouse Ig-Primer Set (Novagen, TB326 Rev.B0503), and then sequenced. Amino acid sequences corresponding to the heavy and light chain variable region DNA sequences of mAb1705, mAb1706, mAb1780, mAb1773, and mAb1779 were obtained (amino acid residues of VH / VL CDRs were determined and annotated according to the Kabat numbering standard).

[0158] mAb1705 mouse heavy chain variable region sequence EVQLVESGGGLVQPGRSLKLSCTASGFTFS HYYMA WVRQAPKKGLEWVT SISYEGDITYYGDSVKG RFTISRDNAKSTLYLQMNSLRSEDTATYYCAS QTLRESFDY WGQGVMVTVSS SEQ ID NO:6

[0159] mAb1705 mouse light chain variable region sequence DIQMTQSPSSMSVSLGDRVTITC RASQDIANYLS WYQQKIARSPKLVIY GTSNLEV GVPSRFSGSRSGSDYSLTINTLESEDTGIYFC LQDKEFPRT FGGGTRLELK SEQ ID NO:7

[0160] mAb1706 mouse heavy chain variable region sequence EVQLVESGGGLVQPGRSLKLSCAASGFTFS HYYMA WVRQAPKKGLEWVT SINYEGNSAYYGDSVKG RFTISRDNAKSTLYLQMDSLRSEDTATYYCAT ETLRESLDY WGQGVMVTVSS SEQ ID NO:8

[0161] mAb1706 mouse light chain variable region sequence DIQMTQSPSSMSVSLGDRVTITC RASQDIGNYLS WYQQKLGKSPKLMIH SASNLEV GVPSRFSGSRSGSDYSLTINTLESEDPGIYFC LQHKQFPRT FGGGTKLELK SEQ ID NO:9

[0162] mAb1780 mouse heavy chain variable region sequence QVKLLQSGAALVKPGDSVKMSCKASDYTFT EYLIH WVKQSQGRSLEWIG YINPYSGGTVYNEKFKS KATLTVDKFSSTAYMEFRRLTFEDSAIYYCAR DGGYSDPLDY WGQGVMVTVSS SEQ ID NO:10

[0163] mAb1780 mouse light chain variable region sequence DTVLTQSPALAVSPGERVSISC RASEGLTSYMH WYQQKPGQQPKLLIY KASNLAS GVPARFSGSGSGTDFTLTIDPVEADDAATYFC QQNWNDPWT FGGGTKLELK SEQ ID NO:11

[0164] mAb1773 mouse heavy chain variable region sequence EVQLQQSLAELVRPGASVTLSCTASGFNIK NTYIH WVKQRPEQGLEWIG RIDPANGDTKHGPKFQG KATITADTSSNTAYLQFSSLTSEDTAIYYCFR YG YPDH WGQGTPLTVSS SEQ ID NO:12

[0165] mAb1773 mouse light chain variable region sequence QIVLTQSPALMSASPGEKVTMTC SASSSVNYIY WYQQKPRSSPKPWIY LTATLAS GVPARFSGSGSGTSFSLTISRMEAEDAATYYC QQWNSYPYT FGGGTKLEIE SEQ ID NO:13

[0166] mAb1779 mouse heavy chain variable region sequence QVKLLQSGAALVKPGDSVKMSCKASGYTFT DYIIH WVKQSHGKSLEWIG YFNPNSGGSNYNENFKR KATLTADKSSSTAYLEFSRVTSEDSAIYYCGR RIAWDHWYFDF WGPGTMVTVSS SEQ ID NO:14

[0167] mAb1779 mouse light chain variable region sequence DIQMTQSPASLSASLGETVSIEC LASEGISNDVAWYQQKSGRSPQLLVY AASRLQD GVPSRFSGSGSGTRYFFKISGMQPEDEADYFC QQGYKTPLT FGSGTKLEIK SEQ ID NO:15

[0168] The CDR sequences of the light and heavy chains of each antibody are shown in Table 1.

[0169] [Table 1]

[0170] The activity detection results by Biacore can be seen in Table 2.

[0171] [Table 2]

[0172] The above results demonstrate that the mouse antibodies of the present disclosure have high affinity for the antigen.

[0173] Example 4: Purification of IL-5-related recombinant proteins and purification of hybridoma and recombinant antibodies 4.1 Purification steps of IL-5-Flag-His recombinant protein: The sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume. An NI-NTA affinity column (QIAGEN, Cat. No. 30721) was equilibrated with PBS and washed with 2-5 column volumes. After removal of impurities, the cell expression supernatant sample was loaded onto the column. The column was washed with PBS until the A280 readings dropped to baseline. The column was washed with PBS to remove contaminating proteins and the sample was collected. The target protein was eluted sequentially with wash buffer (20 mM imidazole) and elution buffer (300 mM imidazole) and the elution peak was collected.

[0174] The collected eluate was further purified by ion exchange (Hiload16 / 600 superdex200 column). The column was equilibrated with approximately two column volumes of PBS to ensure a pH of 7.4. The elution buffer containing the identified target protein was concentrated and loaded, and samples were collected, confirmed by SDS-PAGE and identification by LC-MS, and aliquoted for use.

[0175] 4.2 Purification of hybridoma-expressed antibodies and Fc fusion proteins Cell expression supernatant samples were centrifuged at high speed to remove impurities. Hybridoma expression supernatant was purified by protein G column, and Fc fusion protein expression supernatant was purified by protein A column. The column was washed with PBS until A280 readings dropped to baseline. Target proteins were eluted with 100mM acetic acid at pH 3.0 and neutralized with 1M Tris-HCl at pH 8.0. After appropriate concentration of the eluted samples, the samples were further purified by gel chromatography Superdex200 (GE) equilibrated with PBS, and the aggregate-free peak was collected and aliquoted for use.

[0176] Example 5: Design of humanized anti-human IL-5 monoclonal antibody Humanization of the mouse anti-human IL-5 monoclonal antibody was performed as disclosed in numerous references in the art. Briefly, the constant regions of the mouse antibody were replaced by human constant regions, the CDRs of the mouse antibody were grafted onto the FR human template with the highest homology, and back mutations were generated in the amino acids in the FR regions.

[0177] Heavy and light chain variable region germline sequences with high identity to the amino acid sequences of mAb-1705, mAb-1706, mAb1780, mAb1773 and mAb1779 antibodies, respectively, by aligning against the IMGT human antibody heavy and light chain variable region germline gene database were selected as templates, and the CDRs of the mouse antibodies were grafted onto the corresponding human templates to form variable regions in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Amino acid residues were determined and annotated according to the Kabat numbering standard.

[0178] Selection of human FR regions and amino acid back mutations Based on the typical structure of the obtained mouse antibody VH / VL CDR, the light chain variable region (VL) and heavy chain variable region (VH) homologous sequences were searched from the human germline database and ranked according to FR homology (in descending order); the germline with the highest FR homology was selected as the primary template. After grafting the CDR region of the mouse antibody into the human template, the FR residues were mutated and the amino acid residues were optimized to obtain the final humanized molecule.

[0179] 5.1 Selection of humanized framework for hybridoma clone mAb1705 Regarding h1705, IGHV3-23 * 04 was selected as the template for VH, and IGKV1-12 * VL was selected from the human template mAb1701. The CDRs of mAb1705 were grafted onto the human template; the incorporated residues and those that directly interacted with the CDR regions (found by the software) were backmutated. Various humanized antibody light and heavy chain variable regions were obtained, as shown in Table 3.

[0180] [Table 3] Note: "Grafted" refers to the grafting of a murine antibody CDR onto a human germline FR region sequence. For example, A43S refers to the mutation of the A at position 43 of the "Grafted" back to an S, according to natural numbering of amino acid sequences.

[0181] [Table 4] Note: This table shows the sequences obtained by combining different mutations. As shown by h1705-007, the humanized mouse antibody h1705-007 contains two variants, i.e., light chain h1705_VL.1A and heavy chain h1705_VH.1A, etc.

[0182] The specific sequences of the variable regions of humanized antibody h1705 are as follows: >h1705_VL.1 (SEQ ID NO: 46) DIQMTQSPSSVSASVGDRVTITCRASQDIANYLSWYQQKPGKAPKLLIYGTSNLEVGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDKEFPRTFGGGTKVEIK

[0183] >h1705_VL.1A (SEQ ID NO: 47) DIQMTQSPSSVSASVGDRVTITCRASQDIANYLSWYQQKPGKSPKLLIYGTSNLEVGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQDKEFPRTFGGGTKVEIK

[0184] >h1705_VL.1B (SEQ ID NO: 48) DIQMTQSPSSVSASVGDRVTITCRASQDIANYLSWYQQKPGKSPKLVIYGTSNLEVGVPSRFSGSRSGSDYTLTISSLQPEDFATYFCLQDKEFPRTFGGGTKVEIK

[0185] >h1705_VH.1 (SEQ ID NO: 49) EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVSSISYEGDITYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKQTLRESFDYWGQGTLVTVSS

[0186] >h1705_VH.1A (SEQ ID NO: 50) EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVSSISYEGDITYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASQTLRESFDYWGQGTLVTVSS

[0187] >h1705_VH.1B (SEQ ID NO: 51) EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVTSISYEGDITYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCASQTLRESFDYWGQGTLVTVSS

[0188] Each of the above light chain variable regions was combined with a light chain constant region to form a light chain sequence, and each of the heavy chain variable regions was combined with a heavy chain constant region to form a heavy chain sequence. Exemplary humanized antibody constant region sequences are shown below.

[0189] > Heavy chain IgG1 constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL Y I T R EPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:52 Note: The underlined parts represent the M252Y, S254T, and T256E mutations.

[0190] >Light chain kappa constant region: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:53

[0191] 5.2 Selection of humanized framework for hybridoma clone mAb1706 For h1706, IGHV3-23 * 04 was selected as the template for VH, and IGKV1-12 * 01 was selected as the template for VL. The CDRs of mouse antibody mAb1706 were grafted into the selected humanized template; FR amino acids were backmutated. The humanized antibody light and heavy chain variable regions were obtained, as shown in Table 5.

[0192] [Table 5] Note: "Grafted" refers to the grafting of a murine antibody CDR onto a human germline FR region. For example, A43S refers to the mutation of the A at position 43 of the "Grafted" back to an S, according to natural numbering of the amino acid sequence.

[0193] As shown in Table 6, the designed humanized molecules were combined to form a variety of antibodies as presented in the table below.

[0194] [Table 6]

[0195] The specific sequences of the variable regions of the h1706 humanized antibody are as follows: >h1706_VL.1 (SEQ ID NO: 54) DIQMTQSPSSVSASVGDRVTITCRASQDIGNYLSWYQQKPGKAPKLLIYSASNLEVGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHKQFPRTFGGGTKVEIK

[0196] >h1706_VL.1A (SEQ ID NO: 55) DIQMTQSPSSVSASVGDRVTITCRASQDIGNYLSWYQQKPGKSPKLLIYSASNLEVGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHKQFPRTFGGGTKVEIK

[0197] >h1706_VL.1B (SEQ ID NO: 56) DIQMTQSPSSVSASVGDRVTITCRASQDIGNYLSWYQQKPGKSPKLMIYSASNLEVGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCLQHKQFPRTFGGGTKVEIK

[0198] >h1706_VH.1 (SEQ ID NO: 57) EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVSSINYEGNSAYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKETLRESLDYWGQGTMVTVSS

[0199] >h1706_VH.1A (SEQ ID NO: 58) EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVSSINYEGNSAYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATETLRESLDYWGQGTMVTVSS

[0200] >h1706_VH.1B (SEQ ID NO: 59) EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVTSINYEGNSAYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCATETLRESLDYWGQGTMVTVSS.

[0201] Each of the above light chain variable regions was combined with a light chain constant region to form a light chain sequence, and each of the heavy chain variable regions was combined with a heavy chain constant region to form a heavy chain sequence.

[0202] 5.3 Selection of humanized framework for hybridoma clone mAb1780 Regarding h1780, IGHV1-2 * 02 was selected as the template for VH, and IGKV3-11 * 01 was selected as the template for VL. The CDRs of mouse antibody mAb1780 were grafted into the selected humanized template; FR amino acids were backmutated. The humanized antibody light and heavy chain variable regions were obtained, as shown in Table 7.

[0203] [Table 7] Note: "Grafted" refers to the grafting of a murine antibody CDR onto a human germline FR region. For example, E1D refers to the mutation of the E at position 1 of "Grafted" back to D, according to the natural numbering of the amino acid sequence.

[0204] As shown in Table 8, the designed humanized molecules were combined to form various molecules presented in the table below.

[0205] [Table 8]

[0206] The specific sequences of the variable regions of the h1780 humanized antibody are as follows: >h1780_VL.1 (SEQ ID NO: 60) EIVLTQSPATLSLSPGERATLSCRASEGLTSYMHWYQQKPGQAPRLLIYKASNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQNWNDPWTFGGGTKVEIK

[0207] >h1780_VL.1A (SEQ ID NO: 61) DTVLTQSPATLSLSPGERATLSCRASEGLTSYMHWYQQKPGQAPRLLIYKASNLASGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQNWNDPWTFGGGTKVEIK

[0208] >h1780_VL.1B (SEQ ID NO: 62) DTVLTQSPATLSLSPGERATLSCRASEGLTSYMHWYQQKPGQAPRLLIYKASNLASGVPARFSGSGSGTDFTLTISSLEPEDFATYFCQQNWNDPWTFGGGTKVEIK

[0209] >h1780_VH.1 (SEQ ID NO: 63) EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYLIHWVRQAPGQGLEWMGYINPYSGGTVYNEKFKSRVTMTRDTSISTAYMELSRLRSDDTAVYYCARDGGYSDPLDYWGQGTMVTVSS

[0210] >h1780_VH.1A (SEQ ID NO: 64) EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYLIHWVRQAPGQGLEWMGYINPYSGGTVYNEKFKSRVTLTVDKSISTAYMELSRLRSDDTAVYYCARDGGYSDPLDYWGQGTMVTVSS

[0211] >h1780_VH.1B (SEQ ID NO: 65) EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYLIHWVRQAPGQGLEWIGYINPYSGGTVYNEKFKSRATLTVDKSISTAYMEFSRLRSDDTAVYYCARDGGYSDPLDYWGQGTMVTVSS

[0212] >h1780_VH.1C (SEQ ID NO: 66) EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYLIHWVKQAPGQGLEWIGYINPYSGGTVYNEKFKSKATLTVDKSISTAYMEFSRLRSDDTAVYYCARDGGYSDPLDYWGQGTMVTVSS.

[0213] Each of the above light chain variable regions was combined with a light chain constant region to form a light chain sequence, and each of the heavy chain variable regions was combined with a heavy chain constant region to form a heavy chain sequence.

[0214] 5.4 Selection of humanized framework for hybridoma clone mAb1773 For h1773, IGHV3-73 * 01 was selected as the template for VH, and IGKV1-39 *01 was selected as the template for VL. The CDRs of mouse antibody mAb1773 were grafted into the selected humanized template; amino acids were backmutated. The humanized antibody light and heavy chain variable regions were obtained as shown in Table 9. Furthermore, N in h1773 HCDR2 (RIDPANGDTK HGPKFQG) was mutated to V (i.e., N55V) to form a heavy chain variable region HCDR2 variant (the sequence of the mutated HCDR2 is as shown in SEQ ID NO: 82: RIDPAVGDTKHGPKFQG).

[0215] [Table 9] Note: "Grafted" refers to the grafting of a murine antibody CDR onto a human germline FR region. For example, M4L refers to the mutation of the M at position 4 of "Grafted" back to L according to natural numbering of the amino acid sequence.

[0216] As shown in Table 10, the designed humanized molecules were combined to form various molecules presented in the table below.

[0217] [Table 10]

[0218] The specific sequences of the variable regions of the h1773 humanized antibody are as follows: >h1773_VL.1 (SEQ ID NO: 67) DIQMTQSPSSLSASVGDRVTITCSASSSVNYIYWYQQKPGKAPKLLIYLTATLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWNSYPYTFGGGTKVEIK

[0219] >h1773_VL.1A (SEQ ID NO: 68) DIQLTQSPSSLSASVGDRVTITCSASSSVNYIYWYQQKPGKSPKPWIYLTATLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWNSYPYTFGGGTKVEIK

[0220] >h1773_VH.1 (SEQ ID NO: 69) [ka]

[0221] >h1773_VH.1A (SEQ ID NO: 70) [ka]

[0222] >h1773_VH.1B (SEQ ID NO: 71) [ka]

[0223] Each of the above light chain variable regions was combined with the light chain constant region sequence shown in SEQ ID NO: 53 to form the final complete light chain sequence. Each of the heavy chain variable regions was combined with the heavy chain constant region sequence shown in SEQ ID NO: 52 to form the final complete heavy chain sequence.

[0224] 5.5 Selection of humanized framework for hybridoma clone mAb1779 For h1779, IGHV1-2 * 02 was selected as the template for VH, and IGKV1-33 * 01 was selected as the template for VL. The CDRs of mouse antibody h1779 were grafted into the selected humanized template; amino acids were backmutated. The humanized antibody light and heavy chain variable regions were obtained, as shown in Table 11.

[0225] [Table 11] Note: "Grafted" refers to the grafting of a murine antibody CDR onto a human germline FR region. For example, A43S refers to the mutation of the A at position 43 of the "Grafted" back to an S, according to natural numbering of the amino acid sequence.

[0226] As shown in Table 12, the designed humanized molecules were combined to form various molecules presented in the table below.

[0227] [Table 12]

[0228] The specific sequences of the variable regions of the h1779 humanized antibody are as follows: >h1779_VL.1 (SEQ ID NO: 72) DIQMTQSPSSLSASVGDRVTITCLASEGISNDVAWYQQKPGKAPKLLIYAASRLQDGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGYKTPLTFGQGTKLEIK

[0229] >h1779_VL.1A (SEQ ID NO: 73) DIQMTQSPSSLSASVGDRVTITCLASEGISNDVAWYQQKPGKSPKLLIYAASRLQDGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGYKTPLTFGQGTKLEIK

[0230] >h1779_VL.1B (SEQ ID NO: 74) DIQMTQSPSSLSASVGDRVTITCLASEGISNDVAWYQQKPGKSPKLLVYAASRLQDGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGYKTPLTFGQGTKLEIK

[0231] >h1779_VH.1 (SEQ ID NO: 75) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIIHWVRQAPGQGLEWMGYFNPNSGGSNYNENFKRRVTMTRDTSISTAYMELSRLRSEDTAVYYCARRIAWDHWYFDFWGQGTMVTVSS

[0232] >h1779_VH.1A (SEQ ID NO: 76) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIIHWVRQAPGQGLEWMGYFNPNSGGSNYNENFKRRVTMTADKSISTAYMELSRLRSEDTAVYYCARRIAWDHWYFDFWGQGTMVTVSS

[0233] >h1779_VH.1B (SEQ ID NO: 77) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIIHWVRQAPGQGLEWIGYFNPNSGGSNYNENFKRRATMTADKSISTAYMELSRLRSEDTAVYYCARRIAWDHWYFDFWGQGTMVTVSS

[0234] >h1779_VH.1C (SEQ ID NO: 78) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIIHWVRQAPGQGLEWIGYFNPNSGGSNYNENFKRRATMTADKSISTAYLEFSRLRSEDTAVYYCARRIAWDHWYFDFWGQGTMVTVSS

[0235] >h1779_VH.1D (SEQ ID NO: 79) EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIIHWVKQAPGQGLEWIGYFNPNSGGSNYNENFKRKATMTADKSISTAYLEFSRLRSEDTAVYYCARRIAWDHWYFDFWGQGTMVTVSS

[0236] Each of the above light chain variable regions was combined with the light chain constant region sequence shown in SEQ ID NO:53 to form a light chain sequence. Each of the heavy chain variable regions was combined with the heavy chain constant region sequence shown in SEQ ID NO:52 to form a heavy chain sequence.

[0237] The full-length sequence of an exemplary humanized antibody is as follows: H1705-008 Heavy Chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVTSISYEGDITYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTATYYCASQTLRESFDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:83

[0238] h1705-008 Light Chain: DIQMTQSPSSVSASVGDRVTITCRASQDIANYLSWYQQKPGKSPKLLIYGTSNLEVGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCLQDKEFPRTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:84

[0239] H1706-009 Heavy Chain: EVQLVESGGGLVQPGGSLRLSCAASGFTFSHYYMAWVRQAPGKGLEWVSSINYEGNSAYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATETLRESLDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:85

[0240] h1706-009 Light Chain: DIQMTQSPSSVSASVGDRVTITCRASQDIGNYLSWYQQKPGKSPKLMIYSASNLEVGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCLQHKQFPRTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:86

[0241] h1780-017 heavy chain: EVQLVQSGAEVKKPGASVKVSCKASGYTFTEYLIHWVRQAPGQGLEWIGYINPYSGGTVYNEKFKSRATLTVDKSISTAYMEFSRLRSDDTAVYYCARDGGYSDPLDYWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:87

[0242] h1780-017 Light Chain: DTVLTQSPATLSLSPGERATLSCRASEGLTSYMHWYQQKPGQAPRLLIYKASNLASGVPARFSGSGSGTDFTLTISSLEPEDFATYFCQQNWNDPWTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:88

[0243] h1773-007 heavy chain: EVQLVESGGGLVQPGGSLKLSCAASGFTISNTYIHWVKQASGKGLEWIGRIDPAVGDTKHGPKFQGRFTISADDSKNTAYLQMNSLKTEDTAVYYCFRYGIYPDHWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:89

[0244] h1773-007 light chain: DIQLTQSPSSLSASVGDRVTITCSASSSVNYIYWYQQKPGKSPKPWIYLTATLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQWNSYPYTFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:90

[0245] h1779-014 heavy chain: EVQLVQSGAEVKKPGASVKVSCKASGYTFTDYIIHWVKQAPGQGLEWIGYFNPNSGGSNYNENFKRKATMTADKSISTAYLEFSRLRSEDTAVYYCARRIAWDHWYFDFWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:91

[0246] h1779-014 Light Chain: DIQMTQSPSSLSASVGDRVTITCLASEGISNDVAWYQQKPGKSPKLLVYAASRLQDGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQGYKTPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:92;

[0247] The antibody Hu39D10 against IL5 in WO 2012 / 083370 was used as a positive control in the present disclosure, and its heavy and light chain sequences are shown in SEQ ID NO:80 and SEQ ID NO:81, respectively.

[0248] Hu39D10 heavy chain sequence EVQLVESGGGLVQPGGSLRLSCAVSGLSLTSNSVNWIRQAPGKGLEWVGLIWSNGDTDYNSAIKSRFTISRDTSKSTVYLQMNSLRAEDTAVYYCAREYYGYFDYWGQGTLVTVSS ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK SEQ ID NO:80

[0249] Hu39D10 light chain sequence DIQMTQSPSSLSASVGDRVTITCLASEGISSYLAWYQQKPGKAPKLLIYGANSLQTGVPSRFSGSGSATDYTLTISSLQPEDFATYYCQQSYKFPNTFGQGTKVEVK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Sequence number:81.

[0250] Example 6: Preparation of recombinant chimeric and humanized antibodies 1. Molecular cloning of recombinant chimeric antibodies After obtaining positive antibody molecules by screening hybridomas, the gene sequences encoding the variable regions were obtained by sequencing. Based on the obtained sequences, forward and reverse primers were designed, and the sequenced genes were used as templates to construct each antibody VH / VK gene fragment by PCR, and then inserted into the expression vector pHr (having signal peptide and hIgG1 / hκ constant region gene (CH1-Fc / CL) fragment) by homologous recombination to construct recombinant chimeric antibody full-length expression plasmid VH-CH1-Fc-pHr / VL-CL-pHr, and five chimeric antibodies Ch1705, Ch1706, Ch1780, Ch1773 and Ch1779 were obtained.

[0251] 2. Molecular cloning of humanized antibodies After codon optimization of the humanized antibody sequence, a coding gene sequence with human codon preference was obtained; primers were designed to construct each antibody VH / VK gene fragment by PCR, which was then integrated into the expression vector pHr (with signal peptide and hIgG1 / hκ constant region gene (CH1-Fc / CL) fragment) by homologous recombination to construct the humanized antibody full-length expression plasmid VH-CH1-Fc-pHr / VL-CL-pHr.

[0252] 3. Expression and purification of recombinant chimeric and humanized antibodies Plasmids expressing antibody light or heavy chains were transfected separately into HEK293E cells. After 6 days, the expression supernatants were collected, centrifuged at high speed to remove impurities, and purified by Protein A column. The column was washed with PBS until the A280 readings dropped to baseline. The target proteins were eluted by acidic elution buffer at pH 3.0-pH 3.5 and neutralized by 1M Tris-HCl at pH 8.0-9.0. The eluted samples were appropriately concentrated and further purified by gel chromatography Superdex200 (GE) pre-equilibrated with PBS to remove aggregates, and the monomer peak was collected and aliquoted for use.

[0253] The following test methods were used to confirm the performance and beneficial effects of the antibodies of the present disclosure.

[0254] Biological evaluation of in vitro activity Test Example 1. Binding of mouse IL-5 antibodies to IL-5 from different species by Biacore assay The affinity of the mouse IL-5 antibodies to be tested with human IL-5 was measured by a Biacore T200 (GE) instrument.

[0255] Protein A biosensor chip was used to capture the molecules to be tested by affinity, and then antigen (recombinant human and cynomolgus IL-5 prepared in Example 1) was flowed over the surface of the chip, and the reaction signal was detected in real time by Biacore T200 instrument to obtain binding and dissociation curves. After the dissociation of each experimental cycle was completed, the biosensor chip was washed and regenerated by glycine-HCl regeneration solution (pH 1.5). Using BIA evaluation version 4.1 GE software, the data was fitted to the (1:1) Langmuir model, and affinity values ​​were obtained and shown in Table 13.

[0256] [Table 13]

[0257] This example demonstrates that antibodies mAb1705, mAb1706, mAb1780, mAb1773 and mAb1779 of the present disclosure have high affinity for IL-5 of different species (human, monkey).

[0258] Test Example 2. Affinity of humanized IL-5 antibodies to IL-5 from different species by Biacore assay The affinity of the humanized IL-5 antibodies to be tested with human IL-5 was measured by a Biacore T200 (GE) instrument.

[0259] Protein A biosensor chip was used to capture the molecule to be tested by affinity, and then antigen (prepared in Example 1) was flowed over the surface of the chip, and the reaction signal was detected in real time by Biacore T200 instrument to obtain binding and dissociation curves. After the dissociation of each experimental cycle was completed, the biosensor chip was washed and regenerated by glycine-HCl type regeneration solution (pH 1.5). Using BIA evaluation version 4.1 GE software, the data was fitted to the (1:1) Langmuir model, and the affinity values ​​were obtained and shown in Table 14.

[0260] [Table 14]

[0261] The above results demonstrate that all humanized IL-5 antibodies have high affinity for human IL-5.

[0262] Test Example 3. ELISA-Based Assay of Murine IL-5 Antibodies That Block IL-5 Binding to the IL-5α Receptor To confirm the ability of IL-5 antibodies to block the binding of IL-5 to the extracellular domain of recombinantly expressed IL-5α receptor protein, IL-5 (5 μg / ml in PBS) was applied to ELISA plates and incubated at 37°C for 1 h; the liquid was removed and 200 μl / well of 5% nonfat milk blocking solution diluted with PBS was added and incubated in a 37°C incubator for 2.5 h for blocking. After blocking, the blocking solution was removed and the plate was washed five times with PBST buffer (PBS pH 7.4 containing 0.05% Tween-20); 25 μl of 10 μg / ml IL-5Rα (in 1% BSA) labeled with a biotin labeling kit (Tojin Chemical, LK03) was added, followed by 25 μl of gradient diluted antibodies (the antibodies competed with IL-5Rα for binding to IL-5) and incubated at 37°C for 1 h. After incubation, the reaction solution in the microtiter plate was removed, the plate was washed five times with PBST, and 50 μl / well of streptavidin-peroxidase polymer (Sigma, S2438-250UG) diluted 1:600 ​​with sample dilution solution was added and incubated at 37°C for 1 hour. The plate was washed five times with PBST, and 50 μl / well of TMB chromogenic substrate (KPL, 52-00-03) was added and incubated at room temperature for 3 to 10 minutes; 50 μl / well of 1M H 2 SO 4The reaction was stopped by adding 0.1% ethanol; the absorbance value at 450 nm was read using a NOVOStar Microplate Reader; and the IC50 value of the IL-5 antibody that blocks the binding of IL-5 to IL-5Rα was calculated. The results are shown in Table 15. The antibodies of the present disclosure can effectively inhibit the binding of IL-5 to the IL-5 receptor.

[0263] [Table 15]

[0264] Test Example 4. FACS-Based Assay of IL-5 Antibodies That Block IL-5 Binding to the IL-5 Receptor To confirm that the screened IL-5 antibodies could block the IL-5 receptor on the cell surface, we constructed a CHOS recombinant cell line that highly expresses two receptors, IL-5Rα / β. This experiment demonstrated that the IL-5 antibodies could block the binding of IL-5 to the recombinant IL-5α / β receptor on the surface of the CHOS cell line, respectively.

[0265] The specific method was as follows: CD-CHO containing 100 ng / ml G418 and 25 ng / ml zeozin was used to culture CHO-S-IL-5Rα and β. The concentration in cell culture was 3 × 10 6 Well-conditioned IL-5Rα / β-CHOS cells were centrifuged (1000 rpm for 5 min), washed once with 10% FBS in PBS, the cells were counted, and the cell concentration was adjusted to 4×10 6The cells / ml were adjusted and 25μl was removed and added to a round-bottom 96-well plate. The antibodies to be tested were diluted in PBS solution containing 10% FBS. The initial concentration was 200μg / ml, which was diluted 1:10 to give 8 gradient dilutions. 25μl of IL-5 labeled with biotin labeling kit (Tojin Chemical, LK03) at 100ng / ml was added and mixed thoroughly with 50μl of diluted antibody at each concentration, then added to the 96-well plate with cells and incubated at 4℃ for 1 hour. After incubation, the samples were centrifuged at 4°C (400g, 5 min), the supernatant was removed, and the plate was washed by centrifugation with 200μl pre-chilled PBS, which was repeated twice; PE-Avidin secondary antibody diluted 1:1333 was added, incubated in the dark at 4°C for 40 min, and centrifuged at 4°C (400g, 5 min); the supernatant was removed, 200μl pre-chilled PBS was added, and the cells were pipetted; the plate was washed by centrifugation at 4°C three times; 100μl PBS was added, and the plate was read by machine; The IC50 value of the IL-5 antibody blocking the binding of IL-5 to IL-5Rα / β was calculated by the fluorescence signal value. The results are shown in Table 16 and Figure 1.

[0266] [Table 16]

[0267] The above results demonstrate that antibodies h1705-008, h1706-009, h1780-017 and h1779-014 exhibit potent ability to block IL-5 binding to the IL-5 receptor on the cell surface.

[0268] Test Example 5. IL-5 antibody inhibits IL-5-induced proliferation of TF1 cells IL-5 can induce the proliferation of TF-1 cells, and IL-5 antibodies can prevent IL-5 from stimulating the proliferation of TF-1 cells. Specifically, TF-1 cells (TCC, CRL-2003) were cultured in RPMI1640 containing 10% FBS and 2 ng / mL rhGM-CSF (LINKBio, Catalog No. 96-AF-300-03-20) at 37°C and 5% CO 2 The cells were placed in an incubator and the cell density was 1 x 10 6 Not exceeding cells / ml. For antibody detection, logarithmic growth phase cells were washed 3 times with PBS and centrifuged at 800 rpm for 5 minutes; adjusted cell density to 6000 cells / well / 90μl with RPMI1640 (FBS: 2%, recombinant human IL-5: 10ng / ml); 10μl of gradient diluted antibody to be tested was added to 96-well plate for culturing, and after 3 days of culturing, 30μl of cell titer solution was added and mixed for detection, and IC50 was calculated by measurement. The test results are shown in Table 17 below.

[0269] [Table 17]

[0270] Test Example 6. Test of IL5 antibody that inhibits eosinophil adhesion induced by IL5 IL5 can induce the differentiation, maturation, migration and activation of eosinophils, leading to respiratory inflammation and the development of asthma. This experiment is based on the principle that IL-5 cytokines can promote the adhesion and activation of eosinophils. Eosinophils were collected and purified from human peripheral blood to test the blocking effect of IL-5 specific antibodies on the IL-5 pathway and detect the blocking effect of IL-5 antibodies on eosinophil adhesion mediated by IL5 in vitro.

[0271] Specifically, human peripheral blood was diluted 5-fold with PBS containing 2 mM EDTA, and monocytes and granulocytes were isolated using Percoll™ (density gradient of 1.088). The red blood cell layer containing granulocytes was carefully aspirated, and red blood cells were removed using red blood cell lysis solution; the remaining cells were counted, and magnetic separation beads (Miltenyi Biotec, Cat. No. 130-045-701) with CD16 antibody were added accordingly, incubated for 30 minutes, and passed through a magnetic bead column, and a subpopulation (mainly eosinophils) was passed directly through the column and collected by negative selection. The isolated eosinophils were counted and then plated at approximately 1×10 per well into a 96-well cell culture plate pre-coated with IgG antibody. 4 Add human IL-5 (20 ng / ml) and IL-5 antibody molecule at different concentrations (10 concentration points, 3-fold dilutions starting from 10 μg / ml); and culture the cell culture plate at 37°C in 5% CO. 2 After 1 hour of incubation in an incubator, the culture plate was removed, 0.3% CTAB was added to dissolve the cells, and finally, peroxidase reaction substrate TMB was added to develop color, and OD450 absorption value was read by a microplate reader. The measurement value of the wells added with IL-5 alone was the maximum absorption value; the wells without IL-5 and antibody-type acting substance were taken as background controls; the inhibition value of the antibody-type acting substance at each concentration relative to the maximum adsorption value was calculated=(maximum adsorption value-[antibody-type acting substance]) / (maximum adsorption value-background control value)×100%, and IC50 was calculated. The results are shown in Table 18.

[0272] [Table 18]

[0273] The above results demonstrate that the humanized antibodies of the present disclosure exhibit potent ability to block IL5-mediated eosinophil adhesion.

[0274] Test Example 7. Evaluation of the specificity of humanized IL-5 antibody against Th2 cytokines IL-5 was one of the Th2 cytokines. To confirm that the IL-5 antibody specifically targets only IL-5 and does not cross-react with other cytokines, Fortebio tested IL-5-specific antibodies that share the α receptor with IL-5, including IL2 (R&D, 202-IL-010 / CF), IL4 (R&D, 204-IL-050 / CF), IL-5 (R&D, 205-IL-025 / CF), IFNgamma, IL6 (R&D, 7270-IL-025 / CF), and IL Twelve Th2 and related cytokines were detected, including IL-9 (R&D, 209-IL-010 / CF), IL10 (R&D, 217-IL-025 / CF), IL13 (R&D, 213-ILB-025 / CF), IL25 (R&D, 8134-IL-025 / CF), IL31 (R&D, 2824-IL-010 / CF), and IL3 (203-IL-050 / CF), as well as GMCSF (R&D, 215-GM-010 / CF).

[0275] Specifically, a Protein A biosensor (PALL Fortebio, 18-5010) was used to capture the antibody and record the capture signal, after which 40 nM of each of the cytokines was injected and the binding signal was recorded. Finally, the binding signal with IL-5 was defined as 100%. The binding signals of other cytokines with the antibody were observed, and the results are shown in Figure 2.

[0276] The above results demonstrate that among the 12 related cytokines, only the humanized IL-5 antibodies h1705-008 and h1706-009 specifically bind to IL-5 and have no cross-reactivity with other Th2 cytokines.

[0277] Biological evaluation of in vivo activity Test Example 8. Evaluation of the efficacy of IL-5 antibody in an OVA-induced mouse asthma model This study evaluates the efficacy of an IL-5 antibody in an ovalbumin (OVA) aerosol-induced BALB / c mouse asthma model based on airway inflammatory responses and airway remodeling.

[0278] Mice were randomly divided into seven groups according to body weight, each group containing 10 mice: normal control group (G1); model group (G2); treatment groups of the two antibodies to be tested, h1705-008 (G3 and G4) and h1706-009 (G5 and G6), at two doses (10mpk and 2mpk) of each antibody to be tested; and a positive antibody Hu39D10 control group (G7, 10mpk). On days 1 and 14, all mice were sensitized by intraperitoneal injection of allergen solution. On days 28, 29, and 30, six groups of mice (except the first group) were challenged with aerosol OVA challenge solution for 30 minutes. Two hours before challenge, the second group (G2) was intraperitoneally injected with phosphate buffer, and the third to seventh groups (G3-G7) were intraperitoneally injected with different doses of different antibodies (once a day for three consecutive days). The antibodies to be tested were freshly prepared before each injection, and administration was terminated within 30 minutes of antibody preparation. The first group of mice (normal control group) was challenged with PBS aerosol for 30 minutes, but 2 hours before this challenge, they were intraperitoneally injected with phosphate buffer once a day for three consecutive days.

[0279] Animals were tested for airway hyperresponsiveness using the WBP system on day 31. All animals were challenged with increasing concentrations of methacholine (1.5625 mg / mL, 3.125 mg / mL, 6.25 mg / mL, 12.5 mg / mL, 25 mg / mL, and 50 mg / mL) by aerosol ingestion, and the respiratory enhanced pause values ​​at the corresponding concentrations were measured.

[0280] On the 31st day, 1 hour after detection of airway hyperresponsiveness by the WBP system, a 1.2 mm diameter tracheal tube was inserted into the trachea and fixed, and lung lavage was performed twice, each time with 0.8 ml of phosphate buffer containing 1% BSA and 0.6 mM EDTA. The volume of the lavage fluid recovered was recorded.

[0281] The BALF was centrifuged at 300g for 5 min at 4°C. The supernatant was kept for cytokine analysis. After centrifugation, the cells were resuspended in 1.5 ml of PBS (containing 1% BSA and 0.6 mM EDTA) and the cells were counted. The total number of cells in the BALF was counted using a hemocytometer and trypan blue staining experiment. The cells were smeared on a slide and stained with Wright's staining solution for 1 min, then with Giemsa stain for 7 min to distinguish eosinophils, neutrophils, macrophages and lymphocytes. Counting was done under a light microscope.

[0282] After lavage, lung tissue was collected and stained with 10% neutral formaldehyde solution, and then fixed in 10% neutral formaldehyde solution. Fixed tissue was embedded in paraffin, trimmed, stained with H&E, and scored. The test results are shown in Figure 3, Figure 4A, and Figure 4B.

[0283] The above results show that the antibody molecules h1705-008 and h1706-009 of the present disclosure can significantly improve lung function in a dose-dependent manner, while the positive compound at a high dose (10mpk) cannot improve lung function (see Figure 3). Meanwhile, the two antibodies significantly reduced the eosinophil level and mucosal thickness at the same dose (10mpk), showing a stronger eosinophil reduction ability than that of the positive antibody (see Figures 4A and 4B). In the same type of mouse asthma model, repeated experiments also confirmed that 1mg / ml of h1705-008, h1706-009 and h1780-017 significantly reduced the eosinophil level in BalF compared to that of the positive antibody (see Figure 4C).

[0284] Test Example 9. Evaluation of the in vivo efficacy of IL-5 antibodies in a guinea pig acute asthma model induced by exogenous human IL-5 In this experiment, male guinea pigs were selected to establish an acute asthma model induced by human IL-5, and the inhibitory effects of five IL-5 humanized mAbs of the present disclosure on the increase of eosinophils in bronchial lavage fluid (BALF) of guinea pig lungs induced by human IL-5 were evaluated; hu39D10 was used as the positive antibody. The guinea pigs were divided into nine groups, each containing 8-10 animals: normal control group, model group, hu39D10 (1 mg / kg) group, h1705-008 (1 mg / kg) group, h1706-009 (1 mg / kg) group, h1780-017 (1 mg / kg) group, h1773-007 (1 mg / kg) group and h1779-014 (1 mg / kg) group. Guinea pigs in the model and treatment groups were each injected with 100 μl of human IL5 (containing 5 μg of IL5 antigen) via the trachea for stimulation on the first day; PBS was injected via the trachea of ​​the normal control group. The treatment group was intraperitoneally injected with 1 mg / kg of the above-mentioned IL5 monoclonal antibody at a dose of 5 ml / kg 2 hours before stimulation; the model group was administered with the corresponding IgG antibody; and the normal control group was intraperitoneally administered with PBS solvent. The guinea pigs were anesthetized 24 hours after tracheal injection, and lung bronchial lavage fluid was extracted. The cell concentration was adjusted to 5^10 6 15μl was dropped onto a slide, dried and fixed; HE staining was performed, and the total cell number and eosinophil number were counted under a 400x microscope, and the percentage of eosinophils was calculated. The results are shown in Figures 5A and 5B, which show that the five humanized antibodies of the present disclosure significantly reduce the level of eosinophils in BALF.

[0285] Selection of ingredients in formulations and stability evaluation Exemplary preparation process for antibody pharmaceutical compositions (formulations): Step 1: A certain amount of purified anti-IL-5 antibody solution was taken and replaced (preferably by ultrafiltration) with an antibody-free buffer (such as 30 mM acetic acid-sodium acetate buffer at pH 5.5); at least 6-fold volume was exchanged through ultrafiltration membrane to concentrate the protein to about 120 mg / mL. A certain volume of sucrose stock solution was added and mixed to make the final concentration of sucrose 72 mg / mL. A certain volume of polysorbate 80 stock solution was added and mixed to make the final concentration of polysorbate 80 0.4 mg / mL. The protein concentration was brought to 100 mg / mL by adding 10 mM acetic acid-sodium acetate buffer at pH 5.5 to make a certain volume (other formulations or stable formulations to be tested were also prepared following similar steps).

[0286] The product was filtered and then tested for the absence of pathogenic agents by centralized sampling. The stock solution was passed through a 0.22 μm PVDF filter and the filtrate was collected.

[0287] Step 2: The volume was adjusted to 1.2 ml, the filtrate was filled into stoppered 2 ml vials, and centralized sampling was performed at the beginning, middle, and end of the challenge to detect differences in challenge volume.

[0288] Step 3: The capping machine was started, and the aluminum caps were attached and capping was carried out.

[0289] Step 4: A visual inspection was performed to ensure that the produce was free of defects such as incorrect filling; vial labels were printed and applied; carton labels were printed; cartons were folded and packed; box labels were applied.

[0290] Test Example 10. Screening of buffer systems h1705-008 formulations with a protein concentration of 100 mg / mL were prepared in a series of buffers ranging from pH 5.0 to 6.5, with the shaker sample containing 0.2 mg / mL polysorbate 80 (PS80) and the other samples containing 0.05 mg / mL PS80. The buffer systems were as follows: 10 mM acetic acid-sodium acetate buffer (AA) at pH 5.0, 5.5; 10 mM succinic acid-sodium succinate buffer (SA) at pH 5.0, 5.5, 6.0; 10 mM citric acid-sodium citrate buffer (CA) at pH 5.5, 6.0, 6.5; 10 mM histidine-hydrochloride buffer (His) at pH 5.5, 6.0, 6.5; and 10 mM phosphate buffer (PB) at pH 6.0, 6.5. Each formulation was filtered, filled, stoppered, and capped. The samples were subjected to forced degradation experiments; appearance, SEC and iCIEF were adopted as evaluation indexes.

[0291] The above results are shown in Table 19. The appearance data shows that the shaken (300 rpm, 25°C) and 40°C samples exhibit varying degrees of particle formation. In general, the appearance improves with decreasing pH, with the acetic acid-sodium acetate and succinic acid-sodium succinate buffer systems improving; the SEC data shows that the AA pH 5.5 group is slightly improved at 40°C; the iCIEF data shows that the AA pH 5.5, His pH 5.5, and CA pH 6.5 groups are slightly improved at 40°C; however, taking into account the overall physical and chemical stability, AA at pH 5.5 is preferred.

[0292] [Table 19-1] [Table 19-2] Note: N / A stands for no detection, D stands for days, and T0 stands for day 0.

[0293] Test Example 11. Screening of excipients in formulations A formulation of h1705-008 with a protein concentration of 100 mg / mL was prepared in 10 mM SA (pH 5.0) buffer containing various types of excipients, specifically: 1) 0.1 mg / mL polysorbate 20 (PS20) 2) 0.1 mg / mL polysorbate 80 (PS80) 3) 50mg / mL sucrose + 0.1mg / mL PS80 4) 50mg / mL trehalose + 0.1mg / mL PS80 5) 50mg / mL mannitol + 0.1mg / mL PS80 6) 50mg / mL sorbitol + 0.1mg / mL PS80 7) 8mg / ml arginine (Arg) + 0.1mg / mL PS80 8) 8mg / ml lysine (Lys) + 0.1mg / mL PS80 9) 8mg / ml glycine (Gly) + 0.1mg / mL PS80 10) 8mg / ml methionine (Met) + 0.1mg / mL PS80 11) 8mg / ml proline (Pro) + 0.1mg / mL PS80 12) 8 mg / ml sodium chloride (NaCl) + 0.1 mg / mL PS80. Each formulation was filtered, filled, stoppered, capped and ready for use. Samples were subjected to forced degradation experiments at 40°C, the results of which show the following (Table 20): There are no significant differences in the SEC, CE and iCIEF test results between each sample group, the Arg / Lys / NaCl group has poor appearance, but there are no significant differences between the other groups. Sucrose, trehalose, mannitol, sorbitol, glycine, proline and methionine have a favorable effect on protein stability.

[0294] [Table 20] Note: D stands for number of days.

[0295] Test Example 12. Screening of surfactants A formulation of h1705-008 containing 10 mM SA, pH 5.5, 70 mg / ml sucrose, 0.4 mg / ml PS20 or PS80 was prepared to a protein concentration of 100 mg / ml.

[0296] The samples were placed at 4°C to investigate the stability. The results are shown in Table 21. The results show that the PS80 group has a transparent appearance and no significant changes in SEC, CE and iCIEF over 4 months at 4°C, indicating favorable stability; whereas a large amount of particles is observed in the PS20 group. Therefore, PS80 is better than PS20. Furthermore, it can be seen that the addition of PS80 to the formulation has a better stabilizing effect on h1705-008, improving the stability of the formulation.

[0297] [Table 21] Note: M stands for months; N / A stands for not detected.

[0298] Test Example 13. Design and screening of DOE formulations DOE (Design of Experiments) was performed using pH of 10 mM acetate buffer (AA), protein concentration and Tween concentration as variables; a series of formulations were designed based on the following factors and levels: pH is 5.0-5.8, PS80 concentration is 0.2-0.6 mg / mL, and antibody h1705-008 concentration is 80-120 mg / mL. The formulations are shown in Table 22. The samples were forced to degrade at a high temperature of 40°C. Appearance, SEC, non-reduced CE and iCIEF were used as evaluation indexes. The results are shown in Table 23.

[0299] [Table 22]

[0300] [Table 23] Note: D stands for number of days.

[0301] From the above results, the appearance of each formulation is transparent; SEC, CE and iCIEF are decreased by less than 2%, less than 2% and about 14%, respectively, but within the acceptable range; the stability of the formulation is shown to be favorable; therefore, the formulation has a protein concentration of 80-120 mg / ml, contains 0.2-0.6 mg / ml PS80, and has a pH of 5.0-5.8. The optimal formulation is as follows: 100 mg / ml protein, 0.4 mg / ml PS80, pH 5.5.

[0302] Test Example 14. Stability test A h1705-008 formulation containing 10 mM AA at pH 5.5, 70 mg / ml sucrose, and 0.4 mg / ml PS80 was prepared to a protein concentration of 100 mg / ml; samples were submitted for stability studies at 4° C. and 25° C. The results are shown in Table 24.

[0303] The above results show that under high temperature conditions, the SEC, CE and iCIEF of the h1705-008 formulation are slightly decreased, but this decrease is within the acceptable range; under other conditions, all the indicators have no significant changes. The formulation has favorable stability and can ensure the stability of h1705-008 at 4°C for up to 6 months.

[0304] [Table 24] Note: M stands for number of months.

[0305] Test Example 15. Ionic Strength Screening h1705-008 formulations with 100 mg / mL protein concentration, 70 mg / mL sucrose and 0.4 mg / mL PS80 were prepared in (sodium) acetate buffers with different ionic strengths; the pH of the buffer used for exchange and the pH of the final formulation were measured. The results are shown in Table 25. The results show that the higher the ionic strength, the smaller the pH shift. When the ionic strength is 30 mM, the pH shift is less than 0.1.

[0306] [Table 25]

[0307] Test Example 16. Screening of sugar concentration Formulations of h1705-008 with a protein concentration of 100 mg / mL, 30 mM AA at pH 5.5, and 0.4 mg / ml PS80 were prepared in the buffers described below with different concentrations of sucrose. Osmolality was measured. The results are shown in Table 26.

[0308] The above results show that when the sugar concentration is 70-75 mg / ml, the osmolality is in the optimal isotonic range of 290-310 mosm; the osmolality data for the 70 mg / ml and 73 mg / ml groups show that when the sugar concentration is 72 mg / ml, the osmolality is at its best value of about 300 mosm.

[0309] [Table 26]

[0310] Test Example 17. Stability test of formulation A h1705-008 formulation containing 30 mM AA, 72 mg / ml sucrose, and 0.4 mg / ml PS80 at pH 5.5 with a protein concentration of 100 mg / mL was prepared to investigate the stability at 4° C. and 25° C. The results are shown in Table 27. The results show that the SEC, CE, and IEC of the h1705-008 formulation are slightly decreased under high temperature conditions, but the decrease is within the acceptable range; there is no significant change in all the indexes under 4° C. conditions, indicating that the formulation has favorable stability.

[0311] [Table 27] Note: M stands for months, T stands for hours, and N / A stands for no detection.

[0312] Test Example 18. Further optional formulations Additionally, the present disclosure provides additional formulations for anti-IL-5 antibody pharmaceutical formulations, including, but not limited to, the following: (1) 1 mg / ml anti-IL-5 antibody (h1705-008), 72 mg / ml sucrose, 0.4 mg / ml polysorbate 80, and 10 mM acetic acid-sodium acetate buffer at pH 5.5; (2) 100 mg / ml anti-IL-5 antibody (h1705-008), 72 mg / ml sucrose, 0.4 mg / ml polysorbate 80, and 20 mM acetic acid-sodium acetate buffer at pH 5.5; (3) 120 mg / ml anti-IL-5 antibody (h1705-008), 72 mg / ml sucrose, 0.4 mg / ml polysorbate 80, and 40 mM acetic acid-sodium acetate buffer at pH 5.5; (4) 100 mg / ml anti-IL-5 antibody (h1705-008), 80 mg / ml sucrose, 0.6 mg / ml polysorbate 80, and 30 mM acetic acid-sodium acetate buffer at pH 5.0; (5) 80 mg / ml anti-IL-5 antibody (h1705-008), 75 mg / ml sucrose, 0.6 mg / ml polysorbate 80, and 20 mM acetic acid-sodium acetate buffer at pH 5.4; (6) 100 mg / ml anti-IL-5 antibody (h1705-008), 80 mg / ml sucrose, 0.4 mg / ml polysorbate 80, and 30 mM acetic acid-sodium acetate buffer at pH 5.5; (7) 90 mg / ml anti-IL-5 antibody (h1705-008), 74 mg / ml sucrose, 0.5 mg / ml polysorbate 80 and 25 mM acetic acid-sodium acetate buffer at pH 5.6; (8) 90 mg / ml anti-IL-5 antibody (h1705-008), 76 mg / ml sucrose, 0.3 mg / ml polysorbate 80, and 35 mM acetic acid-sodium acetate buffer at pH 5.4; (9) 80 mg / ml anti-IL-5 antibody (h1705-008), 72 mg / ml sucrose, 0.4 mg / ml polysorbate 80 and 30 mM acetic acid-sodium acetate buffer at pH 5.6; (10) 100 mg / ml anti-IL-5 antibody (h1705-008), 72 mg / ml sucrose, 0.4 mg / ml polysorbate 80 and 40 mM acetic acid-sodium acetate buffer at pH 5.5; (11) 100 mg / ml anti-IL-5 antibody (h1705-008), 80 mg / ml sucrose, 0.4 mg / ml polysorbate 80 and 40 mM acetic acid-sodium acetate buffer at pH 5.5.

[0313] The above experimental results show that the above IL-5 antibody formulation has favorable stability and can be applied to the preparation of IL-5 antibody-type active substances.

[0314] Test Example 19. Freeze-drying of anti-IL-5 antibody preparation The h1705-008 antibody formulation was prepared in 30 mM acetic acid-sodium acetate buffer at pH 5.5, containing 72 mg / ml sucrose, 0.4 mg / ml polysorbate 80, and anti-IL-5 antibody at a concentration of 100 mg / ml. The antibody was filled into 6 mL vials at 2.15 mL / vial and placed in a lyophilization chamber for lyophilization. The lyophilization process included pre-freezing, primary drying, and secondary drying. After the lyophilization process, the vials were vacuumed and stoppered. The reconstituted samples were compared to their pre-lyophilized counterparts. The results showed that the reconstituted solution could maintain favorable performance similar to that of the liquid formulation.

[0315] [Table 28] Note: N / A means the above table is not applicable.

Claims

1. (a) an anti-IL-5 antibody or an antigen-binding fragment thereof; (b) a buffer, the buffer being an acetic acid-sodium acetate buffer or a succinic acid-sodium succinate buffer, having a pH of 5.0 to 6.5 and a concentration of 10 mM to 40 mM; (c) a surfactant, the surfactant being polysorbate 80, the concentration of the polysorbate 80 being 0.1 mg / ml to 0.6 mg / ml; (d) a stabilizer, the stabilizer being sucrose, the concentration of the sucrose being 50 mg / ml to 80 mg / ml; A pharmaceutical composition comprising: The anti-IL-5 antibody or antigen-binding fragment thereof is a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 as set forth in amino acid SEQ ID NOs: 16, 17 and 18, respectively; and A pharmaceutical composition comprising a light chain variable region comprising LCDR1, LCDR2 and LCDR3 as set forth in amino acid sequence numbers 19, 20 and 21, respectively.

2. 2. The pharmaceutical composition of claim 1, wherein the pH of the buffer is 5.0 to 5.

8.

3. 3. The pharmaceutical composition of claim 2, wherein the pH of the buffer is about 5.

5.

4. 2. The pharmaceutical composition of claim 1, wherein the concentration of the buffer is from 20 mM to 30 mM.

5. 2. The pharmaceutical composition of claim 1, wherein the concentration of the buffer is about 30 mM or about 32 mM.

6. The pharmaceutical composition according to claim 1, wherein the concentration of the anti-IL-5 antibody or antigen-binding fragment thereof is from 1 mg / ml to 120 mg / ml.

7. The pharmaceutical composition according to claim 6, wherein the concentration of the anti-IL-5 antibody or antigen-binding fragment thereof is 80 mg / ml to 120 mg / ml.

8. The pharmaceutical composition of claim 7, wherein the concentration of the anti-IL-5 antibody or antigen-binding fragment thereof is about 100 mg / ml.

9. 2. The pharmaceutical composition of claim 1, wherein the concentration of polysorbate 80 is from 0.2 mg / ml to 0.6 mg / ml.

10. 10. The pharmaceutical composition of claim 9, wherein the concentration of polysorbate 80 is about 0.4 mg / ml or about 0.6 mg / ml.

11. 2. The pharmaceutical composition of claim 1, wherein the concentration of the sucrose is from 70 mg / ml to 75 mg / ml.

12. 12. The pharmaceutical composition of claim 11, wherein the concentration of sucrose is about 72 mg / ml.

13. 10. The pharmaceutical composition of claim 1, comprising: 1 mg / ml to 120 mg / ml of an anti-IL-5 antibody or antigen-binding fragment thereof; 10 mM to 40 mM acetic acid-sodium acetate buffer with a pH of 5.0 to 6.5; 0.1 mg / ml to 0.6 mg / ml of polysorbate 80; and 50mg / ml to 80mg / ml sucrose.

14. 80 mg / ml to 120 mg / ml of an anti-IL-5 antibody or an antigen-binding fragment thereof; 10 mM to 30 mM acetic acid-sodium acetate buffer with a pH of 5.0 to 5.8; 0.2 mg / ml to 0.6 mg / ml of polysorbate 80 and 70mg / ml to 80mg / ml sucrose The pharmaceutical composition of claim 1 , comprising:

15. About 100 mg / ml of an anti-IL-5 antibody or antigen-binding fragment thereof; about 30 mM acetic acid-sodium acetate buffer having a pH of about 5.5; Polysorbate 80 at approximately 0.4 mg / ml and Approximately 72 mg / ml sucrose 15. The pharmaceutical composition of claim 14, comprising:

16. The pharmaceutical composition of claim 1, wherein the anti-IL-5 antibody or antigen-binding fragment thereof is a murine antibody, a chimeric antibody or a humanized antibody.

17. The humanized anti-IL-5 antibody comprises a heavy chain variable region as set forth in any one of SEQ ID NOs: 49, 50, and 51, or having 95% sequence identity with any one of SEQ ID NOs: 49, 50, and 51; and A light chain variable region as set forth in any one of SEQ ID NOs: 46, 47, and 48, or having 95% sequence identity with any one of SEQ ID NOs: 46, 47, and 48.

17. The pharmaceutical composition of claim 16, comprising:

18. The pharmaceutical composition of claim 17, wherein the humanized anti-IL-5 antibody comprises a heavy chain variable region set forth in SEQ ID NO: 51 and a light chain variable region set forth in SEQ ID NO:

47.

19. The pharmaceutical composition of claim 1, wherein the anti-IL-5 antibody comprises a human antibody constant region.

20. 20. The pharmaceutical composition of claim 19, wherein the anti-IL-5 antibody comprises a human antibody heavy chain constant region set forth in SEQ ID NO:52 and a human antibody light chain constant region set forth in SEQ ID NO:

53.

21. The pharmaceutical composition of claim 20, wherein the anti-IL-5 antibody comprises a heavy chain set forth in SEQ ID NO:83 and a light chain set forth in SEQ ID NO:

84.

22. 22. A method for preparing the pharmaceutical composition of any one of claims 1 to 21, comprising replacing a stock solution of an anti-IL-5 antibody or an antigen-binding fragment thereof with a buffer, wherein the buffer is an acetic acid-sodium acetate buffer.

23. A lyophilized formulation comprising an anti-IL-5 antibody or an antigen-binding fragment thereof, which is a lyophilized form of the pharmaceutical composition according to any one of claims 1 to 21.

24. A method for obtaining the freeze-dried formulation described in claim 23, comprising sequentially a pre-freezing step, a primary drying step and a secondary drying step.

25. A reconstitution solution comprising an anti-IL-5 antibody or an antigen-binding fragment thereof, which is a reconstituted form of the lyophilized formulation according to claim 23.

26. An article comprising a container containing the pharmaceutical composition according to any one of claims 1 to 21, or the lyophilized formulation according to claim 23, or the reconstitution solution according to claim 25.

27. 22. The pharmaceutical composition according to any one of claims 1 to 21 for treating an IL-5 mediated disease, wherein the IL-5 mediated disease is selected from the group consisting of asthma, chronic pneumonia, allergic rhinitis, allergic bronchopulmonary aspergillosis, eosinophilia, Churg-Strauss syndrome, atopic dermatitis, onchocerciasis dermatitis, intermittent angioedema, eosinophilic myalgia syndrome, eosinophilic gastroenteritis, parasitic infections, Hodgkin's disease, nasal polyps, Loeffler's syndrome, urticaria, hypereosinophilic bronchitis, arteritis nodosa, sinusitis, eosinophilic esophagitis, allergic eosinophilic esophagitis, allergic conjunctivitis, onchocerciasis dermatitis, endometriosis and steroid-dependent eosinophilic bronchitis.

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