CN116271015A

CN116271015A - IgM antibody preparation and application thereof

Info

Publication number: CN116271015A
Application number: CN202310253628.1A
Authority: CN
Inventors: 仇思念; 陈明键; 李澍; 杨雨濛; 杨继; 刘春�; 李莹; 戴新悦
Original assignee: Beijing Dongfangluo Biomedical Technology Co ltd
Current assignee: Beijing Dongfangluo Biomedical Technology Co ltd
Priority date: 2022-12-16
Filing date: 2023-03-16
Publication date: 2023-06-23
Anticipated expiration: 2043-03-16
Also published as: CN116271015B; CN117482225A

Abstract

The present invention relates to an IgM antibody preparation and its use. In particular, the IgM antibody preparation comprises IgM antibodies, which are nanobody fusion proteins and J-chain forming IgM antibodies, which nanobody fusion proteins are fusion proteins comprising nanobodies and Fc fragments, which may optionally be linked by a linker, wherein the preparation further comprises a buffer selected from a citric acid buffer system, optionally the preparation further comprises one or more of a stabilizer, an osmolality regulator and a surfactant.

Description

IgM antibody preparation and application thereof

Technical Field

The invention belongs to the technical field of biological medicine. In particular, the invention relates to an IgM antibody preparation and uses thereof.

Background

The existing vaccine can not completely prevent the infection and transmission of the new coronavirus, and the administration of the injected vaccine can not effectively reach the respiratory tract and has large dosage, and professional administration is needed. Many preclinical studies have now found that intranasal vaccination may be more effective in generating immunity against some respiratory viruses than more traditional injection vaccination, and that administration by nasal spray, with its active ingredient attached to the mucosal surface of the respiratory tract, may allow rapid generation of immunity in the respiratory tract, cross-linking of the virus to form aggregated particles, blocking the binding of viral RBD to ACE 2.

IgM antibodies are the earliest expressed antibodies after exposure of the immune system to an immunogen, a class of immunoglobulins with the greatest molecular weight, and are predominantly distributed in serum. Classical secretory IgM forms pentamers through the J chain, but hexamer forms without J chain also exist. The additional binding sites provide multivalent binding capacity and thus have higher affinity and broader binding capacity than IgG antibodies, and can trigger stronger antigen neutralization and immunomodulation in the body. The J chain in the pentameric IgM structure can act to stabilize the antibody structure, the main function of which is to transport IgM antibodies to mucosal surfaces by interacting with the polymeric immunoglobulin receptor (pIgR). Therefore, the pentameric IgM has potential application value in the prevention and treatment of related respiratory diseases.

The selection of a reasonable formulation prescription is critical to better exert the therapeutic effect of the drug. IgM is an antibody protein, and in order to ensure the biological activity of the protein, the preparation prescription must at least ensure the complete conformation of the amino acid core sequence of the protein, and simultaneously prevent the protein from being degraded or aggregated and other chemical and physical changes. Protein aggregation is an important biological drug detection index, and is required to be strictly controlled in the processes of biopharmaceutical development and production, because the aggregation not only affects the quality of products, but also can cause excessive immune response.

Many factors influence protein aggregation, including protein structure, ambient temperature, production process, buffer composition, and the like. Conventional IgM comprising light and heavy chains has a molecular weight of around 900KD, and even if the antibody variable regions are replaced with nanobodies comprising only heavy chain variable regions, the complete molecular weight can reach 600KD. The huge molecular weight makes IgM molecules extremely unstable, and aggregation, sedimentation and other phenomena are easy to occur. Therefore, research on how to improve the stability of IgM antibody preparations and reduce the aggregation of antibody proteins is of great significance to the research and development and application of IgM antibody medicaments.

Disclosure of Invention

The object of the present application is to provide a recombinant IgM antibody preparation. The preparation has good solubility and stability of IgM antibody proteins, and can effectively reduce aggregation of IgM antibody proteins.

In one aspect, the invention provides an IgM antibody formulation comprising an IgM antibody, the formulation further comprising one or more of a buffer, a stabilizer, an osmolality regulator, and a surfactant.

In some embodiments, the IgM antibody is at a concentration of about 0.1-50mg/mL.

In some embodiments, the buffer is selected from the group consisting of an acetate buffer system, a histidine-hydrochloric acid buffer system, and a citric acid buffer system. In some embodiments, the buffer is at a concentration of about 2-30mM.

In some embodiments, the stabilizing agent is selected from mannitol, trehalose, sorbitol, sucrose, arginine hydrochloride, proline, glycine, and methionine. In some embodiments, the concentration of the stabilizer is about 16-50mg/mL.

In some embodiments, the osmolality adjusting agent is selected from glucose, sodium chloride, sodium sulfate. In some embodiments, the osmolality adjusting agent is at a concentration of about 40 to 200mM.

In some embodiments, the surfactant is selected from polysorbate 20 and polysorbate 80. In some embodiments, the concentration of the surfactant is about 0.04-2mg/mL.

In some embodiments, the formulation has a pH of about 4.7-7.0.

In some embodiments, the formulation comprises:

IgM antibodies about 1-20mg/mL;

about 10-20mM citric acid buffer system;

sorbitol is about 20-35mg/mL;

about 40-60mM NaCl;

about 0.2 to about 0.4mg/mL polysorbate 20;

and water; and is also provided with

The pH of the formulation is about 5.6-6.4.

In some embodiments, the formulation comprises:

IgM antibodies about 1mg/mL or about 5mg/mL;

about 20mM citrate buffer system;

sorbitol about 30mg/mL;

NaCl about 60mM (3.5 mg/mL);

polysorbate 20 is about 0.3 or 0.4mg/mL;

and water; and is also provided with

The pH of the formulation was about 6.0.

In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, which is a fusion protein comprising a nanobody and an Fc fragment, which may optionally be linked by a linker. In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, the nanobody fusion protein being a fusion protein comprising a nanobody and an Fc fragment, wherein the nanobody is a nanobody that specifically binds SARS-CoV-2RBD, the Fc fragment is selected from the Fc fragment of a human IgM, the nanobody and Fc fragment optionally being linked by a linker.

In some embodiments, the formulation is in the form of a nasal spray, oral formulation, suppository, or parenteral formulation.

In one aspect, the invention provides the use of a formulation according to the invention in the manufacture of a medicament for the prophylaxis or treatment of infection by a novel coronavirus, wherein the novel coronavirus is an original strain of SARS-CoV-2 and/or a variant strain of SARS-CoV-2.

Drawings

FIG. 1 is a schematic diagram of IgM structure formed by nanobody fusion proteins.

FIG. 2 is a diagram showing the SDS-PAGE identification of IgM antibodies.

Detailed Description

The technical scheme of the invention is further described by the following specific embodiments. It should be apparent to those skilled in the art that the detailed description is merely provided to aid in understanding the invention and should not be taken as limiting the invention in any way.

In addition, numerous specific details are set forth in the following description in order to provide a better illustration of the invention. It will be understood by those skilled in the art that the present invention may be practiced without some of these specific details. In some embodiments, materials, elements, methods, means, etc. well known to those skilled in the art are not described in detail in order to highlight the gist of the present invention.

IgM antibody preparations

The present invention relates to an IgM antibody formulation comprising an IgM antibody, the formulation further comprising one or more of a buffer, a stabilizer, an osmolality regulator and a surfactant.

In some embodiments, the IgM antibody formulation comprises a buffer, and in optional embodiments, the formulation further comprises one or more of a stabilizer, an osmolality regulator, and a surfactant.

In some embodiments, the IgM antibody formulation comprises a buffer. In some embodiments, the IgM antibody formulation comprises a stabilizer. In some embodiments, the IgM antibody formulation comprises a buffer and a stabilizer. In some embodiments, the IgM antibody formulation comprises a buffer, a stabilizer, and an osmolality regulator. In some embodiments, the IgM antibody formulation comprises a buffer, a stabilizer, an osmolality regulator, and a surfactant.

The components are mutually matched, so that the IgM antibody has good solubility and stability in a prescription, and the preparation formula can effectively reduce the aggregation tendency of antibody proteins and improve the storage validity period. In some embodiments, the osmotic pressure of the IgM antibody formulation approaches the isotonic state of the human body, meets formulation osmotic pressure criteria, e.g., meets nasal formulation osmotic pressure criteria, is more compatible with the physiological environment within the nasal cavity of the human body, is less irritating to the nasal environment, facilitates nasal spray administration to the patient, allows rapid immunity to occur in the respiratory tract, cross-links the virus to form aggregated particles, and blocks the binding of viral RBD to ACE 2.

In some embodiments, the concentration of IgM antibodies is less than or equal to about 50mg/mL. In some embodiments, the IgM antibody is at a concentration of about 0.1-50mg/mL. In some embodiments, the IgM antibody is at a concentration of about 5-50mg/mL. In a preferred embodiment, the IgM antibody is present at a concentration of about 1-50mg/mL. In preferred embodiments, the IgM antibody is at a concentration of about 0.1mg/mL, 0.5mg/mL, 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 23mg/mL, 24mg/mL, 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, 36mg/mL, 37mg/mL, 38mg/mL, 39mg/mL, 40mg/mL, 41mg, 42mg, 43mg, 48mg, 46mg, 47mg/mL and 48 mg/mL. In a preferred embodiment, the concentration of IgM antibodies is less than or equal to about 20mg/mL. In preferred embodiments, the IgM antibody is present at a concentration of about 0.1-20mg/mL, 1-15mg/mL, 1-10mg/mL, 1-9mg/mL, 1-8mg/mL, 1-7mg/mL, 1-6mg/mL, 1-5mg/mL, 1-4mg/mL, 1-3mg/mL, 1-2mg/mL, 5-20mg/mL, 5-15mg/mL, 5-10mg/mL, 5-9mg/mL, 5-8mg/mL, 5-7mg/mL, and 5-6mg/mL. In a more preferred embodiment, the IgM antibody is present at a concentration of about 1-20mg/mL or 5-20mg/mL. In a most preferred embodiment, the IgM antibody is at a concentration of about 1mg/mL or about 5mg/mL.

In some embodiments, the buffer is selected from the group consisting of an acetate buffer system, a histidine-hydrochloric acid buffer system, and a citric acid buffer system. In a preferred embodiment, the buffer is selected from a citric acid buffer system (e.g., citric acid-sodium citrate buffer).

In some embodiments, the concentration of buffer is about 2-30mM. In preferred embodiments, the buffer is at a concentration of about 2mM, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, and 30mM. In preferred embodiments, the buffer is at a concentration of about 2-25mM, 2-20mM, 2-15mM, 2-10mM, 10-30mM, 10-25mM, 10-20mM, 10-15mM, 15-30mM, 15-25mM, 15-20mM, 20-30mM, 20-25mM, and 25-30mM. In a more preferred embodiment, the concentration of buffer is about 10-20mM. In a most preferred embodiment, the concentration of buffer is about 20mM.

In some embodiments, the stabilizing agent is selected from mannitol, trehalose, sorbitol, sucrose, arginine hydrochloride, proline, glycine, and methionine. In a preferred embodiment, the stabilizer is selected from sorbitol, sucrose and trehalose. In a more preferred embodiment, the stabilizer is selected from sorbitol.

In some embodiments, the concentration of the stabilizer is about 1-60mg/mL. In some embodiments, the concentration of the stabilizer is about 16-50mg/mL. In a preferred embodiment of the present invention, the concentration of the stabilizing agent was about 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, 6mg/mL, 7mg/mL, 8mg/mL, 9mg/mL, 10mg/mL, 11mg/mL, 12mg/mL, 13mg/mL, 14mg/mL, 15mg/mL, 16mg/mL, 17mg/mL, 18mg/mL, 19mg/mL, 20mg/mL, 21mg/mL, 22mg/mL, 23mg/mL, 24mg/mL, 25mg/mL, 26mg/mL, 27mg/mL, 28mg/mL, 29mg/mL, 30mg/mL, 31mg/mL, 32mg/mL, 33mg/mL, 34mg/mL, 35mg/mL, 36mg/mL, 37mg/mL, 38mg/mL, 39mg/mL, 40mg/mL, 41mg/mL, 42mg/mL, 43mg/mL, 44mg/mL, 45mg/mL, 46mg/mL, 47mg/mL, 48mg/mL, 49mg/mL, 50mg/mL, 51mg/mL, 52mg/mL, 53mg/mL, 54mg/mL, 55mg/mL, 56mg/mL, 57mg/mL, 58mg/mL, 59mg/mL and 60mg/mL. In preferred embodiments, the concentration of the stabilizing agent is about 1-50mg/mL, 1-40mg/mL, 1-35mg/mL, 1-30mg/mL, 1-25mg/mL, 1-20mg/mL, 1-15mg/mL, 1-10mg/mL, 1-5mg/mL, 10-50mg/mL, 10-40mg/mL, 10-35mg/mL, 10-30mg/mL, 10-25mg/mL, 10-20mg/mL, 10-15mg/mL, 15-50mg/mL, 15-40mg/mL, 15-35mg/mL, 15-30mg/mL, 15-25mg/mL, 15-20mg/mL, 16-50mg/mL, 16-40mg/mL, 16-35mg/mL, 16-30mg/mL, 16-20mg/mL, 20-50mg/mL, 20-40mg/mL, 20-35mg/mL, 20-30mg/mL, 20-25mg/mL, 15-35mg/mL, 15-25mg/mL, 16-50mg/mL, 30mg and 30 mg-30 mg/mL. In a preferred embodiment, the concentration of the stabilizer is about 20-40mg/mL. In a more preferred embodiment, the concentration of the stabilizer is about 20-35mg/mL. In a most preferred embodiment, the concentration of the stabilizer is about 30mg/mL.

In some embodiments, the osmolality adjusting agent is selected from glucose, sodium chloride and sodium sulfate. In a preferred embodiment, the osmolality adjusting agent is selected from sodium chloride.

In some embodiments, the concentration of osmolality adjusting agent is about 40-200mM. In some embodiments, the concentration of osmolality adjusting agent is about 20-200mM. In preferred embodiments, the osmolality adjusting agent is at a concentration of about 20mM, 25mM, 30mM, 35mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM, 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM, 61mM, 62mM, 63mM, 64mM, 65mM, 66mM, 67mM, 68mM, 69mM, 70mM, 75mM, 80mM, 85mM, 90mM, 100mM, 110mM, 120mM, 130mM, 140mM, 150mM, 160mM, 170mM, 180mM, 190mM, and 200mM. In preferred embodiments, the osmolality adjusting agent is at a concentration of about 20-100mM, 20-90mM, 20-80mM, 20-70mM, 20-60mM, 30-90mM, 30-80mM, 30-70mM, 30-60mM, 30-50mM, 30-40mM, 40-80mM, 40-70mM, 40-60mM, 40-50mM, 50-70mM, and 50-60mM. In a more preferred embodiment, the osmolality adjusting agent is at a concentration of about 40-65mM. In a more preferred embodiment, the osmolality adjusting agent is at a concentration of about 55 to 65mM. In a more preferred embodiment, the osmolality adjusting agent is at a concentration of about 40-60mM. In a most preferred embodiment, the concentration of osmolality adjusting agent is about 60mM.

In some embodiments, the IgM antibody formulation has an osmolality of about 300-700mOsm. In some embodiments, the IgM antibody formulation has an osmolality of about 300-356mOsm. In some embodiments, the IgM antibody formulation has an osmolality of about 320-356mOsm. In some embodiments, the formulation of the present invention approaches the isotonic state of the human body, meets the osmotic pressure requirements of nasal spray formulations, facilitates the administration of the nasal spray to the patient, and allows rapid immunity to occur in the respiratory tract, thereby blocking the binding of the RBD of the novel coronavirus to ACE 2.

In some embodiments, the surfactant is selected from polysorbate 20 and polysorbate 80. In a preferred embodiment, the surfactant is selected from polysorbate 20.

In some embodiments, the concentration of surfactant is about 0.04-2mg/mL. In preferred embodiments, the concentration of surfactant is about 0.04mg/mL, 0.05mg/mL, 0.1mg/mL, 0.15mg/mL, 0.2mg/mL, 0.25mg/mL, 0.3mg/mL, 0.35mg/mL, 0.4mg/mL, 0.45mg/mL, 0.5mg/mL, 0.55mg/mL, 0.6mg/mL, 0.65mg/mL, 0.7mg/mL, 0.75mg/mL, 0.8mg/mL, 0.85mg/mL, 0.9mg/mL, 0.95mg/mL, 1mg/mL, 1.1mg/mL, 1.2mg/mL, 1.3mg/mL, 1.4mg/mL, 1.5mg/mL, 1.6mg/mL, 1.7mg/mL, 1.8mg/mL, 1.9mg/mL and 2mg/mL. In preferred embodiments, the concentration of surfactant is about 0.04-1.5mg/mL, 0.04-1mg/mL, 0.04-0.5mg/mL, 0.04-0.4mg/mL, 0.04-0.3mg/mL, 0.04-0.2mg/mL, 0.04-0.1mg/mL, 0.1-1mg/mL, 0.1-0.5mg/mL, 0.1-0.4mg/mL, 0.1-0.3mg/mL, 0.1-0.2mg/mL, 0.2-1mg/mL, 0.2-0.5mg/mL, 0.2-0.4mg/mL, 0.2-0.3mg/mL, 0.3-1mg/mL, 0.3-0.5mg/mL, 0.3-0.4mg/mL, 0.4-1mg/mL and 0.4-0.5mg/mL. In a more preferred embodiment, the concentration of surfactant is about 0.1-0.4mg/mL. In a more preferred embodiment, the concentration of surfactant is about 0.2-0.4mg/mL. In the most preferred embodiment, the concentration of surfactant is about 0.3 or 0.4mg/mL.

In some embodiments, the IgM antibody preparation has a pH of about 4.7-7.0. In preferred embodiments, the pH of the formulation is about 4.7, 4.8, 4.9, 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, 6.6, 6.7, 6.8, 6.9 and 7.0. In a preferred embodiment of the present invention, the pH of the formulation is about 4.9-6.8, 4.9-6.7, 4.9-6.6, 4.9-6.5, 4.9-6.4, 4.9-6.3, 4.9-6.2, 4.9-6.1, 4.9-6.0, 4.9-5.9, 4.9-5.8, 4.9-5.7, 4.9-5.6, 4.9-5.5, 4.9-5.4, 4.9-5.3, 4.9-5.2, 4.9-5.1, 4.9-5.0, 5.3-6.8 5.3-6.7, 5.3-6.6, 5.3-6.5, 5.3-6.4, 5.3-6.3, 5.3-6.2, 5.3-6.1, 5.3-6.0, 5.3-5.9, 5.3-5.8, 5.3-5.7, 5.3-5.6, 5.3-5.5, 5.3-5.4, 5.4-6.6, 5.4-6.5, 5.4-6.4, 5.4-6.3, 5.4-6.2, 5.4-6.1, 5.4-6.0 5.3-6.7, 5.3-6.6, 5.3-6.5, 5.3-6.4, 5.3-6.3, 5.3-6.2, 5.3-6.1, 5.3-6.0, 5.3-5.9, 5.3-5.8, 5.3-5.7 5.3-5.6, 5.3-5.5, 5.3-5.4, 5.4-6.6, 5.4-6.5, 5.4-6.4, 5.4-6.3, 5.4-6.2, 5.4-6.1, 5.4-6.0, 6.1-6.2, 6.2-6.6, 6.2-6.5, 6.2-6.4, 6.2-6.3, 6.3-6.6, 6.3-6.5, 6.3-6.4, 6.4-6.6, 6.4-6.5 and 6.5-6.6. In a preferred embodiment, the pH of the formulation is from about 5.3 to about 6.8. In a more preferred embodiment, the pH of the formulation is from about 5.4 to about 6.6. In a more preferred embodiment, the pH of the formulation is from about 5.6 to about 6.4. In a more preferred embodiment, the pH of the formulation is from about 5.8 to about 6.2. In a more preferred embodiment, the pH of the formulation is from about 6.0 to about 6.4. In a most preferred embodiment, the pH of the formulation is about 6.0.

In some embodiments, the IgM antibody formulation comprises:

IgM antibodies about 0.1-20mg/mL;

about 10-30mM citric acid buffer system;

sorbitol is about 20-35mg/mL;

about 40-65mM NaCl;

about 0.1 to about 0.4mg/mL of polysorbate 20;

and water; and is also provided with

The pH of the formulation is about 5.6-6.4.

In some embodiments, the IgM antibody formulation comprises:

IgM antibodies about 1-20mg/mL;

about 10-20mM citric acid buffer system;

sorbitol is about 20-35mg/mL;

about 40-60mM NaCl;

about 0.2 to about 0.4mg/mL polysorbate 20;

and water; and is also provided with

The pH of the formulation is about 5.6-6.4.

In some embodiments, the IgM antibody formulation comprises:

IgM antibodies about 1mg/mL or about 5mg/mL;

about 20mM citrate buffer system;

sorbitol about 30mg/mL;

NaCl about 60mM (3.5 mg/mL);

polysorbate 20 about 0.3 or about 0.4mg/mL;

and water; and is also provided with

The pH of the formulation was about 6.0.

IgM antibodies

In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, e.g., an IgM antibody formed from a nanobody fusion protein and a J chain, the nanobody fusion protein being a fusion protein comprising a nanobody and an Fc fragment, the nanobody and Fc fragment optionally being linked by a linker. In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, e.g., an IgM antibody is an IgM antibody formed from a nanobody fusion protein and a J chain, the nanobody fusion protein is a fusion protein comprising a nanobody and an Fc fragment, wherein the nanobody is a nanobody that specifically binds SARS-CoV-2RBD, the Fc fragment is selected from the Fc fragment of a human IgM, and the nanobody and the Fc fragment are optionally linked by a linker.

In a preferred embodiment, the nanobody fusion protein has a structure from the N-terminus to the C-terminus as shown in formula (I):

A-L-B(I)

wherein, the liquid crystal display device comprises a liquid crystal display device,

a is a nanobody, e.g., a nanobody that specifically binds SARS-CoV-2 RBD;

b is an Fc fragment of human IgM;

l is (GGGGS) m, wherein m=0, 1, 2, 3 or 4.

In a preferred embodiment, the nanobody (e.g., a nanobody that specifically binds SARS-CoV-2 RBD) comprises the following CDRs: CDR1 with the amino acid sequence shown as SEQ ID NO. 1, CDR2 with the amino acid sequence shown as SEQ ID NO. 2, and CDR3 with the amino acid sequence shown as SEQ ID NO. 3.

In a preferred embodiment, the nanobody (e.g., a nanobody that specifically binds SARS-CoV-2 RBD) further comprises 4 framework regions FR1-4, said FR1-4 being staggered in sequence with said CDR1, CDR2 and CDR3; more preferably, the FR1-4 is shown in SEQ ID NOS.4, 5, 6, 7, respectively.

In a preferred embodiment, the nanobody (e.g., a nanobody that specifically binds SARS-CoV-2 RBD) has the amino acid sequence set forth in SEQ ID NO: 8.

In a preferred embodiment, the Fc fragment of human IgM has the amino acid sequence shown in SEQ ID NO. 10.

In a preferred embodiment, the nanobody fusion protein has the amino acid sequence shown in SEQ ID NO. 9.

In a preferred embodiment, the IgM antibody comprises a J chain having the amino acid sequence shown in SEQ ID NO. 11.

In some embodiments, the IgM antibodies are recombinant IgM pentamers produced by mammalian expression systems. The IgM structure is shown in FIG. 1, and the antibody variable region is different from the traditional H chain and L chain combination, but adopts a novel coronavirus (SARS-CoV-2) alpaca-derived nanobody R14. The nanometer antibody is obtained by simultaneously immunizing alpaca with SARS-CoV-2RBD protein and SARS-CoV-2NTD protein, constructing an antibody library and screening by utilizing phage display technology, and the amino acid sequence of the nanometer antibody is shown as SEQ ID NO. 8 (namely QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGST SYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAG GMDYWGQGTQVTVSS).

After fusion of R14 with the amino acid sequence shown as SEQ ID NO. 8 and Fc fragment of human IgM with the amino acid sequence shown as SEQ ID NO. 10 (i.e. VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY) by homologous recombination, nanobody fusion protein R14-Fc (shown as SEQ ID NO. 9, i.e. QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY) can be obtained.

The nanometer antibody fusion protein R14-Fc and the J chain with the amino acid sequence shown as SEQ ID NO. 11 (i.e. QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFV YHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYG GETKMVETALTPDACYPD) can form a recombinant IgM pentameric antibody by self-assembly in cells.

IgM antibody preparation dosage form

In a preferred embodiment, the nasal spray is selected from the group consisting of aerosols, sprays and powder sprays.

In a preferred embodiment, the oral formulation is selected from the group consisting of tablets, powders, pills, powders, granules, fine granules, soft/hard capsules, film coatings, pellets, sublingual tablets and ointments.

In preferred embodiments, the parenteral formulation is a transdermal, ointment, plaster, topical liquid, injectable or bolus formulation.

Use of IgM antibody preparations

The invention also relates to the use of the formulation of the invention in the manufacture of a product or medicament for the prevention or treatment of infection by a novel coronavirus, wherein the novel coronavirus is a SARS-CoV-2 original strain and/or a SARS-CoV-2 variant strain.

In a preferred embodiment, the SARS-CoV-2 variant strain is Alpha (B.1.1.7), beta (B.1.351), gamma (P.1), kappa (B.1.617.1), delta (B.1.617.2) strain, omicron (B.1.1.529) subtype BA.1 strain, BA.2 strain.

Abbreviation description

Abbreviations	Name of the name
		AA	Acetic Acid (Acetic Acid)
CA	Citric Acid (Citric Acid)
		His	Histidine (Histidine)
HCl	Hydrochloric acid (Hydrochloric acid)
		NaCl	Sodium chloride
PS20	Polysorbate 20
		SEC	Size exclusion chromatography
nrCE-SDS	Non-reducing sodium dodecyl sulfate capillary gel electrophoresis
		LMW	Low molecular weight
HMW	High molecular weight
		Main	Major peak of protein

Formulation prescription detection

In some embodiments of prescription screening of IgM antibody liquid formulations, opalescence as referred to in the observation of visible particles refers to the diffuse reflectance effect of colloidal dispersed phases or ultra-microscopic particles. Slight opalescence refers to colorless clear transparent solution under normal illumination conditions, and is transparent and blue-light under a clarity instrument; severe opalescence refers to opalescence that can be seen under natural light conditions;

the detection items and detection methods in the following embodiments:

(1) Visual appearance visible particle observation: the sample was placed in a visible light and clarity meter and the solution was visually observed.

(2) Purity: size Exclusion Chromatography (SEC) and non-reducing sodium dodecyl sulfate capillary gel electrophoresis (nrCE-SDS).

(3) Visible foreign matter: and detecting by a lamp inspection instrument.

(4) Protein concentration: and detecting by an ultraviolet spectrophotometer, wherein the detection wavelength is 280nm.

(5) Osmotic pressure detection: and detecting by an osmotic molar concentration tester.

The IgM antibodies used in the examples below were IgM in pentameric form formed by MR14 (represented by nanobody fusion protein R14-Fc (amino acid sequence shown in SEQ ID NO: 9) and J chain (amino acid sequence shown in SEQ ID NO: 11), and the structures are shown in FIG. 1. R14-Fc and J chains are expressed by the mammalian host cell CHO K1, and self-assemble within the cell to form the pentameric IgM antibody.

Expressed cell supernatant proteins were purified and then identified by SDS-PAGE. The Native-Page results are shown in FIG. 2, sample1 is cell supernatant, and Sample2 is purified IgM antibody.

Example 1: screening of IgM antibody prescription base buffer System

After the IgM antibody of the present invention was expressed, a stock solution (concentration of about 52 mg/mL) was obtained by purification and ultrafiltration, which was replaced in PBS buffer, and the solution was cloudy although no precipitate was generated during the replacement. The applicant selects 4 buffer systems, prepares 6 prescriptions through pH adjustment, and screens the proper buffer systems through the observation result of visible particles in the appearance during liquid change.

As shown in Table 1, igM antibody molecules were sensitive to buffer systems, 1 drop of raw liquid was added to about 5mL of buffer, and precipitation occurred in the acetate buffer system of F1, the histidine-acetate buffer systems of F2 and F3, and the histidine-hydrochloric acid buffer systems of F4 and F5, in addition to the citric acid buffer system of F6. Further examining F6, applicants found that buffer was more difficult to centrifuge when the ultrafiltration tube was changed, indicating that IgM antibody molecules were sensitive to shear forces. Applicants have attempted to exchange a liquid with a molecular sieve and add different stabilizers to protect the molecules from shear forces. As shown in F7-F11, the citric acid buffer system and different stabilizing agents can be added to protect the molecular prescription, so that the liquid can be changed normally through the molecular sieve system, and the stock solution is clarified after the liquid is changed; the column is blocked when the molecular sieve of the acetic acid buffer system is used for changing liquid.

Based on the observation of the F1-F11 liquid change process, a citric acid buffer system is selected as a basic buffer system of the IgM antibody.

TABLE 1

Example 2: igM antibody prescription stabilizer screening

3.5mg/mL (60 mM) NaCl is added into the prescription of the 20mM citric acid buffer system to increase the ionic strength, and the effect of regulating the osmotic pressure is achieved at the same time, so that each prescription reaches the state of human body isotonicity. Mannitol, trehalose, sorbitol, sucrose, arginine hydrochloride, proline, glycine, methionine and the like are selected as stabilizers, and 0.4mg/mL PS20 is added as a surfactant to prepare the F12-F20 prescription with the pH value of 5.3+/-0.1 and the protein concentration of 50mg/mL. The F12-F20 prescription samples were subjected to accelerated stability studies at 40 ℃ for sampling times Day0, day3, 1 week (Day 7), 2 weeks (Day 14) and 3 weeks (Day 21), and were examined by appearance-visible particles and Size Exclusion Chromatography (SEC), respectively. As shown in Table 2, after 3 weeks of standing at 40℃, the F12 containing 30mg/mL sorbitol, F15 containing 60mg/mL sucrose, and F16 containing 60mg/mL trehalose were found to be relatively stable from visible particle observations. F12, F15 and F16 prescriptions were sampled at Day0, 40 ℃ for one week, and 40 ℃ for two weeks, respectively. Protein purity was measured by SEC and the results are shown in table 3: f12, F15 and F16 were formulated with a 31.5%, 33.4% and 29.8% decrease in protein main peak area compared to Day0, respectively, and exhibited primarily as an increase in high molecular weight aggregates (HMW).

The applicant examined the stabilizing effect of 40mg/mL mannitol, trehalose and sorbitol as stabilizers on 39mg/mL IgM antibody protein of the present invention at pH 5.0 (see prescriptions F21-F23). As shown in table 2, by accelerated stability examination at 40 ℃ for 1 week, it was found that the mannitol-stabilized F21 formulation and the trehalose-stabilized F22 formulation had become cloudy after overnight at 40 ℃, whereas the sorbitol-stabilized F23 formulation only appeared opalescent after overnight at 40 ℃. Therefore, sorbitol has better stabilizing effect on IgM molecules than mannitol and trehalose.

The formulas of F24, F25 and F26 are prepared by taking 30mg/mL sorbitol as a stabilizer, and the formulas are similar to those of F12, wherein the differences of pH and protein concentration are that the sorbitol has better stabilizing effect on IgM molecules as shown by the observation result of visible particles accelerated for one week at 40 ℃.

The results of various stabilizer evaluations indicate that sorbitol as a stabilizer has the best stabilizing effect on IgM antibodies.

TABLE 2

TABLE 3 Table 3

Example 3: igM antibody prescription stabilizer concentration screening

The effect of stabilizer concentration and salt concentration on IgM molecular stability was examined. Sorbitol stabilizer concentrations were set at 30, 35 and 40mg/mL, and osmolality regulator NaCl concentrations were set at 40, 50 and 60mM. F27, F28 and F29 formulations were prepared as in table 4, and the F27, F28 and F29 formulations were placed at 40 ℃ for 2 weeks for accelerated stability studies, and were tested for visible particles, protein concentration, osmotic pressure, size Exclusion Chromatography (SEC) and non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), respectively.

From the results of the visible particle observation and the protein concentration retest (Table 4), the stability effect of the three prescriptions F27, F28 and F29 on the IgM molecules is similar, the protein concentration retest results at different time points have no obvious difference, and the osmotic pressure range is 333-356mOsm. From the results of the purity measurements of nr CE-SDS and SEC (table 5), varying the concentration of the stabilizer had no significant effect on the stability of the IgM protein molecules.

TABLE 4 Table 4

TABLE 5

Example 4: igM antibody prescription pH optimization

The observed visible particles of the F24-F26 formulation in example 2 accelerated for one week at 40 ℃ showed that IgM molecules were more stable at ph=5.3 and no precipitation occurred. Applicants performed pH optimization based on pH=5.3, with the basic recipe for F30-F34 being 20mM CA+3.5mg/mL (60 mM) NaCl+30mg/mL sorbitol+0.4 mg/mL PS20, protein concentration 50mg/mL, pH in the range 5.2-6.0. The respective prescription solutions were subjected to accelerated stability studies at 40℃for three weeks, and evaluated in terms of visible particles, protein concentration, osmotic pressure, size Exclusion Chromatography (SEC), non-reducing sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS), and the like, respectively.

As shown in table 6, the visible particle observations indicate that: igM molecules have differences in stability under different pH conditions, and prescriptions with ph=5.6-6.0 do not show severe opalescence and turbidity. In combination with the accelerated observation of each prescription at 40 ℃ for 3 weeks in table 7, the F32 prescription SEC purity detection main peak area at ph=5.6 decreased by 27.0%, the F33 prescription main peak at ph=5.8 decreased by 22.7%, and the F34 prescription main peak at ph=6.0 decreased by 19.0%, indicating that IgM antibody molecules were more stable in the prescription at ph=6.0.

Applicants further performed pH optimization on a pH=6.0 basis, the basic recipe for F35-F39 was still 20mM CA+3.5mg/mL (60 mM) NaCl+30mg/mL sorbitol+0.4 mg/mL PS20, protein concentration was 20mg/mL, pH range was 6.0-6.8, osmotic pressure range was 320-339.

Based on the visual appearance of the F35-F39 formulations in tables 8 and 9, the results of the particle observations, the protein concentration replicates, the results of the purity measurements of nr CE-SDS and SEC, only the F39 formulation with ph=6.8 was observed to produce moderate particle production (5 < number of visible foreign matter < 20) when accelerated for two weeks at 40 ℃. The major protein peak ratio in each of the formulas F35-F39 decreased in magnitude from 7.1%, 8.4%, 10.1%, 11.8% and 13.6% with increasing pH from 6.0, and the instability of IgM antibodies was progressively increased. The results show that the major peak protein ratio in the pH range of 6.0-6.4 falls by less than 10%, with F35 formulation at ph=6.0 being better.

It can be seen that IgM antibodies have good solubility and stability in the ph=5.3-6.8 range, with ph=6.0 being more preferred.

TABLE 6

TABLE 7

TABLE 8

TABLE 9

Example 5: igM antibody prescribed antibody concentration

IgM antibody concentrations of 5mg/mL, 10mg/mL, 15mg/mL and 20mg/mL were selected and prescriptions for F40, F41, F42, F43, F44 and F45 were formulated as in Table 10.

The particle observations and concentration retests in Table 10 show that the solutions remain clear after the F40, F41, F42, F43, F44 and F45 formulations are accelerated for 2 weeks at 40 ℃, the normal slight opalescence can be seen in the clarity instrument, and the concentration retests at different time points have no significant difference, which indicates that the existing formulation formulations can better ensure good solubility of IgM at different antibody concentrations. The purity detection results of nr CE-SDS and SEC in Table 11 show that the instability of IgM molecules increases with the increase of protein concentration, but the overall main peak drop amplitude is within 10%, which indicates that the existing preparation formula can ensure good stability of IgM. The stabilizing effect of the surfactant and the different concentrations of the surfactant on IgM antibodies is not significantly different.

Table 10

TABLE 11

The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Sequence listing

CDR1 sequence of VHH chain of SEQ ID NO. 1 nanobody R14

GFTLDYYAIG

CDR2 sequence of VHH chain of SEQ ID NO. 2 nanobody R14

CISSSDGSTSYADSVKG

CDR3 sequence of VHH chain of SEQ ID NO. 3 nanobody R14

TPATYYSGRYYYQCPAGGMDY

FR1 sequence of VHH chain of SEQ ID NO. 4 nanobody R14

QVQLQESGGGLVQPGGSLRLSCAVS

FR2 sequence of VHH chain of SEQ ID NO. 5 nanobody R14

WFRQAPGKEREGVS

FR3 sequence of VHH chain of SEQ ID NO. 6 nanobody R14

RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA

FR4 sequence of VHH chain of SEQ ID NO. 7 nanobody R14

WGQGTQVTVSS

Amino acid sequence of VHH chain of SEQ ID NO 8 nanobody R14

QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGS

TSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCP

AGGMDYWGQGTQVTVSS

Amino acid sequence of SEQ ID NO 9 nanobody fusion protein R14-Fc

QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGS

TSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCP

AGGMDYWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQI

QVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVD

HRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISW

TRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQT

ISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPE

KYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKS

TGKPTLYNVSLVMSDTAGTCY

Fc sequence of human IgM antibody of SEQ ID NO 10

VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTD

QVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQ

DTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHP

NATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPP

AREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYF

AHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTA

GTCY

SEQ ID NO. 11 human IgM antibody J chain sequence

QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRF

VYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVY

GGETKMVETALTPDACYPD

Claims

1. An IgM antibody preparation comprising IgM antibodies which are formed by nanobody fusion proteins which are fusion proteins comprising nanobodies and Fc fragments, which can optionally be linked by a linker, and J chains, wherein,

the formulation further comprises a buffering agent selected from the group consisting of citric acid buffer systems,

optionally, the formulation further comprises one or more of a stabilizer, an osmotic pressure regulator, and a surfactant.

2. The formulation of claim 1, wherein the buffer is at a concentration of 2-30mM.

3. The formulation of claim 1 or 2, the stabilizer being selected from the group consisting of mannitol, trehalose, sorbitol, sucrose, arginine hydrochloride, proline, glycine, and methionine;

the osmotic pressure regulator is selected from glucose, sodium chloride and sodium sulfate;

the surfactant is selected from polysorbate 20 and polysorbate 80.

4. The formulation of claim 1 or 2, having a pH of 4.7-7.0.

5. The formulation of claim 1 or 2, comprising:

IgM antibodies: 0.1-20mg/mL;

citric acid buffer system: 10-30mM;

sorbitol: 20-35mg/mL;

NaCl：40-65mM；

polysorbate 20:0.1-0.4mg/mL;

and water; and is also provided with

The pH of the formulation is 5.6-6.4.

6. The formulation of claim 1 or 2, comprising:

IgM antibodies: about 1mg/mL or about 5mg/mL;

citric acid buffer system: about 20mM;

sorbitol: about 30mg/mL;

NaCl: about 60mM;

polysorbate 20: about 0.3 or about 0.4mg/mL;

and water; and is also provided with

The pH of the formulation was about 6.0.

7. The formulation of claim 1 or 2, wherein the nanobody fusion protein has a structure from N-terminus to C-terminus as shown in formula (I):

A-L-B(I)

a is a nanobody;

b is an Fc fragment of human IgM;

l is (GGGGS) m, wherein m = 0, 1, 2, 3 or 4;

wherein the nanobody comprises the following CDRs: CDR1 with the amino acid sequence shown as SEQ ID NO. 1, CDR2 with the amino acid sequence shown as SEQ ID NO. 2, and CDR3 with the amino acid sequence shown as SEQ ID NO. 3;

the nanobody further comprises 4 framework regions FR1-4, wherein the FR1-4 and the CDR1, CDR2 and CDR3 are sequentially staggered; the FR1-4 is shown as SEQ ID NO. 4, 5, 6 and 7 respectively.

8. The formulation of claim 7, wherein the nanobody has an amino acid sequence as shown in SEQ ID No. 8; the Fc fragment of the humanized IgM has an amino acid sequence shown as SEQ ID NO. 10; the nanometer antibody fusion protein has an amino acid sequence shown as SEQ ID NO. 9; the J chain has an amino acid sequence shown in SEQ ID NO. 11.

9. The formulation of claim 1 or 2, wherein the formulation is in the form of a nasal spray, an oral formulation, a suppository or a parenteral formulation.

10. Use of a formulation according to any one of claims 1-9 in the manufacture of a medicament for the prevention or treatment of a new coronavirus infection, wherein the new coronavirus is a SARS-CoV-2 original strain and/or a SARS-CoV-2 variant strain.