A pharmaceutical composition containing a novel coronavirus antibody and its uses

Abstract

A pharmaceutical composition of a humanized monoclonal antibody against the novel coronavirus (2019-nCoV) and its use in medicine. The pharmaceutical composition contains an antibody or its antigen-binding fragment that specifically binds to the 2019-nCoV RBD, a buffer, and may also contain at least one stabilizer, and optionally may also contain a surfactant.

Description

Technical Field

[0001] The present invention relates to the field of therapeutic pharmaceutical compositions. In particular, the present invention relates to the field of pharmaceutical preparations, and the pharmaceutical composition contains a humanized antibody that specifically binds to the novel coronavirus (2019-nCoV, also known as SARS-CoV-2). Background Art

[0002] 2019-nCoV belongs to the coronavirus. The severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV), which also belong to the coronavirus, have caused epidemics in 2002-2003 and 2012 respectively. On January 12, 2020, the World Health Organization officially named this novel coronavirus "2019 novel coronavirus (2019-nCoV)". Subsequently, on February 11-12, 2020, the International Committee on Taxonomy of Viruses (ICTV) announced that the official classification name of the novel coronavirus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). On the same day, the World Health Organization (WHO) announced at the Global Research and Innovation Forum held in Geneva that the official name of the disease caused by this virus is "COVID-19".

[0003] For a virus to infect a cell, it first needs to bind to the receptor of the host through an envelope protein. Antibodies, especially neutralizing antibodies, bind to the envelope protein to block the binding of the virus to the cell receptor, thereby blocking virus infection. At the same time, the antibody binds to the envelope protein, thereby labeling free viruses or infected cells, and recruiting immune cells and immune molecules such as macrophages or complement through the Fc region of the antibody, thereby clearing free viruses and infected cells. Therefore, antibodies targeting the receptor binding domain (RBD) not only have the activity of neutralizing virus infection, but also can play a role through the Fc region to promote the clearance of viruses and infected cells.

[0004] Based on the research on other coronaviruses, especially SARS-CoV and MERS-CoV, the important envelope protein that binds to the receptor is the spike protein (S). S can be further divided into two parts, S1 and S2. The role of S2 is to mediate membrane fusion. Both the N-terminal (NTD) and C-terminal (CTD) of S1 may be the RBD. Through the research on 2019-nCoV, the present invention discovers that the CTD is the RBD of 2019-nCoV, which binds to the receptor ACE2. Therefore, antibodies targeting the RBD and capable of blocking the binding of S to ACE2 may become neutralizing antibodies that inhibit virus infection. The object of the present invention is to provide specific human neutralizing antibodies with a protective effect against 2019-nCoV.

[0005] There is currently no specific drug for 2019-nCoV. Summary of the Invention

[0006] The pharmaceutical composition described in the present invention is a highly stable pharmaceutical composition containing a humanized monoclonal antibody that specifically binds to 2019-nCoV. In particular, the present invention discovers that the humanized monoclonal antibody that specifically binds to 2019-nCoV has unexpected characteristics in a histidine buffer system and a combination of mannitol, sucrose, or trehalose, that is, it has high stability.

[0007] The present invention provides a pharmaceutical composition, comprising: (1) a buffer; (2) a humanized monoclonal antibody or its antigen-binding fragment, wherein the humanized monoclonal antibody specifically binds to 2019-nCoV RBD.

[0008] In some embodiments, the concentration of the humanized monoclonal antibody or its antigen-binding fragment in the pharmaceutical composition is about 1-300 mg / mL, preferably about 10-200 mg / mL, more preferably about 20-150 mg / mL, more preferably about 40-120 mg / mL; even more preferably about 40-100 mg / mL; more preferably, the concentration of the above-mentioned humanized monoclonal antibody or its antigen-binding fragment is about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL or 200 mg / mL, preferably about 40 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 120 mg / mL.

[0009] In some embodiments, the above buffer is selected from one or more of acetate buffer, citrate buffer and histidine buffer.

[0010] In some embodiments, the above buffer is histidine buffer. Preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer, and preferably histidine-hydrochloride buffer.

[0011] In some embodiments, the above-mentioned histidine-hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of 1 - 30 mM of L-histidine and 1 - 30 mM of L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride with a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride with a molar ratio of 1:1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride with a molar ratio of 1:3. In some embodiments, the histidine preparation is a histidine buffer with a pH of 5.5 made of 4.5 mM of L-histidine and 15.5 mM of L-histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of 5.5 made of 7.5 mM of L-histidine and 22.5 mM of L-histidine monohydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of 6.0 made of 10 mM of histidine and 10 mM of histidine hydrochloride. In some embodiments, the histidine preparation is a histidine buffer with a pH of 6.0 made of 15 mM of histidine and 15 mM of histidine hydrochloride.

[0012] In some embodiments, the above-mentioned histidine buffer is a histidine-acetate buffer. Preferably, the molar ratio of the two is 1:1 to 1.5:1. Preferably, the pH of such a buffer is 5.5 ± 0.3, preferably about 5.5. Preferably, such a buffer contains 15 - 20 mM of histidine and 12 - 15 mM of acetic acid.

[0013] In some embodiments, the above-mentioned histidine buffer is a histidine-acetate buffer. Preferably, the molar ratio of the two is 1:1 to 1.5:1. Preferably, the pH of such a buffer is 6.0 ± 0.3, preferably about 6.0. Preferably, such a buffer contains 18 - 22 mM of histidine or 18 - 22 mM of acetic acid.

[0014] In some embodiments, the above-mentioned buffer is an acetate buffer. Preferably, the acetate buffer is an acetic acid-sodium acetate buffer or an acetic acid-potassium acetate buffer, preferably an acetic acid-sodium acetate buffer.

[0015] In some embodiments, the above-mentioned buffer is a citrate buffer. Preferably, the citrate buffer is a citric acid-sodium citrate buffer.

[0016] In some embodiments, the above-mentioned buffer is a succinate buffer. Preferably, the succinate buffer is a succinic acid-sodium succinate buffer.

[0017] In some embodiments, the concentration of the above buffer is about 1 - 200 mM, preferably about 1 - 100 mM, more preferably about 5 - 50 mM, even more preferably about 10 - 30 mM; preferably about 10 - 20 mM; preferably about 20 - 30 mM; non-limiting examples of the buffer concentration are about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, 45 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 105 mM, 110 mM, 115 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM or 180 mM or a range formed by any two values within these ranges as endpoints, preferably 10 mM, 15 mM, 20 mM, 25 mM or 30 mM.

[0018] In some embodiments, the pH of the above buffer is about 5.0 - 6.5, preferably about 5.0 - 6.0, more preferably about 5.5 - 6.5, even more preferably about 5.0 - 5.5, preferably about 5.5 - 6.0, preferably about 6.0 - 6.5, non-limiting examples of the pH of the buffer are about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, preferably about 5.5 or 6.0.

[0019] In some embodiments, the above pharmaceutical composition further comprises a stabilizer.

[0020] In some embodiments, the stabilizer is selected from one or more of L-arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose.

[0021] In some embodiments, the above stabilizer is selected from one or more of mannitol, sucrose and trehalose.

[0022] In some embodiments, the concentration of the above stabilizer is about 10 mM - 400 mM, preferably about 50 mM - 300 mM, more preferably about 100 mM - 300 mM, even more preferably about 200 mM - 300 mM.

[0023] In some embodiments, the above stabilizer is sodium chloride at a concentration of about 30 - 200 mM; or the stabilizer is sodium chloride at a concentration of about 50 - 200 mM; or the stabilizer is mannitol at a concentration of about 100 - 300 mM; or the stabilizer is sorbitol at a concentration of about 100 - 300 mM; or the stabilizer is sucrose at a concentration of about 100 - 300 mM; or the stabilizer is trehalose at a concentration of about 100 - 300 mM; or the stabilizer is L-arginine hydrochloride at a concentration of about 30 - 200 mM; or the stabilizer is proline at a concentration of about 100 - 300 mM; or the stabilizer is glycine at a concentration of about 100 - 300 mM; preferably, the stabilizer is mannitol at a concentration of about 200 - 300 mM, or sucrose at a concentration of about 200 - 300 mM, or trehalose at a concentration of about 200 - 300 mM.

[0024] In some embodiments, the above stabilizer is sodium chloride. In some embodiments, the above stabilizer is sodium chloride at a concentration of about 30 - 200 mM, and the concentration of the sodium chloride is preferably about 50 - 190 mM, preferably about 100 - 180 mM, preferably about 120 - 170 mM, preferably about 130 - 150 mM. Non-limiting examples of the concentration of the sodium chloride are about 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, and preferably 135 mM or 140 mM.

[0025] In some embodiments, the above stabilizer is mannitol. In some embodiments, the above stabilizer is mannitol at a concentration of about 100 - 300 mM, and the concentration of the mannitol is preferably about 150 - 300 mM, preferably about 180 - 280 mM, preferably about 200 - 260 mM. Non-limiting examples of the concentration of the mannitol are about 200 mM, 210 mM, 220 mM, 225 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 235 mM.

[0026] In some embodiments, the above stabilizer is sorbitol. In some embodiments, the above stabilizer is sorbitol at a concentration of about 100 - 300 mM, and the concentration of the sorbitol is preferably about 150 - 300 mM, preferably about 180 - 280 mM, preferably about 200 - 260 mM. Non-limiting examples of the concentration of the sorbitol are about 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 235 mM.

[0027] In some embodiments, the above stabilizer is sucrose. In some embodiments, the above stabilizer is sucrose at a concentration of about 100 - 300 mM, and the concentration of the above sucrose is preferably about 150 - 300 mM, preferably about 180 - 280 mM, preferably about 200 - 260 mM. Non-limiting examples of the above sucrose concentration are about 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 235 mM.

[0028] In some embodiments, the above stabilizer is trehalose. In some embodiments, the above stabilizer is trehalose at a concentration of about 100 - 300 mM, and the concentration of the above trehalose is preferably about 150 - 300 mM, preferably about 180 - 280 mM, preferably about 200 - 260 mM. Non-limiting examples of the above trehalose concentration are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 235 mM.

[0029] In some embodiments, the above stabilizer is L-arginine hydrochloride. In some embodiments, the above stabilizer is L-arginine hydrochloride at a concentration of about 30 - 200 mM, and the concentration of the above L-arginine hydrochloride is preferably about 50 - 190 mM, preferably about 100 - 180 mM, preferably about 120 - 170 mM, preferably about 130 - 150 mM. Non-limiting examples of the above L-arginine hydrochloride concentration are about 100 mM, 110 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, and preferably 135 mM or 140 mM.

[0030] In some embodiments, the above stabilizer is proline. In some embodiments, the above stabilizer is proline at a concentration of about 100 - 300 mM, and the concentration of the above proline is preferably about 150 - 300 mM, preferably about 200 - 280 mM. Non-limiting examples of the above proline concentration are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and preferably 240 mM.

[0031] In some embodiments, the above-mentioned stabilizer is glycine. In some embodiments, the above-mentioned stabilizer is glycine at a concentration of about 100 - 300 mM. The concentration of the above-mentioned glycine is preferably about 150 - 300 mM, more preferably about 200 - 280 mM. Non-limiting examples of the concentration of the above-mentioned glycine are about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, and the preferred one is 260 mM.

[0032] In some embodiments, the above-mentioned pharmaceutical composition further comprises a surfactant, and the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer 188.

[0033] In some embodiments, the above-mentioned surfactant is selected from polysorbate 80.

[0034] In some embodiments, the above-mentioned surfactant is selected from polysorbate 20.

[0035] In some embodiments, calculated by w / v, the concentration of the above-mentioned surfactant is about 0.001% - 0.1%, preferably about 0.01% - 0.1%, more preferably about 0.02% - 0.08%; as non-limiting examples, the concentration of the above-mentioned surfactant is about 0.02%, 0.03%, 0.04%, 0.05%, 0.06% or 0.08%.

[0036] In some embodiments, the above-mentioned humanized monoclonal antibody or its antigen-binding fragment has the amino acid sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain variable region as shown in SEQ ID NO:7, and the amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region as shown in SEQ ID NO:8.

[0037] In some embodiments, the above-mentioned humanized monoclonal antibody or its antigen-binding fragment has HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively.

[0038] In some embodiments, the above-mentioned humanized monoclonal antibody has a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:7, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:8.

[0039] In some embodiments, the above-mentioned humanized monoclonal antibody has a heavy chain amino acid sequence as shown in SEQ ID NO:9 and a light chain amino acid sequence as shown in SEQ ID NO:10.

[0040] In some embodiments, the above-mentioned pharmaceutical composition comprises the components shown in any one of (1)-(6) below, wherein the humanized monoclonal antibody or its antigen-binding fragment is as described in any embodiment of the present invention:

[0041] (1) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 20 mg / mL - 150 mg / mL; (b) a histidine buffer at about 5 - 50 mM with a pH of about 5.0 - 6.5; (c) sodium chloride at about 50 - 200 mM; and (d) polysorbate 80 at about 0.01% - 0.1%; or

[0042] (2) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 20 mg / mL - 150 mg / mL; (b) a histidine buffer at about 5 - 50 mM with a pH of about 5.0 - 6.5; (c) mannitol at about 100 - 300 mM; and (d) polysorbate 80 at about 0.01% - 0.1%; or

[0043] (3) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 20 mg / mL - 150 mg / mL; (b) a histidine buffer at about 5 - 50 mM with a pH of about 5.0 - 6.5; (c) sucrose at about 100 - 300 mM; and (d) polysorbate 80 at about 0.01% - 0.1%; or

[0044] (4) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 20 mg / mL - 150 mg / mL; (b) a histidine buffer at about 5 - 50 mM with a pH of about 5.0 - 6.5; (c) trehalose at about 100 - 300 mM; and (d) polysorbate 80 at about 0.01% - 0.1%; or

[0045] (5) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 20 mg / mL - 150 mg / mL; (b) an acetate buffer at about 10 - 30 mM with a pH of about 5.5 - 6.0; (c) mannitol at about 100 - 300 mM; and (d) polysorbate 80 at about 0.01% - 0.1%; or

[0046] (6) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 20 mg / mL - 150 mg / mL; (b) a citrate buffer at about 10 - 30 mM with a pH of about 5.5 - 6.0; (c) mannitol at about 100 - 300 mM; and (d) polysorbate 80 at about 0.01% - 0.1%.

[0047] Preferably, the pharmaceutical composition comprises the components shown in any one of (7)-(9) below, wherein the humanized monoclonal antibody or its antigen-binding fragment is as described in any embodiment of the present invention:

[0048] (7) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 40 mg / mL - 120 mg / mL; (b) A histidine buffer at about 10 - 30 mM, with a pH of about 5.5 - 6.0; (c) Mannitol at about 200 - 300 mM; (d) And polysorbate 80 at about 0.02% - 0.08%; or

[0049] (8) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 40 mg / mL - 120 mg / mL; (b) A histidine buffer at about 10 - 30 mM, with a pH of about 5.5 - 6.0; (c) Sucrose at about 200 - 300 mM; (d) And polysorbate 80 at about 0.02% - 0.08%; or

[0050] (9) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 40 mg / mL - 120 mg / mL; (b) A histidine buffer at about 10 - 30 mM, with a pH of about 5.5 - 6.0; (c) Trehalose at about 200 - 300 mM; (d) And polysorbate 80 at about 0.02% - 0.08%.

[0051] More preferably, the pharmaceutical composition comprises the components shown in any one of (10)-(16) below, wherein the humanized monoclonal antibody or its antigen-binding fragment is as described in any embodiment of the present invention:

[0052] (10) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 40 mg / mL; (b) A histidine buffer at about 20 mM, with a pH of about 6.0; (c) Mannitol at about 235 mM or mannitol at about 247 mM; (d) And polysorbate 80 at about 0.02%; or

[0053] (11) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 40 mg / mL; (b) A histidine buffer at about 20 mM, with a pH of about 6.0; (c) Sucrose at about 235 mM; (d) And polysorbate 80 at about 0.02%; or

[0054] (12) (a) A humanized monoclonal antibody or its antigen-binding fragment at about 40 mg / mL; (b) A histidine buffer at about 20 mM, with a pH of about 6.0; (c) Trehalose at about 240 mM; (d) And polysorbate 80 at about 0.02%; or

[0055] (13) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at about 80 mg / mL; (b) About 20 mM histidine buffer with a pH of about 6.0; (c) About 235 mM sucrose; (d) And about 0.02% polysorbate 80; or

[0056] (14) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at about 80 mg / mL; (b) About 20 mM histidine buffer with a pH of about 6.0; (c) About 240 mM trehalose; (d) And about 0.02% polysorbate 80; or

[0057] (15) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at about 100 mg / mL; (b) About 20 mM histidine buffer with a pH of about 6.0; (c) About 235 mM sucrose; (d) And about 0.03% polysorbate 80; or

[0058] (16) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at about 100 mg / mL; (b) About 20 mM histidine buffer with a pH of about 6.0; (c) About 235 mM sucrose; (d) And about 0.05% polysorbate 80.

[0059] In some embodiments, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

[0060] In some embodiments, the pharmaceutical composition is a liquid preparation.

[0061] In some embodiments, the above liquid preparation or lyophilized preparation is stable at 2 - 8 °C for at least 3 months, at least 6 months, at least 12 months, at least 18 months or at least 24 months.

[0062] In some embodiments, the above aqueous solution or lyophilized preparation is stable at 40 °C for at least 7 days, at least 14 days or at least 28 days.

[0063] The present invention also provides an injection, which comprises the pharmaceutical composition described in any one of the embodiments herein and 0.9% (w / v) sodium chloride solution; preferably, the concentration of the humanized monoclonal antibody is 1 - 40 mg / mL; preferably, the pH of the injection is 5.5 - 6.0.

[0064] The present invention also provides the use of the above pharmaceutical composition or injection in the preparation of a drug for treating or preventing COVID-19 infection.

[0065] The present invention also provides the use of a histidine buffer, one or more stabilizers selected from mannitol, sucrose, and trehalose, and optionally a surfactant (preferably polysorbate 80) in enhancing the stability of a pharmaceutical formulation of a humanized monoclonal antibody specifically binding to COVID-19, or in the preparation of a pharmaceutical formulation of a humanized monoclonal antibody specifically binding to COVID-19 with enhanced stability. Preferably, the histidine buffer, stabilizer, and surfactant and their amounts are as described in any of the embodiments herein; the enhanced stability is as described in any of the embodiments herein. Brief Description of the Drawings

[0066] Figure 1 : Binding ELISA analysis of humanized monoclonal antibody CB6 (lot number 20200307) with 2019-nCoV RBD protein.

[0067] Figure 2 : Blocking ELISA analysis of humanized monoclonal antibody CB6 (lot number 20200307) with 2019-nCoV RBD protein.

[0068] Figure 3 : Humanized monoclonal antibody CB6 (lot number 20200306) blocks 2019-nCoV infection of Huh-7 cells.

[0069] Figure 4 : Humanized monoclonal antibody CB6 (lot number 20200306) blocks 2019-nCoV infection of Vero E6 cells.

[0070] Figure 5 : Humanized monoclonal antibody CB6 reduces the cytopathic effect of SARS-CoV-2 virus on Vero E6 cells in a dose-dependent manner. Detailed Description of the Embodiments

[0071] Definitions and Explanations

[0072] To facilitate a better understanding of the present invention, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions, or biological systems, and of course, changes can be made thereto. It should also be understood that the terms used in this application are only for describing specific embodiments and are not intended to be limiting.

[0073] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

[0074] The term "pharmaceutical composition" or "formulation" refers to a mixture containing one or more antibodies described herein and other components such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and thus exert biological activity.

[0075] The term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a lyophilized formulation in a heavy suspension. The liquid formulations of the present invention are stable upon storage, and their stability does not depend on lyophilization (or other state change methods such as spray drying).

[0076] The term "aqueous liquid formulation" refers to a liquid formulation that uses water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and / or freezing to maintain stability (such as chemical and / or physical stability and / or biological activity).

[0077] The term "excipient" refers to a reagent that can be added to a formulation to provide desired properties (such as consistency, enhanced stability) and / or adjust the osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

[0078] As used herein, "about" in reference to a measurable value such as an amount, duration, etc. is intended to cover variations of ±20% or ±10% relative to the specific value, including ±5%, ±1%, and ±0.1%, because such variations are suitable for performing the disclosed methods.

[0079] The term "buffer with a pH of about 5.0 - 6.5" refers to a reagent that, through the action of its acid / base conjugate components, enables a solution containing the reagent to resist pH changes. The buffer used in the formulations of the present invention may have a pH in the range of about 5.0 to about 6.5, or in the range of about 5.5 to about 6.5, or in the range of about 5.0 to about 6.0.

[0080] Examples of "buffers" that control the pH within this range herein include acetate (such as sodium acetate), succinic acid, succinate (such as sodium succinate), gluconic acid, histidine, histidine hydrochloride, methionine, citric acid, citrate, phosphate, citrate / phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.

[0081] "Histidine buffer solution" refers to a buffer solution containing histidine ions. Examples of histidine buffer solutions include histidine and salts of histidine, such as histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, etc., such as a histidine buffer solution containing histidine and histidine hydrochloride; the histidine buffer solution of the present invention also includes a histidine buffer solution containing histidine and acetate (such as sodium salt or potassium salt).

[0082] "Citric acid buffer solution" is a buffer solution containing citrate ions. Examples of citric acid buffer solutions include citric acid - sodium citrate, citric acid - potassium citrate, citric acid - calcium citrate, citric acid - magnesium citrate, etc. The preferred citrate buffer solution is citric acid - sodium citrate buffer solution.

[0083] "Acetic acid buffer solution" is a buffer solution containing acetate ions. Examples of acetic acid buffer solutions include acetic acid - sodium acetate, acetic acid - potassium acetate, acetic acid - calcium acetate, acetic acid - magnesium acetate, etc. The preferred acetate buffer solution is acetic acid - sodium acetate buffer solution.

[0084] "Succinic acid buffer solution" is a buffer solution containing succinate ions. Examples of succinate buffer solutions include succinic acid - sodium succinate, succinic acid - potassium succinate, succinic acid - calcium succinate, succinic acid - magnesium succinate, etc. The preferred succinate buffer solution is succinic acid - sodium succinate buffer solution.

[0085] The term "w / v" represents mass - volume concentration. For example, in "0.9% (w / v) sodium chloride solution", "0.9%" means "0.9 g of solute is contained in 100 mL of solution".

[0086] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols, and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines, alanylaminopine, octopine, strombine, and N-oxide of trimethylamine (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin, and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizers specifically used in the present invention are selected from one or more of polyols, amino acids, salts, and sugars. Preferred sugars are sucrose and trehalose, and preferred polyols are mannitol. Preferred amino acids are arginine or its salts (such as arginine hydrochloride), glycine, and proline. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, more preferably arginine hydrochloride, sodium chloride-sucrose, arginine hydrochloride-mannitol, arginine hydrochloride-sucrose, and even more preferably arginine hydrochloride-sucrose. In a particularly preferred embodiment, the stabilizers used in the present invention are selected from one or more of mannitol, sucrose, and trehalose.

[0087] The term "surfactant" generally includes reagents that protect proteins such as antibodies from stress induced by the air / solution interface and solution / surface interface to reduce antibody aggregation or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymer, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkyl phenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymer (poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). In a particularly preferred embodiment, the surfactant used in the present invention is polysorbate 80.

[0088] The term "isotonic" means that the preparation has an osmotic pressure substantially the same as human blood. Isotonic preparations generally have an osmotic pressure of about 250 to 350 mOsm. Osmotic pressure can be measured using a vapor pressure or freezing point depression osmometer.

[0089] A "stable" preparation is one in which the antibody therein substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or upon storage. A pharmaceutical preparation can be stable even if the contained antibody does not maintain its 100% chemical structure or biological function after storage for a certain period of time. In some cases, after storage for a certain period of time, maintaining about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the antibody structure or function can also be considered "stable". Various analytical techniques for measuring protein stability are available in the art and are reviewed in "Peptide and Protein Drug Delivery" 247 - 301, edited by Vincent Lee, Marcel Dekker, Inc., New York, N.Y., Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29 - 90 (both incorporated by reference).

[0090] After a preparation has been stored at a certain temperature for a certain period of time, its stability can be measured by determining the percentage of the remaining native antibody therein (among other methods). Among other methods, the percentage of the native antibody can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC - HPLC]), where "native" means non - aggregated and non - degraded. In some protocols, the stability of a protein is determined by the percentage of monomeric protein in a solution with a low percentage of degraded (e.g., fragmented) and / or aggregated protein. In some protocols, the preparation can be stably stored at room temperature, about 25 - 30 °C or 40 °C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer, with no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the antibody in aggregated form.

[0091] By measuring the percentage of the antibody (“acidic form”) that migrates in a fraction that is more acidic than the main fraction of the antibody (“main charged form”) during ion exchange (and other methods), stability can be measured, where stability is inversely proportional to the percentage of the acidic form of the antibody. Among other methods, the percentage of “acidified” antibody can be measured by ion exchange chromatography (such as cation exchange high performance liquid chromatography [CEX-HPLC]). In some embodiments, an acceptable level of stability means that when the formulation is stored at a certain temperature for a certain period of time, the detectable acidic form of the antibody is at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. The certain period of time stored before measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer. When evaluating stability, the certain temperature at which the pharmaceutical formulation is allowed to be stored can be any temperature within the range of about -80°C to about 45°C, such as stored at about -80°C, about -30°C, about -20°C, about 0°C, about 2-8°C, about 5°C, about 25°C, or about 40°C.

[0092] An antibody “retains its physical stability” in the pharmaceutical composition if it does not substantially show signs of, for example, aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or by measurement by UV light scattering, or by size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the extent of forming visible precipitates.

[0093] The stability of a formulation, such as physical stability, can be evaluated by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is in part an inherent property of the protein dissolved in the solution and is typically measured by nephelometry and quantified in nephelometric turbidity units (NTU).

[0094] Turbidity levels that vary with, for example, the concentration of one or more components in a solution (such as protein and / or salt concentration) are also referred to as the "opalescence" or "opalescent appearance" of a formulation. Turbidity levels can be calculated by reference to a standard curve generated using a suspension of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia standards (European Pharmacopoeia, Fourth Edition, "Directorate for the Quality of Medicine of the Council of Europe" (EDQM), Strasbourg, France). According to the European Pharmacopoeia standards, a clear solution is defined as a solution having a turbidity lower than or equal to that of a reference suspension having a turbidity of approximately 3 according to the European Pharmacopoeia standards. Turbidity measurements by nephelometry can detect Rayleigh scattering in the absence of association or non-ideal effects, which generally varies linearly with concentration. Other methods for assessing physical stability are known in the art.

[0095] An antibody "maintains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time point is such that the antibody is considered to still retain its biological activity as defined hereinafter. Chemical stability can be evaluated, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical alterations can include size alterations (such as truncation), which can be evaluated using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge alterations (such as those occurring as a result of deamidation or oxidation), which can be evaluated by, for example, ion exchange chromatography.

[0096] An antibody "maintains its biological activity" in a pharmaceutical composition if the antibody is biologically active for its intended purpose. For example, if after storage of a formulation at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (such as 1 to 12 months), the affinity of the humanized monoclonal antibody contained in the formulation for binding to COVID-19 is at least 90%, 95% or more of the antibody binding affinity before such storage, then the formulation of the present invention can be considered stable. Binding affinity can also be determined using, for example, ELISA or surface plasmon resonance techniques.

[0097] In the context of the present invention, a "therapeutically effective amount" or "effective amount" of an antibody, in a pharmacological sense, is an amount that is effective in the prevention, treatment, or alleviation of the symptoms of a disorder that the antibody can effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes the regression of a disease, as evidenced by a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or the prevention of injury or disability caused by the affliction of the disease. The ability of a drug to promote the regression of a disease can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems predictive of human efficacy, or by measuring the activity of the agent in in vitro assays. A therapeutically effective amount of a drug includes a "preventively effective amount", i.e., any amount of the drug that inhibits the development or recurrence of a disease when administered alone or in combination with other therapeutic agents to a subject at risk of developing the disease or subject to recurrence of the disease.

[0098] The terms "subject" or "patient" are intended to include mammalian organisms. Examples of subjects / patients include humans and non-human mammals such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a particular embodiment of the present invention, the subject is a human.

[0099] The terms "administer", "give", and "treat" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. Routes of administration of a humanized monoclonal antibody that specifically binds to 2019-nCoV or an antigen-binding fragment thereof include intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral routes of administration such as injection or infusion. "Parenteral administration" refers to a mode of administration other than enteral or topical administration, typically by injection, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intramammary, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intradural, and intrasternal injection and infusion, as well as in vivo electroporation.

[0100] Antibody

[0101] The term "antibody" as used herein should be understood to include intact antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to 2019-nCoV (2019 novel coronavirus) or an epitope thereof.

[0102] As used herein, the term "full-length antibody" or "intact antibody molecule" refers to an immunoglobulin molecule containing four peptide chains, two heavy (H) chains (about 50-70 kDa when full-length) and two light (L) chains (about 25 kDa when full-length), which are interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into complementarity-determining regions (CDRs) with high variability and intervening regions that are more conserved and are called framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0103] As used herein, the term "CDR" refers to the complementarity-determining regions within the variable sequences of an antibody. There are three CDRs present in each of the variable regions of the heavy and light chains, which are named HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each of the heavy and light chain variable regions. The exact boundaries of these CDRs are defined differently according to different systems.

[0104] The precise amino acid sequence boundaries of the CDRs of the variable regions of the antibodies of the present invention can be determined using any one of a number of well-known schemes, including the Kabat scheme described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD" ("Kabat" numbering scheme) and the IMGT scheme described by Lefranc M.-P. et al. (1999 Nucleic Acids Research, 27, 209-212).

[0105] As used herein, "antigen-binding fragment" includes fragments or derivatives of antibodies, and generally includes at least one fragment of the antigen-binding region or variable region (such as one or more CDRs) of the parental antibody, which retains at least some of the binding specificities of the parental antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies formed from antibody fragments, and multispecific antibodies. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or its derivative generally retains at least 10% of the antigen-binding activity of the parental antibody. Preferably, the binding fragment or its derivative retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen-binding affinity of the parental antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as "conservative variants" or "functionally conservative variants" of the antibody).

[0106] When referring to a ligand / receptor, antibody / antigen or other binding pair, "specific" binding refers to determining the presence of the protein in a heterogeneous population of proteins and / or other biological reagents. For example, the binding reaction of the monoclonal antibody of the present invention with the 2019-nCoV RBD protein. Thus, under the specified conditions, a particular ligand / antigen binds to a particular receptor / antibody and does not bind to other proteins present in the sample in significant amounts.

[0107] The humanized monoclonal antibodies or their antigen-binding fragments described herein include any of the humanized monoclonal antibodies or their antigen-binding fragments described in Application No. CN202010114283.8, the entire content of which is incorporated herein by reference. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the present invention include the CDR sequences of the antibody CB6 described in CN202010114283.8. In some embodiments, the CDR sequences of the antibodies used in the methods and compositions of the present invention include the variable region sequences of the antibody CB6 described in CN202010114283.8. In some embodiments, the antibodies used in the methods and compositions of the present invention are humanized monoclonal antibodies obtained by constructing an expression vector by conventional techniques and expressing it in cells from the variable region sequences of the antibody CB6 described in CN202010114283.8.

[0108] In the embodiments of the present text, the non-limiting, exemplary antibody used is the humanized antibody CB6 described in CN202010114283.8, which is capable of specifically binding to the 2019-nCoV RBD. Among them, antibody CB6 has HCDR1, HCDR2, and HCDR3 composed of the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region with the amino acid sequence shown in SEQ ID NO:7, and LCDR1, LCDR2, and LCDR3 composed of the LCDR1, LCDR2, and LCDR3 of the light chain variable region with the amino acid sequence shown in SEQ ID NO:8; according to the "Kabat" numbering scheme, antibody CB6 has HCDR1, HCDR2, and HCDR3 with the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and LCDR1, LCDR2, and LCDR3 with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively; according to the IMGT scheme, antibody CB6 has HCDR1, HCDR2, and HCDR3 with the amino acid sequences shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13 respectively, and LCDR1, LCDR2, and LCDR3 with the amino acid sequences shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 respectively; preferably, antibody CB6 has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:7 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:8; preferably, antibody CB6 has a heavy chain amino acid sequence shown in SEQ ID NO:9 and a light chain amino acid sequence shown in SEQ ID NO:10.

[0109] The HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:7 and the heavy chain amino acid sequence shown in SEQ ID NO:9, according to the "Kabat" numbering scheme, are respectively shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; according to the IMGT scheme, they are respectively shown in SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:13.

[0110] The LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:8 and the light chain amino acid sequence shown in SEQ ID NO:10, according to the "Kabat" numbering scheme, are shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively; according to the IMGT scheme, they are shown in SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16 respectively.

[0111] Pharmaceutical preparation

[0112] The pharmaceutical composition of the present invention is a highly stable pharmaceutical composition containing a humanized antibody that specifically binds to 2019-nCoV. In particular, the present invention discovers that the combination of a histidine buffer system and mannitol, sucrose, or trehalose has high stability.

[0113] The present invention provides a pharmaceutical composition comprising: (1) a buffer; (2) a humanized monoclonal antibody or an antigen-binding fragment thereof, wherein the humanized monoclonal antibody specifically binds to 2019-nCoV RBD.

[0114] The humanized monoclonal antibody in the pharmaceutical composition of the present invention is as described in any one of the embodiments in the "Antibody" section of this application.

[0115] For example, in some embodiments, the humanized monoclonal antibody in the pharmaceutical composition of the present invention has the amino acid sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO:7, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO:8; or has the amino acid sequences of HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and the amino acid sequences of LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively; preferably, the humanized monoclonal antibody in the pharmaceutical composition of the present invention has a heavy chain variable region with the amino acid sequence shown in SEQ ID NO:7 and a light chain variable region with the amino acid sequence shown in SEQ ID NO:8; more preferably, the humanized monoclonal antibody in the pharmaceutical composition of the present invention has the heavy chain amino acid sequence shown in SEQ ID NO:9 and the light chain amino acid sequence shown in SEQ ID NO:10 respectively.

[0116] In the pharmaceutical composition of the present invention, the concentration of the humanized monoclonal antibody or its antigen-binding fragment is about 1-300 mg / mL, preferably about 10-200 mg / mL, more preferably about 20-150 mg / mL, more preferably about 40-120 mg / mL, more preferably about 40-100 mg / mL; more preferably, the concentration of the above-mentioned humanized monoclonal antibody or its antigen-binding fragment is about 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL or 200 mg / mL, preferably about 40 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, 105 mg / mL, 110 mg / mL, 120 mg / mL.

[0117] The buffer in the pharmaceutical composition of the present invention can be selected from acetate buffer, citrate buffer and histidine buffer, so as to provide a pH of about 5.0 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.5-6.0, and more preferably about 6.0 for the pharmaceutical composition of the present invention. On the other hand, the pH of the buffer used in the pharmaceutical composition of the present invention is about 5.0-6.5, preferably about 5.0-6.0, more preferably about 5.5-6.0, and more preferably about 6.0.

[0118] A particularly preferred buffer in the pharmaceutical composition of the present invention is a histidine buffer, including a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer. More preferably, the histidine-hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of 1-20 mM of L-histidine and 1-20 mM of L-histidine monohydrochloride. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1:1 to 1:4. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1:1. In some embodiments, the histidine buffer is made of histidine and histidine hydrochloride in a molar ratio of 1:3. In some embodiments, the histidine buffer is a histidine buffer with a pH of about 5.5 made of 4.5 mM of L-histidine and 15.5 mM of L-histidine monohydrochloride. In some embodiments, the histidine buffer is a histidine buffer with a pH of about 5.5 made of 7.5 mM of L-histidine and 22.5 mM of L-histidine monohydrochloride. In some embodiments, the histidine buffer is a histidine buffer with a pH of about 6.0 made of 15 mM of L-histidine and 15 mM of L-histidine monohydrochloride. In some embodiments, the histidine buffer is a histidine buffer with a pH of about 6.0 made of 10 mM of L-histidine and 10 mM of L-histidine monohydrochloride.

[0119] Therefore, the pharmaceutical composition of the present invention may contain: a histidine-histidine hydrochloride buffer with a pH of about 5.5-6.0, and its concentration in the pharmaceutical composition is about 10-30 mM; and a humanized monoclonal antibody or its antigen-binding fragment described in any of the previous embodiments, preferably about 40-100 mg / mL, especially the CB6 antibody or its antigen-binding fragment described herein, at a concentration of about 40-120 mg / mL.

[0120] In some embodiments, the pharmaceutical composition of the present invention further contains a stabilizer. Preferably, the stabilizer is selected from one or more of L-arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose. Preferably, the stabilizer in the pharmaceutical composition is selected from mannitol, sucrose, and trehalose. The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10 mM - 400 mM, preferably 50 mM - 300 mM, more preferably 100 mM - 300 mM. In some embodiments, the stabilizer is sodium chloride at a concentration of about 30 - 200 mM; or the stabilizer is mannitol at a concentration of about 100 - 300 mM, preferably about 200 - 300 mM; or the stabilizer is sucrose at a concentration of about 100 - 300 mM, preferably about 200 - 300 mM; or the stabilizer is trehalose at a concentration of about 100 - 300 mM, preferably about 200 - 300 mM.

[0121] Thus, in some embodiments, the pharmaceutical composition of the present invention contains: a histidine - histidine hydrochloride buffer with a pH of about 5.5 - 6.0, and its concentration in the pharmaceutical composition is about 10 - 30 mM; a humanized monoclonal antibody or an antigen - binding fragment thereof described in any of the previous embodiments at about 40 - 120 mg / mL, preferably about 40 - 100 mg / mL, especially the CB6 antibody or an antigen - binding fragment thereof described herein; and a stabilizer at about 100 mM - 300 mM. Preferably, the stabilizer includes one of mannitol, sodium chloride, sucrose, and trehalose, preferably mannitol at about 100 - 300 mM, sucrose at about 100 - 300 mM, and trehalose at about 100 - 300 mM. In some embodiments, the stabilizer is sucrose at about 200 - 300 mM. In some embodiments, the stabilizer is trehalose at about 200 - 300 mM. In some embodiments, the stabilizer is mannitol at about 200 - 300 mM.

[0122] In some embodiments, the pharmaceutical composition of the present invention further includes a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20, and poloxamer 188. The most preferred surfactant is polysorbate 80. Calculated on a w / v basis, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% - 0.1%, preferably about 0.02% - 0.08%. As non - limiting examples, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, or 0.08%.

[0123] Thus, in some embodiments, the pharmaceutical composition of the present invention comprises: a histidine - histidine hydrochloride buffer with a pH of about 5.5 - 6.0, and its concentration in the pharmaceutical composition is about 10 - 30 mM; a humanized monoclonal antibody or an antigen - binding fragment thereof as described in any of the previous embodiments at 40 - 120 mg / mL, preferably about 40 - 100 mg / mL, particularly the CB6 antibody or an antigen - binding fragment thereof as described herein; a stabilizer at about 100 mM - 300 mM, preferably, the stabilizer is about 100 - 300 mM of sucrose, or about 100 - 300 mM of mannitol, or about 100 - 300 mM of trehalose; and polysorbate 80 at about 0.02% - 0.08% w / v.

[0124] The pharmaceutical composition of the present invention can be a liquid preparation or a lyophilized preparation.

[0125] Medical uses and methods

[0126] The present invention also provides a pharmaceutical composition or an injection as described in any of the embodiments of the present invention for treating or preventing diseases related to 2019 - nCoV infection, the use of the pharmaceutical composition or injection as described in any of the embodiments of the present invention in the preparation of a drug for treating or preventing diseases related to 2019 - nCoV infection, and a method of administering a therapeutically effective amount of the pharmaceutical composition or injection as described in any of the embodiments of the present invention to an individual or patient in need to treat or prevent diseases related to 2019 - nCoV infection.

[0127] In the present invention, diseases related to 2019 - nCoV infection refer to diseases caused by the occurrence and development of 2019 - nCoV infection.

[0128] The present invention will be described below by way of specific examples. It should be understood that these examples are merely illustrative and are not intended to limit the scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention. The methods and materials used in the examples are conventional methods and materials in the art unless otherwise specified.

[0129] Example 1: Screening experiments on buffer system, pH, excipients, and protein concentration

[0130] In liquid pharmaceutical compositions, the buffer system, pH, excipients, and protein concentration closely affect the stability of antibodies. Each antibody with unique physical and chemical properties has the most suitable buffer type, pH, and excipient conditions. This example aims to screen an optimal buffer system, pH, and excipients to enable the humanized antibody disclosed in the present invention to have the best stability for clinical application.

[0131] This example was carried out with antibody CB6 at concentrations of approximately 20 mg / mL and 40 mg / mL. The samples were ultrafiltered, concentrated, and buffer-exchanged using a Millipore Pellicon 3 0.11 m 2 membrane. After buffer exchange, the samples were in the corresponding formulations and placed in sealed centrifuge tubes for buffer screening. Acetate buffer, citrate buffer, and histidine buffer were screened for the buffer system, with the pH ranging from 5.5 to 6.0 (as shown in Table 1). For the excipients, sodium chloride, sucrose, trehalose, or mannitol were screened for comparative testing. That is, the above different excipients were separately added to the buffer containing antibody CB6 at a concentration of approximately 20 mg / mL or 40 mg / mL, and the specific formulation information is shown in Table 1. The samples were placed in an environment of 40 ± 2 °C and taken out for analysis and testing at week 0, week 2, and week 4 respectively.

[0132] The main pathways of protein degradation are the formation of aggregates, cleavage products, and charged variants. Size-exclusion chromatography (SEC-HPLC) was used to determine the percentages of the native form (protein monomer) and the aggregated form, and cation-exchange chromatography (CEX-HPLC) was used to determine the percentages of acidic and basic form antibodies. Using the SEC-HPLC monomer content and the CEX-HPLC main peak content after four weeks of storage (4W), a straight line was fitted and the decline slope (% / week) was calculated to investigate the effects of different buffer systems, pH, excipients, and protein concentrations on the stability of antibody CB6.

[0133] The stability was evaluated by the following parameters: (1) visual appearance and visible foreign matters; (2) protein content determined by ultraviolet spectrophotometry; (3) content of antibody monomers, aggregates, or fragments measured by SEC-HPLC; (4) content of main charge, acidic charge, or basic charge of antibodies measured by CEX-HPLC; (5) molecular weight of antibodies detected by NR-CE-SDS method; (6) molecular weight of antibodies detected by R-CE-SDS method; (7) binding activity of antibodies detected by ELISA method.

[0134] Table 1: Formulation information in the buffer system, pH, excipient, and protein concentration screening experiments

[0135]

[0136] The experimental results are shown in Table 2.

[0137] Formulation 1 and Formulation 5 had heavy opalescence at a protein concentration of 40 mg / ml, and there was no abnormality at a concentration of 20 mg / ml. For other formulations, there were no abnormalities in the protein content and appearance at concentrations of 20 mg / ml and 40 mg / ml, indicating that the sodium chloride and pH 6.0 citrate buffer systems are not suitable for the high-concentration stability of this product.

[0138] In the SEC-HPLC experiment, under the accelerated condition of 40 ± 2°C, the monomer content of Prescription 5 decreased rapidly, while the monomer content of Prescriptions 2, 3, and 4 decreased at a relatively lower rate, with an average decrease rate of 0.4% / week. The protein concentration had little effect on the purity at 20 mg / ml and 40 mg / ml.

[0139] The results of the CEX-HPLC decrease rate showed that under the accelerated condition of 40 ± 2°C, the CEX main peak content of Prescriptions 5 and 6 decreased rapidly. The CEX main peak content of Prescriptions 1, 2, 3, and 4 decreased at a relatively lower rate. The protein concentration had little effect on the purity at 20 mg / ml and 40 mg / ml.

[0140] The binding activity (ELISA method) and the purity of NR-CE-SDS were normal, while the purity of R-CE-SDS decreased slightly.

[0141] Table 2: Experimental results of buffer system, pH, excipients, and protein concentration screening

[0142]

[0143] Based on the comprehensive test data, by comparing Prescriptions 1 to 4, it was found that the excipients mannitol, sucrose, and trehalose were better than sodium chloride. By comparing Prescriptions 2 and 5, it was concluded that the histidine buffer system was better than the citrate buffer system, as the latter had heavier opalescence. The SEC-HPLC purity and the CEX main peak of the pH 5.5 acetate buffer system containing the excipient mannitol (Prescription 6) decreased rapidly. Therefore, the histidine buffer system at pH 6.0 containing the excipients mannitol, sucrose, and trehalose (Prescriptions 2 / 3 / 4) was not sensitive to the protein concentration and had better overall stability.

[0144] According to the above screening results, the histidine buffer system at pH 6.0 with the excipients mannitol, sucrose, and trehalose was selected for subsequent research.

[0145] Example 2: Investigation of the formulation prescription stability

[0146] 2.1 Freeze-thaw stability

[0147] The antibody stock solution was selected to prepare the formulation prescriptions as shown in Table 3, with the antibody CB6 concentration of 40 mg / mL. It was manually filled aseptically into 2 ml vials, 2.0 mL per vial, and subjected to three cycles of freezing and thawing from -40°C to room temperature. The stability was investigated by measuring the content of antibody monomers, aggregates, or fragments through appearance and SEC-HPLC.

[0148] Table 3: Prescription information for stability investigation

[0149]

[0150]

[0151] Repeated freeze-thaw cycles: After being placed at ≤ -40°C for more than 4 hours until completely frozen, take it out and allow it to completely thaw at room temperature (25 ± 2°C). After repeating the freeze-thaw cycles three times, the appearance is normal, and Formulations 7, 8, and 9 have no significant effect on the monomer purity of SEC-HPLC. See Table 4 for details.

[0152] Table 4: Formulation screening - Repeated freeze-thaw data

[0153]

[0154] Select the antibody stock solution and prepare the formulated products as shown in Table 3, with the antibody CB6 concentration being 40 mg / mL. Manually fill it aseptically into 125 ml polycarbonate bottles (Thermo Fisher, Part No: 3030-42, made of polycarbonate), 60 mL per bottle, and perform three repeated freeze-thaw cycles from -80°C to room temperature. Examine the stability by measuring the contents of antibody monomers, polymers, or fragments through appearance and SEC-HPLC.

[0155] Repeated freeze-thaw cycles: After being placed at -80°C for more than 4 hours until completely frozen, take it out and allow it to completely thaw at room temperature (25 ± 2°C). After repeating the freeze-thaw cycles three times, the appearance is normal. See Table 5 for details.

[0156] Table 5: Formulation screening - Repeated freeze-thaw cycles in 125 ml polycarbonate bottles

[0157]

[0158] 2.2 Vibration stability

[0159] Select the antibody stock solution and prepare the formulated products as shown in Table 3, with the antibody CB6 concentration being 40 mg / mL. Manually fill it aseptically into 2 ml vials, 2.0 mL per vial. After continuous shaking at 80 rpm or 150 rpm at 25 ± 2°C, examine the stability by measuring the contents of antibody monomers, polymers, or fragments through appearance and SEC-HPLC. See Table 6 for specific information.

[0160] Table 6: Vibration stability investigation

[0161]

[0162] After continuous shaking at 80 rpm or 150 rpm at 25 ± 2°C, there are no significant changes in the appearance and SEC-HPLC purity of the three formulations, showing good stability. See Table 7 for details.

[0163] Table 7: Formulation screening - Shaking

[0164]

[0165]

[0166] 2.3 Saline Dilution Stability

[0167] Select the antibody stock solution and prepare the formulated products as shown in Table 3, with the antibody CB6 concentration being 40 mg / mL. Dilute the samples to different concentrations with saline (0.9% NaCl), detect them after standing at 25 ± 2°C for 8 hours, investigate the sample stability, confirm the formulation conditions, and the screening results are shown in Table 8.

[0168] Table 8: Screening Results of Formulation Stability

[0169]

[0170] The three formulated antibody products have good stability under saline dilution conditions and good compatibility with infusion tubes and infusion bags.

[0171] 2.4 Light Stability

[0172] Select the antibody stock solution and prepare the formulated products as shown in Table 3, with the antibody CB6 concentration being 40 mg / mL. Manually fill 4.0 mL into 6 mL vials aseptically and expose them to light. Investigate the stability by measuring appearance, SEC-HPLC, and CEX-HPLC. The specific information is shown in Table 9.

[0173] Table 9: Light Stability Results

[0174]

[0175]

[0176] 2.5 Long-Term / Accelerated Stability

[0177] Select the antibody stock solution and prepare the formulated products as shown in Table 3, with the antibody CB6 concentration being 40 mg / mL. Manually fill 4.0 mL into 6 mL vials aseptically and conduct long-term / accelerated stability investigations. The specific information is shown in Table 10 and Table 11.

[0178] Table 10: Results of Stability Experiments at 25°C

[0179]

[0180]

[0181]

[0182] Table 11: Results of Stability Experiments at 4°C

[0183]

[0184]

[0185] By conducting stability investigation experiments on the sample under this formulation condition, including repeated freeze-thaw cycles, shaking, dilution with normal saline, and ultraviolet irradiation, there is no significant impact on the monomer content, indicating good stability of the formulation.

[0186] By investigating different buffer systems, different pH conditions, different antibody concentrations, and different excipient compositions, the stability of the humanized antibody CB6 was explored and studied, and the relatively optimal aqueous injection formulation was determined. For antibody CB6, histidine and histidine hydrochloride buffer were selected to adjust the pH, mannitol, sucrose, or trehalose were used to adjust the osmotic pressure of the formulation, and polysorbate 80 was added to increase the solubility of the formulation.

[0187] Example 3: Influence Factors and Stability Study of High-Concentration Formulation

[0188] Based on the above screening results, 20 mM histidine buffer (pH 6.0) and 235 mM sucrose were selected as excipients to conduct research on a high-concentration formulation (concentration approximately 100 mg / ml). The original formulation product was named Drug Substance (DS), and the formulated product obtained by filtering and filling after one freeze-thaw cycle of DS was named Drug Product (DP).

[0189] 3.1 Materials and Methods

[0190] 3.1.1 Freeze-Thaw Stability Study of DS Small Model

[0191] The sample was stored at 2 - 8°C, and ultrafiltration concentration and buffer exchange (UFDF) were performed using Millipore Pellicon3 0.57 m 2 membrane and MilliporePellicon3 1.14 m 2 membrane. After buffer exchange, the sample was in the DS formulation. The DS formulation was: 100 mg / ml antibody CB6, 20 mM histidine buffer (histidine - histidine hydrochloride), 235 mM sucrose, 0.05% polysorbate 80 or 0.03% polysorbate 80, pH 6.0. 30 ml of DS was aseptically filled by hand into 50 ml bags (Millipore), and freeze-thaw stability was studied by SEC-HPLC, CEX-HPLC, CE-SDS, pH, and appearance. The freeze-thaw conditions were: -40°C - 2 - 8°C or room temperature air bath.

[0192] 3.1.2 Freeze-Thaw Stability Study of DS Large Model

[0193] The sample was stored at 2 - 8°C, and Millipore Pellicon3 0.57 m2 Membrane and Millipore Pellicon3 1.14m 2 Ultrafiltration concentration and buffer exchange (UFDF) were performed using the membrane. After buffer exchange, the sample was in the DS formulation. DS formulation: 100 mg / ml antibody CB6, 20 mM histidine buffer, 235 mM sucrose, 0.05% polysorbate 80, pH 6.0. 4 L of DS was filled into a 5 L PC bottle (Nalgene) for freeze-thaw stability study. Freeze-thaw conditions: -80 °C / RT.

[0194] 3.1.3 DS Stability Study

[0195] The sample was stored at 2 - 8 °C and ultrafiltration concentration and buffer exchange (UFDF) were performed using Millipore Pellicon3 0.57m 2 Membrane and Millipore Pellicon3 1.14m 2 Ultrafiltration concentration and buffer exchange (UFDF) were performed using the membrane. After buffer exchange, the sample was in the DS formulation. DS formulation: 100 mg / ml antibody CB6, 20 mM histidine buffer, 235 mM sucrose, 0.05% polysorbate 80, pH 6.0. 10 mL of DS was filled into a 20 mL PC bottle (Nalgene) for stability study at temperatures from -80 °C to 25 °C.

[0196] 3.1.4 DP Forced Degradation Study

[0197] The sample was stored at 2 - 8 °C and ultrafiltration concentration and buffer exchange (UFDF) were performed using Millipore Pellicon3 0.57m 2 Membrane and Millipore Pellicon3 1.14m 2 Ultrafiltration concentration and buffer exchange (UFDF) were performed using the membrane. After buffer exchange, the sample was in the DS formulation. The DS formulation was: 100 mg / ml antibody CB6, 20 mM histidine buffer, 235 mM sucrose, 0.05% polysorbate 80, pH 6.0. After the DS was subjected to one freeze-thaw cycle (freeze-thaw conditions: -40 °C - 2 - 8 °C) and filtration (filter: 0.22 μm, KVGLG10TH1, Millipore), it was aseptically filled by hand into 6R vials (Type I glass, Ompi, Schott), with 2 ml of DS filled into each vial to obtain the DP product. Shaking, light exposure (visible light Vis: 4500 Lux; ultraviolet UV: 90 μw / cm2), and high temperature (40 °C) experiments were carried out respectively, and forced degradation studies were performed by SEC-HPLC, CEX-HPLC, CE-SDS, binding / blocking Elisa, pH, Uv-vis, and appearance.

[0198] 3.1.5 DP Stability Study

[0199] The sample was stored at 2 - 8°C and ultrafiltration concentration and buffer exchange (UFDF) were performed using Millipore Pellicon3 0.57m 2 membrane and Millipore Pellicon3 1.14m 2 membrane. After buffer exchange, the sample was in the DS formulation. The DS formulation was: 100 mg / ml antibody CB6, 20 mM histidine buffer, 235 mM sucrose, 0.05% polysorbate 80, pH 6.0. The DS was subjected to one freeze - thaw cycle (freeze - thaw conditions: - 40°C - 2 - 8°C) and filtration (filter: 0.22 μm, KVGLG10TH1, Millipore), and then aseptically filled by hand into 6R vials (Type I glass, Ompi, Schott), with 2 ml of DS filled into each vial to obtain the DP product; the DP product was placed at 25°C and stability studies were carried out by SEC - HPLC, CEX - HPLC, CE - SDS, binding / blocking ELISA, pH, Uv - vis and appearance

[0200] 3.2 Experimental results

[0201] 3.2.1 Results of freeze - thaw cycle (F - T cycle) of DS small model

[0202] After the freeze - thaw cycle of DS, there were no obvious effects on SEC - HPLC, CEX - HPLC, CE - SDS, pH, etc., showing good stability. The specific results are shown in Table 12

[0203] Table 12: Results of freeze - thaw cycle of DS

[0204]

[0205]

[0206]

[0207] Note: NGHC refers to the antibody isomer without glycosylated heavy chain

[0208] 3.2.2 Freeze - thaw stability of DS large model

[0209] The experimental results of the freeze - thaw stability of the DS large model are shown in Table 13. The results show that there were no significant changes in SEC - HPLC, CE - SDS, pH, Uv - vis and appearance of the sample, showing good stability

[0210] Table 13: Results of freeze - thaw stability study of DS large model

[0211]

[0212]

[0213] Note: "NGHC" refers to the antibody isomer with non-glycosylated heavy chain.

[0214] 3.2.3 DS Stability Study

[0215] The experimental results of the DS stability study are shown in Table 14. The results show that there are no significant changes in SEC-HPLC, CE-SDS, pH, Uv-vis and appearance of the samples, indicating good stability.

[0216] Table 14: Results of the Temperature Study of DS at -80°C to 25°C

[0217]

[0218]

[0219]

[0220] Note: NGHC refers to the antibody isomer with non-glycosylated heavy chain.

[0221] 3.2.4 Results of the DP Forced Degradation Study

[0222] After the DP samples were subjected to shaking, light and high temperature experiments, there were no significant changes in SEC-HPLC, CEX-HPLC, CE-SDS, binding / blocking Elisa, pH, Uv-vis and appearance, indicating good stability. The specific results are shown in Tables 15 - 17.

[0223] Table 15: Results of the DP Shaking Experiment

[0224]

[0225] Note: "NGHC" refers to the antibody isomer with non-glycosylated heavy chain; "NA" indicates that it is a non-key inspection item at this time point and is not detected at this sampling point.

[0226] Table 16: Results of the DP Light Experiment

[0227]

[0228]

[0229] Note: "NGHC" refers to the antibody isomer with non-glycosylated heavy chain; "NA" indicates that it is a non-key inspection item at this time point and is not detected at this sampling point.

[0230] Table 17: Results of the DP High Temperature Experiment

[0231]

[0232]

[0233]

[0234] Note: "NGHC" refers to the antibody isomer with non-glycosylated heavy chain; "NA" indicates that it is a non-key test item at this time point and will not be detected at this sampling point.

[0235] 3.2.5 DP Stability Study Results

[0236] The DP samples showed no significant changes in SEC-HPLC, CEX-HPLC, CE-SDS, binding / blocking ELISA, pH, Uv-vis and appearance, indicating good stability. The specific stability study results are shown in Table 18.

[0237] Table 18: DP Stability Study Results

[0238]

[0239]

[0240] Note: "NGHC" refers to the antibody isomer with non-glycosylated heavy chain; "NA" indicates that it is a non-key test item at this time point and will not be detected at this sampling point.

[0241] Example 4: Binding Specificity and High Binding Activity of Monoclonal Antibody Preparation to RBD of 2019-nCoV Virus S Protein

[0242] Dilute the recombinant SARS-CoV-2 (COVID-19) S protein RBD (Novoprotein, catalog number DRA32) to 3.0 μg / mL for coating, and shake on a microplate shaker for 2 h. Wash the plate and block it with 2% skim milk. Add different concentrations of control antibody (isotype control antibody of IgG1 subtype) and CB6 antibody (diluted 4-fold from 40 μg / mL to 0.009537 ng / mL, prepared according to Prescription 2), incubate for 1 hour and wash the plate. Then incubate with a 1:5000 diluted goat anti-human IgG (Fc-specific) peroxidase antibody (Sigma, catalog number A0170) for 1 hour, and then incubate with HRP substrate TMB (Sigma, catalog number T2885) for 15 minutes for color development to detect the binding signal of the antibody to the RBD of COVID-19 virus S protein. Use the slope curve fitting of logarithm (agonist) to response variable (GraphPad Prism) to fit the EC50.

[0243] The experimental results are shown in Figure 1。By Binding ELISA assay, the CB6 antibody has high binding specificity and binding activity with the recombinant SARS-CoV-2 S protein RBD, and the EC50 is 21.7 ng / mL.

[0244] Example 5: The monoclonal antibody preparation can effectively block the binding of the RBD of the 2019-nCoV virus S protein to its receptor ACE2

[0245] Dilute recombinant human ACE2 (C-6His) (Novoprotein, catalog number C419) to 3.0 μg / mL and coat the plate, incubate at 37 °C for 90 min. Wash the plate and block it with 2% skim milk. Dilute the recombinant SARS-CoV-2 S protein RBD (C-mFc) (Novoprotein, catalog number DRA32) to 1.0 μg / mL with 2% skim milk, and then use the control antibody (isotype control antibody of IgG1 subtype) and CB6 antibody (2-fold serial dilution from 400 μg / mL to 0.2 μg / mL, prepared according to Prescription 2). Add the mixture to the plate and incubate for 1 hour, then wash the plate. Incubate with peroxidase-labeled goat anti-mouse Fc fragment secondary antibody (Sigma, catalog number A2554) diluted 1:5000 for 1 hour, then add TMB (Sigma, catalog number T2885) and incubate for 20 minutes. Use the software GraphPad Prism to fit the IC 50 。

[0246] The experimental results are shown in Figure 2 。By Blocking ELISA assay, the CB6 antibody can effectively block the binding of the RBD of the 2019-nCoV virus S protein to its receptor ACE2, and the IC 50 is 22.8 μg / mL, indicating that the CB6 antibody can inhibit the binding of 2019-nCoV RBD to the coated ACE2 receptor fusion protein.

[0247] Example 6: The monoclonal antibody preparation can effectively block the infection of target cells by pseudovirus

[0248] Mix the 2019-nCoV pseudovirus expressing the full-length 2019-nCoV spike protein and luciferase reporter gene (final concentration of 10000 TCID 50 / ml) with the control antibody (anti-KLH antibody, LALA) or serially diluted CB6 antibody (5-fold serial dilution from 10 μg / ml to 0.128 ng / ml, prepared according to Prescription 2) at a ratio of 1:1 and incubate for 1 h.

[0249] Seed Huh-7 cells at 5×10 per well 4Cells were seeded in a clear-bottom 96-well white wall plate, and then 100 μl of a mixture of antibody and pseudovirus was added to the cells and incubated in an incubator at 37 °C for 24 hours. After incubation, 70 μl of One-Glo TM Firefly luciferase substrate was added to each well, and fluorescence detection was performed using a microplate reader (PerkinElmer / Envision).

[0250] Vero E6 cells were seeded in a clear-bottom 96-well white wall plate at a density of 1×10 4 cells per well, and then placed in an incubator at 37 °C for 3 hours until the cells adhered to the plate. After the cells adhered, 100 μl of a mixture of antibody and pseudovirus was added to the cells and incubated in an incubator at 37 °C for 22 hours. After incubation, 70 μl of One-Glo TM Firefly luciferase substrate was added to each well, and fluorescence detection was performed using a microplate reader (PerkinElmer / Envision).

[0251] Inhibition rate = [1 - (average fluorescence intensity of experimental group - average of blank control) / (average fluorescence intensity of negative control group - average of blank control)] × 100%. The software GraphPad Prism was used to plot the graph and fit the IC 50 .

[0252] The CB6 antibody can effectively inhibit the infection of Huh-7 and Vero E6 cells by the 2019-nCoV pseudovirus, and the IC 50 values are 0.1831 nM (0.02747 μg / ml) and 0.07628 nM (0.01144 μg / ml) respectively. The detailed results are shown in Figure 3 and Figure 4 .

[0253] Example 7 Detection of the neutralization of 2019-nCoV live virus by the antibody of the present invention

[0254] In this study, the neutralizing effect of the CB6 antibody on the 2019-nCoV (SARS-CoV-2) live virus was evaluated by an in vitro neutralization assay.

[0255] 7.1 Reagents

[0256]

[0257] 7.2 Experimental method

[0258] Vero E6 cells were seeded in a clear-bottom 96-well plate at a density of 1×10 5Inoculate at a density into a 96-well culture plate and use after culturing at 37°C for 24 hours. In a 96-well tissue culture plate with DMEM medium, add 50 μl of serially 2-fold diluted CB6 antibody (from 48.8 ng / mL to 100 μg / mL, prepared according to Prescription 3). Then add an equal volume of a SARS-CoV-2 working stock solution containing 200 TCID 50 of SARS-CoV-2, with a final virus titer of 100 TCID50. Incubate the antibody-virus mixture at 37°C for 1 h, then transfer it to a 96-well microtiter plate containing octuplet-fused Vero E6 cells and culture in a CO 2 incubator at 37°C for 3 days. Cells infected with 100 TCID50 of SARS-CoV-2 or cells cultured with control medium (DMEM + 10% FBS) were used as positive control or negative uninfected control, respectively. Observe and record the cytopathic effect (CPE) in each well before and after infection. Perform virus retitration to evaluate the correct virus titer used in the experiment. Calculate the 50% neutralizing dose (ND 50 ) using Prism software. All experiments were conducted in an approved biosafety level 3 facility according to standard operating procedures.

[0259] 7.3 Results and Conclusions

[0260] The neutralizing function of CB6 was evaluated by co-culturing cells with live SARS-CoV-2 virus in the presence of different concentrations of CB6. As Figure 5 shown, CB6 reduced the cytopathic effect of SARS-CoV-2 virus on Vero E6 cells in a dose-dependent manner. The 50% neutralizing dose (ND 50 ) was 5.56 nM.

[0261] CB6 can neutralize live SARS-CoV-2 virus and alleviate the pathological damage of the virus to cells. Sequence Listing <110> Shanghai Junshi Biosciences Co., Ltd. Suzhou Junmeng Biosciences Co., Ltd. <120> Pharmaceutical Composition of a Novel Coronavirus Antibody and Its Use <130> 204497 1PCWO <150> CN 202010535907.3 <151> 2020-06-12 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 1 Ser Asn Tyr Met Ser 1 5 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 2 Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys Gly 1 5 10 15 <210> 3 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 3 Val Leu Pro Met Tyr Gly Asp Tyr Leu Asp Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 4 Arg Ala Ser Gln Ser Ile Ser Arg Tyr Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 5 Ala Ala Ser Ser Leu Gln Ser 1 5 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 6 Gln Gln Ser Tyr Ser Thr Pro Pro Glu Tyr Thr 1 5 10 <210> 7 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> VH <400> 7 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Leu Phe Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Leu Pro Met Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> VL <400> 8 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Glu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 9 <211> 449 <212> PRT <213> Artificial Sequence <220> <223> HC <400> 9 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Tyr Ser Gly Gly Ser Thr Phe Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Met Asn Thr Leu Phe Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Leu Pro Met Tyr Gly Asp Tyr Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 10 <211> 216 <212> PRT <213> Artificial Sequence <220> <223> LC <400> 10 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85 90 95 Glu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn 145 150 155 160 Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser 165 170 175 Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys 180 185 190 Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr 195 200 205 Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 11 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> HCDR1 <400> 11 Gly Phe Thr Val Ser Ser Asn Tyr 1 5 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> HCDR2 <400> 12 Ile Tyr Ser Gly Gly Ser Thr 1 5 <210> 13 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> HCDR3 <400> 13 Ala Arg Val Leu Pro Met Tyr Gly Asp Tyr Leu Asp Tyr 1 5 10 <210> 14 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> LCDR1 <400> 14 Gln Ser Ile Ser Arg Tyr 1 5 <210> 15 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> LCDR2 <400> 15 Ala Ala Ser 1 <210> 16 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> LCDR3 <400> 16 Gln Gln Ser Tyr Ser Thr Pro Pro Glu Tyr Thr 1 5 10

Claims

1. A pharmaceutical composition comprising: (1) A buffer solution, wherein, the buffer solution is a histidine buffer solution with a concentration of 10 - 30 mM and a pH value of 5.5 - 6.0; and (2) A humanized monoclonal antibody or an antigen-binding fragment thereof with a concentration of 20 - 150 mg / mL, wherein the humanized monoclonal antibody specifically binds to 2019-nCoV RBD and has HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 respectively, and LCDR1, LCDR2, and LCDR3 with amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 respectively; (3) A stabilizer, which is mannitol with a concentration of 200 - 300 mM, or sucrose with a concentration of 200 - 300 mM, or trehalose with a concentration of 200 - 300 mM; and (4) Polysorbate 80, wherein, calculated by w / v, the concentration of polysorbate 80 is 0.02% - 0.08%.

2. The pharmaceutical composition according to claim 1, wherein, the humanized monoclonal antibody has a heavy chain variable region with an amino acid sequence shown in SEQ ID NO:7 and a light chain variable region with an amino acid sequence shown in SEQ ID NO:

8.

3. The pharmaceutical composition according to claim 1, wherein, the humanized monoclonal antibody has a heavy chain amino acid sequence shown in SEQ ID NO:9 and a light chain amino acid sequence shown in SEQ ID NO:

10.

4. The pharmaceutical composition according to claim 1, wherein the concentration of the humanized monoclonal antibody or its antigen-binding fragment is 40 - 120 mg / mL.

5. The pharmaceutical composition according to claim 1, wherein the concentration of the humanized monoclonal antibody or its antigen-binding fragment is 40 - 100 mg / mL.

6. The pharmaceutical composition according to claim 1, which comprises the components shown in any one of the following (1) - (3): (1) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 40 mg / mL - 120 mg / mL; (b) A 10 - 30 mM histidine buffer solution with a pH of 5.5 - 6.0; (c) 200 - 300 mM of mannitol; (d) and 0.02% - 0.08% of polysorbate 80; or (2) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 40 mg / mL - 120 mg / mL; (b) A 10 - 30 mM histidine buffer solution with a pH of 5.5 - 6.0; (c) 200 - 300 mM of sucrose; (d) and 0.02% - 0.08% of polysorbate 80; or (3) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 40 mg / mL - 120 mg / mL; (b) A 10 - 30 mM histidine buffer solution with a pH of 5.5 - 6.0; (c) 200 - 300 mM of trehalose; (d) and 0.02% - 0.08% of polysorbate 80.

7. The pharmaceutical composition according to claim 1, which comprises the components shown in any one of the following (1) to (7): (1) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 40 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Mannitol at 235 mM or 247 mM; (d) And 0.02% polysorbate 80; or (2) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 40 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Sucrose at 235 mM; (d) And 0.02% polysorbate 80; or (3) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 40 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Trehalose at 240 mM; (d) And 0.02% polysorbate 80; or (4) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 80 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Sucrose at 235 mM; (d) And 0.02% polysorbate 80; or (5) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 80 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Trehalose at 240 mM; (d) And 0.02% polysorbate 80; or (6) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 100 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Sucrose at 235 mM; (d) And 0.03% polysorbate 80; or (7) (a) A humanized monoclonal antibody or an antigen-binding fragment thereof at 100 mg / mL; (b) 20 mM histidine buffer with a pH of 6.0; (c) Sucrose at 235 mM; (d) And 0.05% polysorbate 80.

8. An injection, which contains the pharmaceutical composition according to any one of claims 1 to 7 and 0.9% sodium chloride solution.

9. The injection according to claim 8, wherein, the concentration of the humanized monoclonal antibody is 1 to 40 mg / mL.

10. The injection according to claim 8, wherein, the pH of the injection is 5.5 to 6.

0.

11. Use of the pharmaceutical composition according to any one of claims 1 to 7 or the injection according to any one of claims 8 to 10 in the preparation of a drug for treating or preventing 2019-nCoV infection.

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