WO2021249548A1

WO2021249548A1 - Pharmaceutical composition of novel coronavirus antibody and use thereof

Info

Publication number: WO2021249548A1
Application number: PCT/CN2021/099780
Authority: WO
Inventors: 刘洪川; 刘沛想; 吴纯; 李园园; 潘隽; 张静; 李理; 周岳华; 冯辉; 姚盛
Original assignee: 上海君实生物医药科技股份有限公司; 苏州君盟生物医药科技有限公司
Priority date: 2020-06-12
Filing date: 2021-06-11
Publication date: 2021-12-16
Also published as: CN115666649A; CN113797333A

Abstract

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

Description

A new type of coronavirus antibody pharmaceutical composition and its use

Technical field

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

Background technique

As of June 9, 2020, the global confirmed cases of the new coronavirus COVID-19 (2019-nCoV) have exceeded 7 million, and the total number of deaths has exceeded 400,000, posing a major threat to the life and health of the public.

However, there is currently no specific medicine for this virus.

2019-nCoV is a coronavirus. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), both of which are coronaviruses, also caused epidemics in 2002-2003 and 2012, respectively. According to World Health Organization (WHO) statistics, SARS-CoV caused a total of 8,000 infections and 794 deaths (https://www.who.int/). Since 2012, MERS-CoV infection cases have continued to increase. As of the end of 2019, there were 2,499 confirmed cases of infection and 861 deaths worldwide. On January 12, 2020, the World Health Organization officially named the new type of coronavirus "2019-nCoV (2019-nCoV)", and then on February 11-12, 2020, the International Committee on Taxonomy (International Committee on Taxonomy) of Viruses, ICTV) announced that the official classification of the new type of coronavirus is named severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), and the World Health Organization (WHO) held a global study in Geneva on the same day He Innovation Forum announced that the official name of the disease caused by this virus is "COVID-19".

In order for a virus to infect a cell, it first needs to bind to the host's receptor through an envelope protein. Antibodies, especially neutralizing active antibodies, bind to envelope proteins to block the binding of the virus to cell receptors, thereby blocking viral infection. At the same time, the antibody binds to the envelope protein to label free viruses or infected cells, and recruit immune cells and immune molecules such as macrophages or complement through the Fc region of the antibody, thereby eliminating free viruses and infected cells. Infected cells. Therefore, antibodies targeting the receptor binding region (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.

Based on 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 RBD. Through research on 2019-nCoV, the present invention found that CTD is the RBD of 2019-nCoV, which binds to the receptor ACE2. Therefore, an antibody that targets RBD, and is an antibody that blocks the binding of S to ACE2, may become a neutralizing antibody that inhibits viral infection. The purpose of the present invention is to provide specific human-derived neutralizing antibodies with protective effects against 2019-nCoV.

Summary of the invention

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

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

In some aspects, the concentration of the humanized monoclonal antibody or antigen-binding fragment thereof 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; more preferably about 40-100 mg/mL; more preferably, the concentration of the above-mentioned humanized monoclonal antibody or antigen-binding fragment thereof is about 5 mg/mL, 10 mg/mL, 15 mg /mL, 20mg/mL, 25mg/mL, 30mg/mL, 35mg/mL, 40mg/mL, 45mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL , 110mg/mL, 120mg/mL, 130mg/mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL or 200mg/mL, preferably about 40mg/mL, 60mg/mL, 70mg/ mL, 80mg/mL, 90mg/mL, 95mg/mL, 100mg/mL, 105mg/mL, 110mg/mL, 120mg/mL.

In some embodiments, the above-mentioned buffer is selected from one or more of acetate buffer, citrate buffer, and histidine buffer.

In some solutions, the above-mentioned buffer is a histidine buffer, preferably, the histidine buffer is selected from histidine-hydrochloride buffer or histidine-acetate buffer, preferably histidine Acid-hydrochloride buffer.

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

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

In some schemes, the above-mentioned histidine buffer is a histidine-acetate buffer, preferably, the molar ratio of the two is 1:1 to 1.5:1, and preferably, the pH of such a buffer is 6.0 ±0.3, preferably about 6.0, preferably, this type of buffer contains 18-22 mM histidine or 18-22 mM acetic acid.

In some solutions, 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.

In some solutions, the above-mentioned buffer is a citric acid buffer, and preferably, the citric acid buffer is a citric acid-sodium citrate buffer.

In some solutions, the above-mentioned buffer is a succinic acid buffer, and preferably, the succinic acid buffer is a succinic acid-sodium succinate buffer.

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

In some embodiments, the pH of the aforementioned buffer is about 5.0-6.5, preferably about 5.0-6.0, preferably about 5.5-6.5, preferably about 5.0-5.5, preferably about 5.5-6.0, preferably about 6.0-6.5 , A non-limiting example of the pH of the above buffer is 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.

In some aspects, the aforementioned pharmaceutical composition further includes a stabilizer.

In some aspects, the stabilizer is selected from one or more of arginine hydrochloride, proline, glycine, sodium chloride, mannitol, sorbitol, sucrose, maltose, xylitol, and trehalose.

In some aspects, the aforementioned stabilizer is selected from one or more of mannitol, sucrose and trehalose.

In some embodiments, the concentration of the aforementioned stabilizer is about 10 mM to 400 mM, preferably about 50 mM to 300 mM, more preferably about 100 mM to 300 mM, more preferably about 200 mM to 300 mM.

In some embodiments, the stabilizer is sodium chloride with a concentration of about 30-200 mM; or the stabilizer is sodium chloride with a concentration of about 50-200 mM; or the stabilizer is mannitol with a concentration of about 100-300 mM; Or the stabilizer is sorbitol with a concentration of about 100-300 mM; or the stabilizer is sucrose with a concentration of about 100-300 mM; or the stabilizer is trehalose with a concentration of about 100-300 mM; or the stabilizer is 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 at a concentration of About 200-300 mM mannitol, or about 200-300 mM sucrose, or about 200-300 mM trehalose.

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

In some aspects, the aforementioned stabilizer is mannitol. In some embodiments, the aforementioned stabilizer is mannitol at a concentration of about 100-300 mM. The concentration of the aforementioned mannitol is preferably about 150-300 mM, preferably about 180-280 mM, preferably about 200-260 mM. Limiting examples 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, preferably 235 mM.

In some aspects, the aforementioned stabilizer is sorbitol. In some embodiments, the aforementioned stabilizer is sorbitol at a concentration of about 100-300 mM. The concentration of the aforementioned sorbitol is preferably about 150-300 mM, preferably about 180-280 mM, preferably about 200-260 mM. Limiting examples are about 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 235 mM.

In some aspects, the aforementioned stabilizer is sucrose. In some embodiments, the aforementioned stabilizer is sucrose at a concentration of about 100-300 mM. The concentration of the aforementioned sucrose is preferably about 150-300 mM, preferably about 180-280 mM, preferably about 200-260 mM. The above-mentioned sucrose concentration is a non-limiting implementation Examples are about 200 mM, 210 mM, 220 mM, 230 mM, 235 mM, 240 mM, 245 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 235 mM.

In some aspects, the aforementioned stabilizer is trehalose. In some embodiments, the aforementioned stabilizer is trehalose at a concentration of about 100-300 mM. The concentration of the aforementioned trehalose is preferably about 150-300 mM, preferably about 180-280 mM, preferably about 200-260 mM. Limiting examples 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, preferably 235 mM.

In some aspects, the aforementioned stabilizer is arginine hydrochloride. In some embodiments, the aforementioned stabilizer is arginine hydrochloride at a concentration of about 30-200 mM, and the concentration of the aforementioned arginine hydrochloride is preferably about 50-190 mM, preferably about 100-180 mM, preferably about 120-170 mM, preferably about It is 130-150mM, the non-limiting example of the above-mentioned arginine hydrochloride concentration is about 100mM, 110mM, 120mM, 125mM, 130mM, 135mM, 140mM, 145mM, 150mM, 155mM, 160mM, 170mM, 180mM, 190mM, 200mM, preferably 135mM or 140mM.

In some aspects, the aforementioned stabilizer is proline. In some embodiments, the aforementioned stabilizer is proline at a concentration of about 100-300 mM. The concentration of the aforementioned proline is preferably about 150-300 mM, preferably about 200-280 mM. A non-limiting example of the aforementioned proline concentration It is about 180 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM, 260 mM, 270 mM, 280 mM, preferably 240 mM.

In some aspects, the aforementioned stabilizer is glycine. In some embodiments, the aforementioned stabilizer is glycine at a concentration of about 100-300 mM. The concentration of the aforementioned glycine is preferably about 150-300 mM, preferably about 200-280 mM. A non-limiting example of the aforementioned glycine concentration is about 180 mM, 200 mM. , 210mM, 220mM, 230mM, 240mM, 250mM, 260mM, 270mM, 280mM, preferably 260mM.

In some aspects, the above-mentioned pharmaceutical composition further includes a surfactant, and the surfactant is selected from polysorbate 80, polysorbate 20, or poloxamer 188.

In some aspects, the above-mentioned surfactant is selected from polysorbate 80.

In some aspects, the above-mentioned surfactant is selected from polysorbate 20.

In some solutions, calculated by w/v, the concentration of the above-mentioned surfactant is about 0.001%-0.1%, preferably about 0.01%-0.1%, preferably about 0.02%-0.08%; as a non-limiting example, the above-mentioned surfactant concentration The concentration of the surfactant is about 0.02%, 0.03%, 0.04%, 0.05%, 0.06% or 0.08%.

In some schemes, the aforementioned humanized monoclonal antibody or antigen-binding fragment thereof has the amino acid sequence of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 7, and the amino acid sequence of HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 8. The amino acid sequences of LCDR1, LCDR2 and LCDR3 of the light chain variable region are shown.

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

In some aspects, the above-mentioned humanized monoclonal antibody has a heavy chain variable region whose amino acid sequence is shown in SEQ ID NO: 7 and a light chain variable region whose amino acid sequence is shown in SEQ ID NO: 8.

In some aspects, the aforementioned 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.

In some embodiments, the above-mentioned pharmaceutical composition comprises the following components shown in any one of (1) to (6), wherein the humanized monoclonal antibody or antigen-binding fragment thereof is as described in any one of the embodiments of the present invention :

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

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

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

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

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

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

Preferably, the pharmaceutical composition comprises the following components shown in any one of (7) to (9), wherein the humanized monoclonal antibody or antigen-binding fragment thereof is as described in any of the embodiments of the present invention:

(7) (a) Humanized monoclonal antibody or its antigen-binding fragment of about 40mg/mL-120mg/mL; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) About 200-300mM mannitol; (d) and about 0.02%-0.08% polysorbate 80; or

(8) (a) Humanized monoclonal antibody or its antigen-binding fragment of about 40mg/mL-120mg/mL; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) About 200-300mM sucrose; (d) and about 0.02%-0.08% polysorbate 80; or

(9) (a) Humanized monoclonal antibody or its antigen-binding fragment of about 40mg/mL-120mg/mL; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) About 200-300mM trehalose; (d) and about 0.02%-0.08% polysorbate 80.

More preferably, the pharmaceutical composition comprises the following components shown in any one of (10) to (16), wherein the humanized monoclonal antibody or antigen-binding fragment thereof is as described in any of the embodiments of the present invention :

(10) (a) About 40 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM mannitol or about 247 mM Mannitol; (d) and about 0.02% polysorbate 80; or

(11) (a) About 40 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM sucrose; (d) And about 0.02% polysorbate 80; or

(12) (a) About 40 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 240 mM trehalose; (d) ) And about 0.02% polysorbate 80; or

(13) (a) about 80mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) about 20mM histidine buffer, pH about 6.0; (c) about 235mM sucrose; (d) And about 0.02% polysorbate 80; or

(14) (a) About 80 mg/mL humanized monoclonal antibody or antigen-binding fragment thereof; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 240 mM trehalose; (d) ) And about 0.02% polysorbate 80; or

(15) (a) About 100 mg/mL humanized monoclonal antibody or antigen-binding fragment thereof; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM sucrose; (d) And about 0.03% Polysorbate 80; or

(16) (a) About 100 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM sucrose; (d) And about 0.05% polysorbate 80.

In some aspects, the pharmaceutical composition is a liquid formulation or a lyophilized formulation.

In some aspects, the pharmaceutical composition is a liquid formulation.

In some scenarios, the above-mentioned liquid formulation or lyophilized formulation 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.

In some protocols, the above-mentioned aqueous solution or lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

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

The invention also provides the use of the above-mentioned pharmaceutical composition or injection in the preparation of a medicine for treating or preventing COVID-19 infection.

The present invention also provides a histidine buffer and one or more stabilizers selected from mannitol, sucrose and trehalose, and optional surfactants (preferably polysorbate 80) to improve the specificity of COVID-19 Application in the stability of a pharmaceutical preparation of a sexually bound humanized monoclonal antibody, or application in the preparation of a pharmaceutical preparation of a humanized monoclonal antibody that specifically binds to COVID-19 with improved stability. Preferably, the histidine buffer, stabilizer, surfactant and the amount thereof are as described in any embodiment herein; the stability improvement is as described in any embodiment herein.

Description of the drawings

Figure 1: Binding ELISA analysis of humanized monoclonal antibody CB6 (batch number 20200307) and 2019-nCoV RBD protein.

Figure 2: Blocking ELISA analysis of humanized monoclonal antibody CB6 (batch number 20200307) and 2019-nCoV RBD protein.

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

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

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

Definition and description

To make it easier to understand 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 belongs. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in this application are only used to describe specific implementations and are not intended to be limiting.

Unless the content is clearly stated otherwise, the singular forms "a", "an" and "the" used in this specification and the appended claims include plural references. Thus, for example, reference to "a polypeptide" includes a combination of two or more polypeptides and the like.

The term "pharmaceutical composition" or "formulation" means a mixture containing one or more of the antibodies described herein and other components, such as physiologically pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredients and then exert the biological activity.

The term "liquid formulation" refers to a formulation in a liquid state, and is not intended to refer to a lyophilized formulation that is resuspended. The liquid formulation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state change methods, such as spray drying).

The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some aspects, an aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and/or freezing to maintain stability (e.g., chemical and/or physical stability and/or biological activity).

The term "excipient" refers to an agent that can be added to a formulation to provide desired characteristics (e.g., consistency, increased stability) and/or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

The "about" used in this application when referring to a measurable value (such as amount, duration, etc.) is intended to cover a variation of ±20% or ±10% relative to a specific value, including ±5%, ±1%, and ±0.1 %, because these changes are suitable for carrying out the disclosed method.

The term "buffer pH is about 5.0-6.5" refers to a reagent whose acid/base conjugated component makes the solution containing the reagent resistant to pH changes. The buffer used in the formulation of the present invention may have a pH in the range of about 5.0 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

As used herein, examples of "buffer" for controlling the pH within this range 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.

"Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, etc., such as containing histidine The histidine buffer of acid and histidine hydrochloride; the histidine buffer of the present invention also includes histidine buffer containing histidine and acetate (such as sodium salt or potassium salt).

"Citrate buffer" is a buffer that includes citrate ions. Examples of the citric acid buffer include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. The preferred citrate buffer is citric acid-sodium citrate buffer.

"Acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. The preferred acetate buffer is acetic acid-sodium acetate buffer.

"Succinate buffer" is a buffer that includes succinate ions. Examples of the succinate buffer include succinate-sodium succinate, succinate-potassium succinate, succinate-calcium succinate, succinate-magnesium succinate, and the like. The preferred succinate buffer is succinate-sodium succinate buffer.

The term "w/v" means mass volume concentration, such as "0.9%" in "0.9% (w/v) sodium chloride solution" means "100 mL of solution contains 0.9 g of solute".

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 manufacture, 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 Acid, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines, alanine, octopine, glycinine (strombine) and trimethylamine N- Oxide (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 stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. The preferred sugars are sucrose and trehalose, and the preferred polyol is 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 is more preferably arginine hydrochloride-sucrose. In a particularly preferred embodiment, the stabilizer used in the present invention is selected from one or more of mannitol, sucrose and trehalose.

The term "surfactant" generally includes agents that protect proteins, such as antibodies, from stress induced by the air/solution interface, solution/surface-induced stress to reduce antibody aggregation or minimize the formation of particulates 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 copolymers, poly Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ether, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (Pluronic), sodium dodecyl sulfate (SDS). In a particularly preferred embodiment, the surfactant used in the present invention is polysorbate 80.

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

The term "stable" formulation is a formulation in which the antibody substantially maintains its physical and/or chemical stability and/or biological activity during the manufacturing process and/or during storage. Even if the contained antibody fails to maintain 100% of its chemical structure or biological function after a certain period of storage, the pharmaceutical preparation can be stable. In some cases, after a certain period of storage, it can maintain about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the structure or function of the antibody, which can also be considered as " stable". Various analytical techniques for measuring protein stability are available in this technical field, and are reviewed in "Peptide and Protein Drug Delivery" 247-301, Vincent Lee Editor-in-Chief, Marcel Dekker, Inc. ., New York, NY, Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both are incorporated by reference).

After the preparation has been stored at a certain temperature for a certain period of time, its stability can be measured by determining the percentage of natural antibodies remaining in it (and other methods). Among other methods, the percentage of natural antibodies can be measured by size exclusion chromatography (such as size exclusion high performance liquid chromatography [SEC-HPLC]), where "natural" refers to unaggregated and undegraded. In some schemes, the stability of the protein is determined in terms of the percentage of monomeric protein in a solution that has a low percentage of degradation (e.g., fragmentation) and/or aggregated protein. In some scenarios, the formulation can be stored stably 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, no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% antibody in aggregate form at most.

By determining the percentage (and other methods) of antibody (and other methods) that migrate in the more acidic fraction of this antibody main fraction ("mainly charged form") during ion exchange, stability can be measured, where stability is the same as The percentage of acidic form of antibody is inversely proportional. Among other methods, the percentage of "acidified" antibodies can be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC]). In some embodiments, an acceptable degree of stability means that when the formulation is stored at a certain temperature for a certain period of time, the acidic form of the antibody that can be detected therein does not exceed about 49%, 45%, 40%, 35 %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%. The certain period of storage 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, a certain temperature that allows the storage of pharmaceutical preparations can be any temperature in the range of about -80°C to about 45°C, for example, storage at about -80°C, about -30°C, about -20°C, or about 0°C , About 2-8°C, about 5°C, about 25°C, or about 40°C.

If the antibody does not substantially show signs of aggregation, precipitation, and/or denaturation during visual inspection of color and/or clarity or by UV light scattering or by pore exclusion chromatography, then the antibody is in the drug combination The substance "maintains its physical stability". Aggregation is the process by which single molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation can proceed to the point where visible precipitates are formed.

The stability of the formulation, such as physical stability, can be evaluated by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurement is related to the turbidity of the formulation. The turbidity of a formulation is partly an inherent property of the protein dissolved in the solution, and is usually measured by turbidimetry and measured in turbidity units (NTU).

The level of turbidity that varies with, for example, the concentration of one or more components in the solution (eg, protein and/or salt concentration) is also referred to as the "opacity" or "opaque appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard used to determine the turbidity level of the pharmaceutical composition can be based on the "European Pharmacopoeia" standard ("European Pharmacopoeia", fourth edition, "Directorate for the Quality of Medicine" of the Council of Europe (EDQM), Strasbourg, France). According to the "European Pharmacopoeia" standard, a clear solution is defined as a solution with a turbidity lower than or equal to the turbidity of a reference suspension of about 3 according to the "European Pharmacopoeia" standard. Turbidity measurement by turbidimetry can detect Rayleigh scattering in the absence of association or non-ideal effects, which usually varies linearly with concentration. Other methods for assessing physical stability are well known in the art.

If the chemical stability of the antibody at a given point in time is such that the antibody is considered to retain its biological activity as defined below, the antibody "retains its chemical stability" in the pharmaceutical composition. The chemical stability can be assessed by, for example, detecting or quantifying the form of chemical changes in the antibody. Chemical changes can include size changes (e.g., cropping), which can be assessed 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 changes include charge changes (for example, occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

If the antibody in the pharmaceutical composition is biologically active for its intended purpose, the antibody "retains its biological activity" in the pharmaceutical composition. For example, if the preparation is stored at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (for example, 1 to 12 months), the binding affinity of the humanized monoclonal antibody contained in the preparation to COVID-19 is as described If the binding affinity of the antibody is at least 90%, 95% or more before storage, it can be considered that the preparation of the present invention is stable. The binding affinity can also be determined using techniques such as ELISA or plasmon resonance.

In the context of the present invention, in a pharmacological sense, the "therapeutically effective amount" or "effective amount" of the antibody refers to an amount effective in preventing or treating or alleviating the symptoms of a disorder that the antibody can effectively treat. In the present invention, the "therapeutically effective dose" or "therapeutically effective dose" of the drug is any amount of drug that protects the subject from the onset of disease or promotes the regression of the disease when used alone or in combination with another therapeutic agent. The regression of the disease is evidenced by a reduction in the severity of the symptoms of the disease, an increase in the frequency and duration of the asymptomatic period of the disease, or the prevention of injury or disability caused by the pain of the disease. The ability of drugs to promote disease regression 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 that predict human efficacy, or by in vitro assays The activity of the agent is determined. A therapeutically effective amount of a drug includes a "prophylactically effective amount", that is, any amount that inhibits the development or recurrence of the disease when administered to a subject at risk or a subject who has a recurrence of the disease, alone or in combination with other therapeutic drugs Drug.

The term "subject" or "patient" is 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 invention, the subject is a human.

The terms "administering", "administering" and "treating" refer to the introduction of a composition containing a therapeutic agent into a subject using any of various methods or delivery systems known to those skilled in the art. The administration route of the humanized monoclonal antibody or its antigen-binding fragment to which 2019-nCoV specifically binds includes intravenous, intramuscular, subcutaneous, peritoneal, spinal, or other parenteral administration routes, such as injection or infusion. "Parenteral administration" refers to administration methods usually by injection other than enteral or local administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, and intrasaccular , Intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspine, intradural and intrasternal injection and infusion, and intracorporeal electroporation.

antibody

The term "antibody" as used herein should be understood to include intact antibody molecules and antigen-binding fragments thereof. As used herein, the term “antigen-binding portion” or “antigen-binding fragment” (or simply “antibody portion” or “antibody fragment”) of an antibody refers to the antibody that retains 2019-nCoV (2019-novel coronavirus) or its One or more fragments of epitope-specific binding ability.

The term "full-length antibody" or "complete antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70kDa at full length) and two light (L) chains (Approximately 25kDa at full length) are connected to each other by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region (abbreviated as CH herein). The heavy chain constant region is composed of three structural domains CH1, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The constant region of the light chain consists of a domain CL. The VH and VL regions can be further subdivided into complementarity determining regions (CDR) with high variability and more conservatively spaced regions called framework regions (FR). Each VH or VL region consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the antibody can mediate the binding of immunoglobulins to host tissues or factors (including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system).

As used herein, the term "CDR" refers to the complementarity determining region within the variable sequence of an antibody. There are 3 CDRs in each variable region of the heavy chain and light chain, which are named HCDR1, HCDR2, and HCDR3 or LCDR1, LCDR2, and LCDR3 for each variable region of the heavy chain and light chain. The precise boundaries of these CDRs have different definitions according to different systems.

The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the present invention can be determined using any one of many well-known schemes, including Kabat et al. (1991), "Sequences of Proteins of Immunological Interest, No. 5 Version, Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering plan) described Kabat scheme and Lefranc M.-P. et al. described IMGT scheme (1999 Nucleic Acids Research, 27, 209-212).

As used herein, "antigen-binding fragment" includes antibody fragments or derivatives thereof, and generally includes at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody, which retains at least some of the parent antibody Binding specificity. 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 (nanobody) And multispecific antibodies. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or derivative thereof generally retains at least 10% of the antigen binding activity of the parent antibody. Preferably, the binding fragment or derivative thereof retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or more of the antigen binding affinity of the parent antibody. It is also expected 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).

When referring to ligand/receptor, antibody/antigen or other binding pairs, "specific" binding refers to determining whether the protein is present in a heterogeneous population of proteins and/or other biological agents. For example, the binding reaction between the monoclonal antibody of the present invention and the 2019-nCoV RBD protein. Therefore, under the specified conditions, a specific ligand/antigen binds to a specific receptor/antibody, and does not bind to other proteins present in the sample in a significant amount.

The humanized monoclonal antibodies or antigen-binding fragments thereof described herein include any one of the humanized monoclonal antibodies or antigen-binding fragments described in the application number CN202010114283.8, and the entire contents disclosed herein are incorporated herein Ways are included in this article. In some aspects, the CDR sequences of the antibodies used in the methods and compositions of the present invention include CDR sequences from the antibody CB6 described in CN202010114283.8. In some aspects, the CDR sequences of the antibodies used in the methods and compositions of the invention include the variable region sequences from the antibody CB6 described in CN202010114283.8. In some schemes, the antibodies used in the methods and compositions of the present invention are derived from the variable region sequence of the antibody CB6 described in CN202010114283.8. The expression vector is constructed by conventional techniques and expressed in cells to prepare the obtained human Sourced monoclonal antibody.

The non-limiting and exemplary antibody used in the examples herein is selected from the humanized antibody CB6 described in CN202010114283.8, which can specifically bind to 2019-nCoV and RBD. Among them, the antibody CB6 has an amino acid sequence of HCDR1, HCDR2, and HCDR3 consisting of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 7, and a light chain having an amino acid sequence of SEQ ID NO: 8 consisting of HCDR1, HCDR2, and HCDR3. The LCDR1, LCDR2, and LCDR3 of the variable region are composed of LCDR1, LCDR2, and LCDR3; according to the "Kabat" numbering scheme, the antibody CB6 has amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6 respectively; according to the IMGT scheme, antibody CB6 has amino acid sequences as shown in SEQ ID NO: 11. HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 12 and SEQ ID NO: 13, and LCDR1, LCDR2 shown in SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively. And LCDR3; preferably, the antibody CB6 has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8; preferably, the antibody CB6 has the amino acid sequence shown in SEQ ID The heavy chain amino acid sequence shown in NO: 9 and the light chain amino acid sequence shown in SEQ ID NO: 10.

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

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

Medicinal preparations

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

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

For example, in some scenarios, the humanized monoclonal antibody antibody in the pharmaceutical composition of the present invention has the amino acid sequence of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 7, and the amino acid sequence as shown in SEQ ID The amino acid sequence of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in NO: 8; or HCDR1, HCDR2 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively. And HCDR3, and LCDR1, LCDR2 and LCDR3 as 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 amino acids The sequence of the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8; more preferably, the humanized monoclonal antibody in the pharmaceutical composition of the present invention has differences The heavy chain amino acid sequence as shown in SEQ ID NO: 9 and the light chain amino acid sequence as shown in SEQ ID NO: 10.

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

The buffer in the pharmaceutical composition of the present invention can be selected from acetate buffer, citrate buffer and histidine buffer, to provide about 5.0 to 6.5, preferably about 5.0 to 6.0, more preferably about 5.0 to 6.5 for the pharmaceutical composition of the present invention. A pH of 5.5-6.0, more preferably about 6.0. 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, more preferably about 6.0.

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

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

In some aspects, the pharmaceutical composition of the present invention also contains a stabilizer. Preferably, the stabilizer is selected from one or more of 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 to 400 mM, preferably 50 mM to 300 mM, more preferably 100 mM to 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 at a concentration of about 100-300 mM , Preferably about 200-300 mM sucrose; or the stabilizer is about 100-300 mM, preferably about 200-300 mM trehalose.

Therefore, 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; About 40-120 mg/mL, preferably about 40-100 mg/mL, the humanized monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, especially the CB6 antibody or antigen-binding fragment thereof described herein; and About 100mM-300mM stabilizer, preferably, the stabilizer includes one of mannitol, sodium chloride, sucrose and trehalose, preferably about 100-300mM mannitol, about 100-300mM sucrose and about 100 -300mM trehalose. In some embodiments, the stabilizer is about 200-300 mM sucrose. In some embodiments, the stabilizer is about 200-300 mM trehalose. In some embodiments, the stabilizer is about 200-300 mM mannitol.

In some aspects, the pharmaceutical composition of the present invention also includes a surfactant. Preferred surfactants are selected from polysorbate 80, polysorbate 20 and poloxamer 188. The most preferred surfactant is polysorbate 80. 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 a non-limiting example, 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%.

Therefore, 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; 40-120 mg/mL, preferably about 40-100 mg/mL, the humanized monoclonal antibody or antigen-binding fragment thereof according to any one of the preceding embodiments, especially the CB6 antibody or antigen-binding fragment thereof described herein; about 100 mM -300 mM stabilizer, preferably, the stabilizer is about 100-300 mM sucrose, or about 100-300 mM mannitol, or about 100-300 mM trehalose; and, on a w/v basis, about 0.02% -0.08% Polysorbate 80.

The pharmaceutical composition of the present invention may be a liquid preparation or a lyophilized preparation.

Medical uses and methods

The present invention also provides the pharmaceutical composition or injection according to any embodiment of the present invention for the treatment or prevention of 2019-nCoV infection-related diseases. The pharmaceutical composition or injection according to any embodiment of the present invention is used for preparing treatment or Use in drugs for preventing 2019-nCoV infection-related diseases, and a method for administering a therapeutically effective amount of the pharmaceutical composition or injection according to any embodiment of the present invention to an individual or patient in need to treat or prevent 2019-nCoV infection-related diseases .

In the present invention, diseases related to 2019-nCoV infection refer to diseases that occur and develop due to 2019-nCoV infection.

Hereinafter, the present invention will be explained in the form of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the present invention. Within the scope of protection. Unless otherwise specified, the methods and materials used in the examples are conventional methods and materials in the art.

Example 1: Buffer system, pH, excipients, protein concentration screening experiment

In the liquid pharmaceutical composition, the buffer system, pH, excipients, and protein concentration closely affect the stability of the antibody. Each antibody with unique physical and chemical properties has the most suitable buffer type, pH and excipient conditions. The purpose of this example is to screen an optimal buffer system, pH and auxiliary materials, so that the humanized antibody disclosed in the present invention has the best stability for clinical application.

This example was performed with antibody CB6 at concentrations of approximately 20 mg/mL and 40 mg/mL. The sample was concentrated by ultrafiltration with Millipore Pellicon3 0.11m ² membrane and the solution was exchanged. After the solution was changed, the sample was placed in the corresponding prescription, and the sample was placed in a closed centrifuge tube for buffer screening. The buffer system screened acetate buffer, citrate buffer and histidine buffer, pH from 5.5 to 6.0 (as shown in Table 1). The excipients were screened with sodium chloride, sucrose, trehalose or mannitol for comparative testing. That is, the above-mentioned different excipients were added to the buffer solution containing the antibody CB6 at a concentration of about 20 mg/mL or 40 mg/mL. The specific prescription information is shown in Table 1. Place the samples in an environment of 40±2°C, and take them out at the 0th week, the 2nd week, and the 4th week for analysis and testing.

The main pathway of protein degradation is the formation of aggregates, lysate products and charged variants. Size exclusion chromatography (SEC-HPLC) was used to determine the percentage of natural form (protein monomer) and aggregate form, and cation exchange chromatography (CEX-HPLC) was used to determine the percentage of acid and basic forms of antibodies. The SEC-HPLC monomer content and the CEX-HPLC main peak content of four weeks (4W) were used to fit a straight line and calculate the decline slope (%/week) to investigate the effects of different buffer systems, pH, excipients, and protein concentrations on the stability of antibody CB6 Influence.

The stability is evaluated by the following parameters: (1) visual appearance and visible foreign matter; (2) UV spectrophotometric method to determine protein content; (3) SEC-HPLC to measure the content of antibody monomers, aggregates or fragments; (4) CEX -HPLC to measure the main charge, acid charge or basic charge content of the antibody; (5) NR-CE-SDS method to detect the molecular weight of the antibody; (6) R-CE-SDS method to detect the molecular weight of the antibody; (7) ELISA method to detect the antibody Binding activity.

Table 1: Prescription information in the screening experiment of buffer system, pH, excipients and protein concentration

The experimental results are shown in Table 2.

Recipe 1 and Recipe 5 have heavier opalescence under the condition of protein concentration of 40mg/ml, and the concentration of 20mg/ml has no abnormality. The protein content and appearance of other prescriptions of 20mg/ml and 40mg/ml have no abnormality, indicating sodium chloride and pH6 .0 The citric acid buffer system is not suitable for the high concentration stability of this product.

In the SEC-HPLC test, under the accelerated condition of 40±2℃, the monomer content of prescription 5 declined faster, and the monomer content of

prescriptions

2, 3 and 4 decreased relatively slowly, with an average rate of decrease of 0.4%/week. Protein concentration at 20mg/ml and 40mg/ml has little effect on purity.

The results of the CEX-HPLC decline rate showed that under the accelerated conditions of 40±2°C, the content of the main peak of CEX in prescription 5 and prescription 6 decreased rapidly. The decrease rate of CEX main peak content of

prescription

1, 2, 3 and 4 is relatively low, and the protein concentration has little effect on the purity under the conditions of 20mg/ml and 40mg/ml.

The binding activity (Elisa method) and the purity of NR-CE-SDS were not abnormal, and the purity of R-CE-SDS was slightly decreased.

Table 2: Buffer system, pH, excipients and protein concentration screening experiment results

Based on various test data, comparing prescription 1 to prescription 4, it is found that the excipients mannitol, sucrose and trehalose are better than sodium chloride. The comparison of prescription 2 and prescription 5 shows that the histidine buffer system is better than the citrate buffer system. The latter has heavier opalescence; the pH5.5 acetic acid buffer system contains the excipient mannitol (prescription 6), and the SEC-HPLC purity and CEX main peak decline faster. Therefore, the pH6.0 histidine buffer system contains excipients Under the conditions of mannitol, sucrose and trehalose (prescription 2/3/4), it is insensitive to protein concentration and has good overall stability.

Based on the above screening results, a histidine buffer system with pH 6.0, mannitol, sucrose and trehalose as excipients was selected for follow-up research.

Example 2: Investigation of formulation stability

2.1 Freeze-thaw stability

The antibody stock solution was selected to prepare the prescription preparation shown in Table 3, and the antibody CB6 concentration was 40 mg/mL. Manually aseptically fill into a 2ml vial of 2.0 mL per vial, carry out -40°C to room temperature, repeat freezing and thawing three times, and measure the content of antibody monomers, aggregates or fragments by appearance and SEC-HPLC for stability investigation.

Table 3: Stability study prescription information

Repeated freezing and thawing. After placing it at ≤-40℃ for more than 4 hours and completely freezing, take it out to room temperature (25±2℃) to completely thaw. After repeated freezing and thawing three times, the appearance is normal. Prescription 7, Prescription 8, and Prescription 9 are SEC- The monomer purity of HPLC has no obvious influence, see Table 4 for details.

Table 4: Prescription screening-repeated freezing and thawing data

The antibody stock solution was selected to prepare the prescription preparation shown in Table 3, and the antibody CB6 concentration was 40 mg/mL. Manually aseptically fill into 125ml polycarbonate bottles (Thermo Fisher, Part No: 3030-42, material is polycarbonate) 60mL per bottle, carry out -80℃ to room temperature, repeat freezing and thawing three times, and pass the appearance and SEC-HPLC Measure the content of antibody monomers, aggregates or fragments for stability investigation.

Repeated freezing and thawing. After placing it at -80°C for more than 4 hours and completely freezing it, take it out to room temperature (25±2°C) to completely thaw. After repeated freezing and thawing three times, the appearance is normal. See Table 5 for details.

Table 5: Prescription screening-125ml polycarbonate bottle repeatedly freeze-thaw

2.2 Shaking stability

The antibody stock solution was selected to prepare the prescription preparation shown in Table 3, and the antibody CB6 concentration was 40 mg/mL. Manually aseptically fill into a 2ml vial of 2.0 mL per vial, and after continuous shaking at 80rpm or 150rpm, 25±2℃, measure the content of antibody monomers, aggregates or fragments by appearance and SEC-HPLC for stability investigation. See Table 6 for specific information.

Table 6: Investigation of shaking stability

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

Table 7: Prescription Screening-Shake

2.3 Dilution stability of normal saline

The antibody stock solution was selected to prepare the prescription preparation shown in Table 3, and the antibody CB6 concentration was 40 mg/mL. The samples were diluted with physiological saline (0.9% NaCl) to different concentrations, and tested after being placed at 25±2°C for 8 hours to investigate the stability of the samples and confirm the prescription conditions. The screening results are shown in Table 8.

Table 8: Prescription stability screening results

The three prescription antibody preparations are stable under the condition of normal saline dilution, and have good compatibility with the infusion tube and the infusion bag.

2.4 Light stability

The antibody stock solution was selected to prepare the prescription preparation shown in Table 3, and the antibody CB6 concentration was 40 mg/mL. Manually aseptically filled into a 6ml vial with 4.0 mL per bottle, irradiated with light, and investigated the stability through appearance, SEC-HPLC and CEX-HPLC measurements. See Table 9 for specific information.

Table 9: Light stability results

2.5 Long-term/accelerated stability

The antibody stock solution was selected to prepare the prescription preparation shown in Table 3, and the antibody CB6 concentration was 40 mg/mL. Manually aseptically fill into a 6ml vial of 4.0mL per bottle, and conduct long-term/accelerated stability inspection. See Table 10 and Table 11 for specific information.

Table 10: 25℃ stability test results

Table 11: 4℃ stability test results

Through repeated freezing and thawing, shaking, diluting with physiological saline, and UV irradiation, the stability of the sample under the conditions here is not significantly affected by the monomer content, showing good stability of the formulation.

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 the relatively optimal formulation of hydro-injection preparations was determined. Antibody CB6 selects histidine and histidine hydrochloride buffers to adjust pH, mannitol, sucrose or trehalose to adjust the osmotic pressure of the preparation, and polysorbate 80 is added to increase the solubility of the preparation.

Example 3: Research on influencing factors and stability of high-concentration prescription

Based on the above screening results, 20mM histidine buffer (pH 6.0) and 235mM sucrose were selected as excipients to carry out the research of high-concentration prescription (concentration about 100mg/ml). The drug substance product is named Drug Substance (DS), and the preparation product obtained by filtering and filling DS after a freeze-thaw cycle is named Drug Product (DP).

3.1 Materials and methods

3.1.1 Research on freeze-thaw stability of DS small model

The samples were stored at 2-8°C, and the Millipore Pellicon3 0.57m ² membrane and Millipore Pellicon3 1.14m ² membrane were used for ultrafiltration concentrated fluid exchange (UFDF). After fluid replacement, the sample was placed in the DS prescription. DS prescription is: 100mg/ml antibody CB6, 20mM histidine buffer (histidine-histidine hydrochloride), 235mM sucrose, 0.05% polysorbate 80 or 0.03% polysorbate 80, pH 6.0 . Fill 30ml of DS manually aseptically into a 50ml bag (Millipore), and conduct freeze-thaw stability study by SEC-HPLC, CEX-HPLC, CE-SDS, pH and appearance. Freeze-thaw conditions: -40℃-2～ 8℃ or room temperature wind bath.

3.1.2 Research on freeze-thaw stability of DS large model

The samples were stored at 2-8°C, and the Millipore Pellicon3 0.57m ² membrane and Millipore Pellicon3 1.14m ² membrane were used for ultrafiltration concentrated fluid exchange (UFDF). After fluid replacement, the sample was placed in the DS prescription. DS prescription: 100mg/ml antibody CB6, 20mM histidine buffer, 235mM sucrose, 0.05% polysorbate 80, pH 6.0. Put 4L of DS into a 5L PC bottle (Nalgene) for freeze-thaw stability study, freeze-thaw conditions: -80°C/RT.

3.1.3 DS stability study

The samples were stored at 2-8°C, and the Millipore Pellicon3 0.57m ² membrane and Millipore Pellicon3 1.14m ² membrane were used for ultrafiltration concentrated fluid exchange (UFDF). After fluid replacement, the sample was placed in the DS prescription. DS prescription: 100mg/ml antibody CB6, 20mM histidine buffer, 235mM sucrose, 0.05% polysorbate 80, pH 6.0. Put 10mL of DS into a 20mL PC bottle (Nalgene) for stability study at a temperature of -80°C to 25°C.

3.1.4 Research on DP forced degradation

The samples were stored at 2-8°C, and the Millipore Pellicon3 0.57m ² membrane and Millipore Pellicon3 1.14m ² membrane were used for ultrafiltration concentrated fluid exchange (UFDF). After fluid replacement, the sample was placed in the DS prescription. DS prescription is: 100mg/ml antibody CB6, 20mM histidine buffer, 235mM sucrose, 0.05% polysorbate 80, pH 6.0. After a freeze-thaw cycle (freeze-thaw conditions: -40℃-2～8℃) and filtration (filter: 0.22μm, KVGLG10TH1, Millipore), DS was manually aseptically filled into 6R vials (Type I glass, Ompi) ,Schott), each vial is filled with 2ml DS to obtain DP products, which are shaken, light (Visible Vis: 4500 Lux; Ultraviolet UV: 90μw/cm2) and high temperature (40°C) experiments, pass SEC-HPLC, CEX-HPLC , CE-SDS, binding/blocking Elisa, pH, Uv-vis and appearance for forced degradation studies.

3.1.5 DP stability study

The samples were stored at 2-8°C, and the Millipore Pellicon3 0.57m ² membrane and Millipore Pellicon3 1.14m ² membrane were used for ultrafiltration concentrated fluid exchange (UFDF). After fluid replacement, the sample was placed in the DS prescription. DS prescription is: 100mg/ml antibody CB6, 20mM histidine buffer, 235mM sucrose, 0.05% polysorbate 80, pH 6.0. After a freeze-thaw cycle (freeze-thaw conditions: -40℃-2～8℃) and filtration (filter: 0.22μm, KVGLG10TH1, Millipore), DS was manually aseptically filled into 6R vials (Type I glass, Ompi) ,Schott), each vial is filled with 2ml DS to obtain DP product; the DP product is placed at 25°C, through SEC-HPLC, CEX-HPLC, CE-SDS, binding/blocking Elisa, pH, Uv-vis and appearance Stability study

3.2 Experimental results

3.2.1 DS small model freeze-thaw cycle (F-T cycle) result

After the DS freeze-thaw cycle, SEC-HPLC, CEX-HPLC, CE-SDS, pH, etc. have no obvious influence, showing good stability. The specific results are shown in Table 12.

Table 12: DS freeze-thaw cycle results

Note: NGHC refers to the antibody isomer without glycosylation modified heavy chain.

3.2.2. Freeze-thaw stability of DS large model

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

Table 13: Research results of freeze-thaw stability of DS large model

Note: "NGHC" refers to the antibody isomer with no glycosylation modified heavy chain.

3.2.3 DS stability study

The results of the DS stability study experiment are shown in Table 14. The results showed that there were no significant changes in the SEC-HPLC, CE-SDS, pH, Uv-vis and appearance of the samples, showing good stability.

Table 14: Research results of DS placed at -80℃～25℃

3.2.4 DP compulsory degradation research results

After the DP sample undergoes shaking, light and high temperature experiments, its SEC-HPLC, CEX-HPLC, CE-SDS, binding/blocking Elisa, pH, Uv-vis and appearance have no significant changes, showing good stability , And the specific results are shown in Table 15-17.

Table 15: DP shaking experimental research results

Note: "NGHC" refers to the antibody isomer with no glycosylation modified heavy chain; "NA" means that it is a non-critical inspection item at this time point, and no detection is performed at this sampling point.

Table 16: DP illumination experiment research results

Table 17: DP high temperature experimental research results

3.2.5 DP stability study results

The DP samples have undergone SEC-HPLC, CEX-HPLC, CE-SDS, binding/blocking Elisa, pH, Uv-vis, and appearance without significant changes, showing good stability. The specific stability study results are shown in Table 18.

Table 18: DP stability study results

Example 4: The binding specificity and high binding activity of the monoclonal antibody preparation to the RBD of the 2019-nCoV virus S protein

Dilute the recombinant SARS-CoV-2 (COVID-19) S protein RBD (Novoprotein, article number DRA32) to a coating of 3.0 μg/mL, and shake on a microplate shaker for 2 hours. The plates are washed and sealed with 2% skimmed milk. Add different concentrations of control antibody (a peer control antibody of IgG1 subtype) and CB6 antibody (from 40μg/mL to 0.009537ng/mL, 4-fold serial dilution, according to prescription 2 configuration), incubate for 1 hour and wash the plate. Then incubate with 1:5000 diluted goat anti-human IgG (Fc specific) peroxidase antibody (Sigma, Catalog No. A0170) for 1 hour, and then incubate with HRP substrate TMB (Sigma, Catalog No. T2885) for 15 minutes to develop color. Detect the binding signal of the antibody to the RBD of the COVID-19 virus S protein. The EC50 was fitted by curve fitting of the slope of the logarithm (agonist) to the response variable (GraphPad Prism).

The experimental results are shown in Figure 1. Measured by Binding ELISA, CB6 antibody and recombinant SARS-CoV-2S protein RBD have higher binding specificity and binding activity, with EC50 of 21.7ng/mL.

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

Recombinant human ACE2 (C-6His) (Novoprotein, catalog number C419) was diluted to 3.0 μg/mL coated plate, and incubated at 37° C. for 90 min. The plates are washed and sealed with 2% skimmed milk. Dilute the recombinant SARS-CoV-2S protein RBD (C-mFc) (Novoprotein, catalog number DRA32) with 2% skimmed milk to 1.0μg/mL, and then use the control antibody (a peer control antibody of IgG1 subtype) and CB6 antibody ( From 400μg/mL to 0.2μg/mL, 2-fold gradient dilution, according to prescription 2 configuration). The mixture was added to the plate and incubated for 1 hour and the plate was washed. By incubating with a 1:5000 diluted peroxidase-labeled goat anti-mouse Fc fragment secondary antibody (Sigma, product number A2554) for 1 hour, then adding TMB (Sigma, product number T2885) and incubating for 20 minutes. Using GraphPad Prism software fitting JS016 the IC _50.

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

Example 6: Monoclonal antibody preparations can effectively block the effect of pseudoviruses infecting target cells

The 2019-nCoV pseudovirus expressing the full sequence of 2019-nCoV spike protein and luciferase reporter gene (final concentration is 10000TCID ₅₀ /ml) and the control antibody (anti-KLH antibody, LALA) or the CB6 antibody after serial dilution ( From 10μg/ml to 0.128ng/ml, 5-fold gradient dilution, according to prescription 2) 1:1 mixed and incubated for a total of 1h.

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

Vero E6 cells were seeded in a 96-well plate with a white wall at a rate of 1×10 ⁴ cells per well, and then placed in an incubator at 37°C for 3 hours until the cells adhered. After the cells adhere to the wall, add 100 μl of the antibody and pseudovirus mixture to the cells, and incubate them in a 37°C incubator for 22 hours. After the incubation, 70μl One-Glo ^TM firefly luciferase substrate was added to each well, and fluorescence detection was performed with a microplate reader (PerkinElmer/Envision).

Inhibition rate=[1-(average fluorescence intensity of experimental group-average blank control)/(average fluorescence intensity of negative control group-average blank control)]×100%. And plotted using GraphPad Prism software fitting the IC _50.

CB6 antibody can effectively inhibit 2019-nCoV pseudovirus infection of Huh-7 and VeroE6 cells, with IC ₅₀ of 0.1831nM (0.02747μg/ml) and 0.07628nM (0.01144μg/ml), detailed results are shown in Figure 3 and Figure 4.

Example 7 Detection of neutralization of 2019-nCoV live virus by antibodies of the present invention

This study evaluated the neutralization effect of CB6 antibody on 2019-nCoV (SARS-CoV-2) live virus through in vitro neutralization experiments.

7.1 Reagents

7.2 Experimental method

The Vero E6 cells were seeded on a 96-well culture plate at a density of ^{1×10 5 per well, and incubated at 37°C for 24 hours before use.} In a 96-well tissue culture plate of DMEM medium, add 50 μl of CB6 antibody (from 48.8 ng/mL to 100 μg/mL, according to prescription 3), which were continuously diluted twice. Then add an equal volume of _{SARS-CoV-2 working stock solution containing 200TCID 50} SARS-CoV-2, and the final viral load is 100TCID50. The antibody-virus mixture was incubated at 37°C for 1 h, then transferred to a 96-well microtiter plate containing octasome fused Vero E6 cells, _{and cultured in a CO 2} incubator at 37°C for 3 days. The 100TCID50 SARS-CoV-2 infected cells or the control medium (DMEM+10% FBS) cultured cells were used as a positive control or a negative uninfected control, respectively. The cytopathic effect (CPE) in each well was observed and recorded before and after infection. Perform a virus back titration to evaluate the correct virus titration used in the experiment. The 50% neutralization dose (ND ₅₀ ) was calculated using Prism software. All experiments were carried out in approved biosafety level 3 facilities in accordance with standard operating procedures.

7.3 Results and conclusions

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

CB6 can neutralize the live SARS-CoV-2 virus and reduce the pathological damage of the virus to cells.

Claims

A pharmaceutical composition comprising:

(1) Buffer; and

(2) A humanized monoclonal antibody or an antigen-binding fragment thereof, wherein the humanized monoclonal antibody specifically binds 2019-nCoV RBD.
The pharmaceutical composition according to claim 1, wherein the buffer is selected from one or more of acetate buffer, citrate buffer and histidine buffer; preferably, the buffer is histidine Acid buffer; more preferably, the histidine buffer is histidine-hydrochloride buffer.
The pharmaceutical composition according to claim 1 or 2, wherein the concentration of the buffer is about 1-100 mM, preferably about 5-50 mM, more preferably about 10-30 mM.
The pharmaceutical composition according to any one of claims 1 to 3, wherein the pH of the buffer is about 5.0-6.5, preferably about 5.5-6.0.
The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition further comprises a stabilizer; preferably, the stabilizer is selected from the group consisting of arginine hydrochloride, proline, glycine, chlorinated One or more of sodium, mannitol, sorbitol, sucrose, maltose, xylitol and trehalose; more preferably, the stabilizer is selected from one or more of mannitol, sucrose and trehalose.
The pharmaceutical composition of claim 5, wherein the concentration of the stabilizer is about 10 mM to 400 mM, preferably about 50 mM to 300 mM, more preferably about 200 mM to 300 mM.
The pharmaceutical composition of claim 5, wherein 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 at a concentration of About 100-300 mM sucrose; or the stabilizer is trehalose with a concentration of about 100-300 mM; preferably, the stabilizer is mannitol with a concentration of about 200-300 mM, or sucrose with a concentration of about 200-300 mM, or About 200-300mM trehalose.
The pharmaceutical composition according to any one of claims 1-7, wherein the pharmaceutical composition further comprises a surfactant; preferably, the surfactant is selected from polysorbate 80, polysorbate 20 Or Poloxamer 188.
The pharmaceutical composition according to claim 8, wherein the concentration of the surfactant is about 0.01%-0.1%, preferably about 0.02%-0.08% on a w/v basis.
The pharmaceutical composition of any one of claims 1-9, wherein the humanized monoclonal antibody has:

(1) The amino acid sequence of HCDR1, HCDR2, and HCDR3 of the heavy chain variable region shown in SEQ ID NO: 7, and the amino acid sequence of LCDR1, LCDR2, and LCDR3 of the light chain variable region shown in SEQ ID NO: 8 Sequence; or

(2) The amino acid sequence of HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, and the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5 and The LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 6.
The pharmaceutical composition according to any one of claims 1-9, wherein the humanized monoclonal antibody has an amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 7, and an amino acid sequence of SEQ ID NO: 7 ID NO: the light chain variable region shown in 8;

Preferably, the 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.
The pharmaceutical composition according to any one of claims 1-11, wherein the concentration of the humanized monoclonal antibody or antigen-binding fragment thereof is about 1-300 mg/mL, preferably about 10-200 mg/mL, and more It is preferably about 20-150 mg/mL, more preferably about 40-120 mg/mL, and more preferably about 40-100 mg/mL.
The pharmaceutical composition according to any one of claims 1-12, which comprises the following components shown in any one of (1)-(16):

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

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

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

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

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

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

(7) (a) Humanized monoclonal antibody or its antigen-binding fragment of about 40mg/mL-120mg/mL; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) About 200-300mM mannitol; (d) and about 0.02%-0.08% polysorbate 80; or

(8) (a) Humanized monoclonal antibody or its antigen-binding fragment of about 40mg/mL-120mg/mL; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) About 200-300mM sucrose; (d) and about 0.02%-0.08% polysorbate 80; or

(9) (a) Humanized monoclonal antibody or its antigen-binding fragment of about 40mg/mL-120mg/mL; (b) about 10-30mM histidine buffer, pH about 5.5-6.0; (c) About 200-300mM trehalose; (d) and about 0.02%-0.08% polysorbate 80; or

(10) (a) About 40 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM or about 247 mM mannitol ; (D) and about 0.02% of polysorbate 80; or

(11) (a) About 40 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM sucrose; (d) And about 0.02% polysorbate 80; or

(12) (a) About 40 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 240 mM trehalose; (d) ) And about 0.02% polysorbate 80; or

(13) (a) about 80mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) about 20mM histidine buffer, pH about 6.0; (c) about 235mM sucrose; (d) And about 0.02% polysorbate 80; or

(14) (a) About 80 mg/mL humanized monoclonal antibody or antigen-binding fragment thereof; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 240 mM trehalose; (d) ) And about 0.02% polysorbate 80; or

(15) (a) About 100 mg/mL humanized monoclonal antibody or antigen-binding fragment thereof; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM sucrose; (d) And about 0.03% Polysorbate 80; or

(16) (a) About 100 mg/mL humanized monoclonal antibody or its antigen-binding fragment; (b) About 20 mM histidine buffer, pH about 6.0; (c) About 235 mM sucrose; (d) And about 0.05% polysorbate 80.
An injection containing the pharmaceutical composition according to any one of claims 1-13 and a 0.9% sodium chloride solution; preferably, the concentration of the humanized monoclonal antibody is 1-40 mg/mL; preferably In particular, the pH of the injection is 5.5-6.0.
Use of the pharmaceutical composition according to any one of claims 1-13 or the injection according to claim 14 in the preparation of a medicament for the treatment or prevention of 2019-nCoV infection.