An igm antibody formulation and uses thereof

Abstract

The present invention relates to an IgM antibody formulation and uses thereof. In particular, the IgM antibody formulation comprises an IgM antibody, which is an IgM antibody formed by a nanobody fusion protein and a J chain, the nanobody fusion protein is a fusion protein comprising a nanobody and an Fc fragment, which are optionally linked by a linker, wherein the formulation further comprises a buffer selected from a citrate buffer system, optionally the formulation further comprises one or more of a stabilizer, an osmotic pressure regulator and a surfactant.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 202310253628.1, filed on March 16, 2023, entitled "An IgM Antibody Preparation and Its Use Thereof". Technical Field

[0002] This invention belongs to the field of biomedical technology. Specifically, this invention relates to an IgM antibody preparation and its uses. Background Technology

[0003] IgM antibodies are among the first antibodies expressed by the immune system after exposure to immunogens and are the largest class of immunoglobulins, primarily distributed in serum. Classical secretory IgM forms a pentamer via its J chain, but a hexamer form without the J chain also exists. The additional binding site provides multivalent binding capacity, thus exhibiting higher affinity and a broader binding spectrum compared to IgG antibodies, enabling stronger antigen neutralization and opsonization. The J chain in the pentamer IgM structure plays a role in stabilizing the antibody structure, and its main function is to transport IgM antibodies to mucosal surfaces through interaction with polymeric immunoglobulin receptors (pIgR). Therefore, pentamer IgM has potential applications in the prevention and treatment of related respiratory diseases.

[0004] Choosing a suitable formulation is crucial for maximizing drug efficacy. IgM is an antibody protein, and to ensure its biological activity, the formulation must at least maintain the conformational integrity of its core amino acid sequence while preventing chemical or physical changes such as degradation or aggregation. Protein aggregation, as an important biopharmaceutical detection indicator, requires strict control during biopharmaceutical development and manufacturing, as aggregation can not only affect product quality but also potentially lead to excessive immune responses.

[0005] Many factors influence protein aggregation, including protein structure, environmental temperature, production process, and buffer composition. Conventional IgM molecules containing both light and heavy chains have a molecular weight of around 900 kDa. Even if the variable region of an antibody is replaced with a nanobody containing only the heavy chain variable region, its intact molecular weight can still reach 600 kDa. This large molecular weight makes IgM molecules extremely unstable, prone to aggregation and sedimentation. Therefore, researching how to improve the stability of IgM antibody formulations and reduce antibody protein aggregation is of great significance for the development and application of IgM antibody drugs. Summary of the Invention

[0006] The purpose of this application is to provide a recombinant IgM antibody formulation. This formulation provides the IgM antibody protein therein with good solubility and stability, and can effectively reduce the aggregation of IgM antibody protein.

[0007] On one hand, the present invention provides an IgM antibody formulation comprising an IgM antibody, and the formulation further comprising one or more of a buffer, a stabilizer, an osmotic pressure regulator, and a surfactant.

[0008] In some implementations, the concentration of the IgM antibody is about 0.1-50 mg / mL.

[0009] In some embodiments, the buffer is selected from acetate buffer systems, histidine-acetidine buffer systems, histidine-hydrochloric acid buffer systems, and citrate buffer systems. In some embodiments, the concentration of the buffer is about 2-30 mM.

[0010] In some embodiments, the stabilizer is selected from mannitol, trehalose, sorbitol, sucrose, arginine hydrochloride, proline, glycine, and methionine. In some embodiments, the concentration of the stabilizer is about 16-50 mg / mL.

[0011] In some embodiments, the osmotic pressure regulator is selected from glucose, sodium chloride, and sodium sulfate. In some embodiments, the concentration of the osmotic pressure regulator is about 40-200 mM.

[0012] In some embodiments, the surfactant is selected from polysorbate 20 and polysorbate 80. In some embodiments, the concentration of the surfactant is about 0.04-2 mg / mL.

[0013] In some embodiments, the pH of the formulation is about 4.7-7.0.

[0014] In some embodiments, the formulation comprises:

[0015] IgM antibody concentration is approximately 1-20 mg / mL;

[0016] The citrate buffer system is approximately 10-20 mM;

[0017] Sorbitol is approximately 20-35 mg / mL;

[0018] NaCl is approximately 40-60 mM;

[0019] Polysorbate 20 is approximately 0.2-0.4 mg / mL;

[0020] and water; and

[0021] The pH of the formulation is approximately 5.6-6.4.

[0022] In some embodiments, the formulation comprises:

[0023] IgM antibody is approximately 1 mg / mL or approximately 5 mg / mL;

[0024] The citrate buffer system is approximately 20 mM.

[0025] Sorbitol is approximately 30 mg / mL;

[0026] NaCl approximately 60 mM (3.5 mg / mL);

[0027] Polysorbate 20 is approximately 0.3 or 0.4 mg / mL;

[0028] and water; and

[0029] The pH of the formulation is approximately 6.0.

[0030] In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, wherein the nanobody fusion protein comprises a nanobody and an Fc fragment, and the nanobody and Fc fragment are optionally linked by a linker. In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, wherein the nanobody is a nanobody that specifically binds to SARS-CoV-2 RBD, and the Fc fragment is selected from the Fc fragment of human IgM, and the nanobody and Fc fragment are optionally linked by a linker.

[0031] In some embodiments, the formulation is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation.

[0032] On the one hand, the present invention provides the use of the formulation described herein in the preparation of a medicament for the prevention or treatment of COVID-19 infection, wherein the COVID-19 virus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the IgM structure formed by the nanobody fusion protein.

[0034] Figure 2 The image shows the SDS-PAGE results of IgM antibody identification. Detailed Implementation

[0035] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the specific embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention.

[0036] Furthermore, to better illustrate the present invention, numerous specific details are provided in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In some embodiments, materials, elements, methods, and means well known to those skilled in the art are not described in detail in order to highlight the spirit of the invention.

[0037] IgM antibody preparations

[0038] This invention relates to an IgM antibody formulation comprising an IgM antibody, and further comprising one or more of a buffer, stabilizer, osmotic pressure regulator and surfactant.

[0039] In some embodiments, the IgM antibody formulation includes a buffer, and in optional embodiments, the formulation also includes one or more of a stabilizer, an osmotic pressure regulator, and a surfactant.

[0040] In some embodiments, the IgM antibody formulation includes a buffer. In some embodiments, the IgM antibody formulation includes a stabilizer. In some embodiments, the IgM antibody formulation includes both a buffer and a stabilizer. In some embodiments, the IgM antibody formulation includes a buffer, a stabilizer, and an osmotic pressure regulator. In some embodiments, the IgM antibody formulation includes a buffer, a stabilizer, an osmotic pressure regulator, and a surfactant.

[0041] This application achieves good solubility and stability of the IgM antibody in the formulation through the synergistic effect of the aforementioned components. The formulation of this invention can effectively reduce the tendency of antibody protein aggregation and extend shelf life. In some embodiments, the osmotic pressure of the IgM antibody formulation is close to the isotonic state of the human body, meeting the osmotic pressure standards for formulations, such as those for nasal formulations. This makes it more compatible with the physiological environment of the human nasal cavity, causing less irritation to the nasal environment, facilitating nasal spray administration, and enabling rapid respiratory immunity. It also cross-links with the virus to form aggregated particles, blocking the binding of viral RBD to ACE2. In some embodiments, the IgM antibody formulation of this invention has great application potential in the prevention and treatment of COVID-19 infection.

[0042] In some embodiments, the concentration of the IgM antibody is less than or equal to about 50 mg / mL. In some embodiments, the concentration of the IgM antibody is about 0.1-50 mg / mL. In some embodiments, the concentration of the IgM antibody is about 5-50 mg / mL. In a preferred embodiment, the concentration of the IgM antibody is about 1-50 mg / mL. In a preferred embodiment, the concentration of the IgM antibody is about 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 2... The concentrations are 4 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, 34 mg / mL, 35 mg / mL, 36 mg / mL, 37 mg / mL, 38 mg / mL, 39 mg / mL, 40 mg / mL, 41 mg / mL, 42 mg / mL, 43 mg / mL, 44 mg / mL, 45 mg / mL, 46 mg / mL, 47 mg / mL, 48 mg / mL, 49 mg / mL, and 50 mg / mL. In a preferred embodiment, the concentration of the IgM antibody is less than or equal to about 20 mg / mL. In a preferred embodiment, the concentration of the IgM antibody is about 0.1-20 mg / mL, 1-20 mg / mL, 1-15 mg / mL, 1-10 mg / mL, 1-9 mg / mL, 1-8 mg / mL, 1-7 mg / mL, 1-6 mg / mL, 1-5 mg / mL, 1-4 mg / mL, 1-3 mg / mL, 1-2 mg / mL, 5-20 mg / mL, 5-15 mg / mL, 5-10 mg / mL, 5-9 mg / mL, 5-8 mg / mL, 5-7 mg / mL, and 5-6 mg / mL. In a more preferred embodiment, the concentration of the IgM antibody is about 1-20 mg / mL or 5-20 mg / mL. In the most preferred embodiment, the concentration of the IgM antibody is about 1 mg / mL or about 5 mg / mL.

[0043] In some embodiments, the buffer is selected from acetate buffer systems, histidine-acetidine buffer systems, histidine-hydrochloric acid buffer systems, and citrate buffer systems. In a preferred embodiment, the buffer is selected from citrate buffer systems (e.g., citrate-sodium citrate buffer).

[0044] In some embodiments, the concentration of the buffer is about 2-30 mM. In preferred embodiments, the concentration of the buffer is about 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, and 30 mM. In a preferred embodiment, the concentration of the buffer is about 2-25 mM, 2-20 mM, 2-15 mM, 2-10 mM, 10-30 mM, 10-25 mM, 10-20 mM, 10-15 mM, 15-30 mM, 15-25 mM, 15-20 mM, 20-30 mM, 20-25 mM, and 25-30 mM. In a more preferred embodiment, the concentration of the buffer is about 10-20 mM. In the most preferred embodiment, the concentration of the buffer is about 20 mM.

[0045] In some embodiments, the stabilizer 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.

[0046] In some embodiments, the concentration of the stabilizer is about 1-60 mg / mL. In some embodiments, the concentration of the stabilizer is about 16-50 mg / mL. In preferred embodiments, the concentration of the stabilizer is about 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, etc. g / 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 a preferred embodiment, the concentration of the stabilizer is approximately 1-50 mg / mL, 1-40 mg / mL, 1-35 mg / mL, 1-30 mg / mL, 1-25 mg / mL, 1-20 mg / mL, 1-15 mg / mL, 1-10 mg / mL, 1-5 mg / mL, 10-50 mg / mL, 10-40 mg / mL, 10-35 mg / mL, 10-30 mg / mL, 10-25 mg / mL, 10-20 mg / mL, 10-15 mg / mL, 15-50 mg / mL, 15-40 mg / mL, 15-35 mg / mL, 15-30 mg / mL. / mL, 15-25mg / mL, 15-20mg / mL, 16-50mg / mL, 16-40mg / mL, 16-35mg / mL, 16-30mg / mL, 16-25mg / mL, 16-20mg / mL, 20-50mg / mL, 20-40mg / m L, 20-35mg / mL, 20-30mg / mL, 20-25mg / mL, 25-50mg / mL, 25-40mg / mL, 25-30mg / mL, 27-33mg / mL, 30-50mg / mL, 30-40mg / mL and 30-35mg / mL.In a preferred embodiment, the concentration of the stabilizer is about 20-40 mg / mL. In a more preferred embodiment, the concentration of the stabilizer is about 20-35 mg / mL. In the most preferred embodiment, the concentration of the stabilizer is about 30 mg / mL.

[0047] In some embodiments, the osmotic pressure regulator is selected from glucose, sodium chloride, and sodium sulfate. In a preferred embodiment, the osmotic pressure regulator is selected from sodium chloride.

[0048] In some embodiments, the concentration of the osmotic pressure regulator is about 40-200 mM. In some embodiments, the concentration of the osmotic pressure regulator is about 20-200 mM. In preferred embodiments, the concentration of the osmotic pressure regulator is about 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 41 mM, 42 mM, 43 mM, 44 mM, 45 mM, 46 mM, 47 mM, 48 mM, 49 mM, 50 mM, 51 mM, 52 mM, 53 mM, 54 mM, 55 mM, 56 mM, 57 mM, 58 mM, 59 mM. 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 a preferred embodiment, the concentration of the osmotic pressure regulator is about 20-100 mM, 20-90 mM, 20-80 mM, 20-70 mM, 20-60 mM, 30-90 mM, 30-80 mM, 30-70 mM, 30-60 mM, 30-50 mM, 30-40 mM, 40-80 mM, 40-70 mM, 40-60 mM, 40-50 mM, 50-70 mM, and 50-60 mM. In a more preferred embodiment, the concentration of the osmotic pressure regulator is about 40-65 mM. In a more preferred embodiment, the concentration of the osmotic pressure regulator is about 55-65 mM. In a more preferred embodiment, the concentration of the osmotic pressure regulator is about 40-60 mM. In the most preferred embodiment, the concentration of the osmotic pressure regulator is about 60 mM.

[0049] In some embodiments, the osmotic pressure of the IgM antibody preparation is approximately 300-700 mOsm. In some embodiments, the osmotic pressure of the IgM antibody preparation is approximately 300-356 mOsm. In some embodiments, the osmotic pressure of the IgM antibody preparation is approximately 320-356 mOsm. In some embodiments, the preparation of the present invention is close to the isotonic state of the human body, meeting the osmotic pressure requirements of nasal spray preparations, facilitating patient administration via nasal spray, enabling rapid respiratory tract immunity, thereby blocking the binding of the SARS-CoV-2 RBD to ACE2.

[0050] In some embodiments, the surfactant is selected from polysorbate 20 and polysorbate 80. In a preferred embodiment, the surfactant is selected from polysorbate 20.

[0051] In some implementations, the concentration of the surfactant is approximately 0.04-2 mg / mL. In a preferred embodiment, the concentration of the surfactant is approximately 0.04 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL, 1 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL, and 2 mg / mL. In a preferred embodiment, the concentration of the surfactant is about 0.04-1.5 mg / mL, 0.04-1 mg / mL, 0.04-0.5 mg / mL, 0.04-0.4 mg / mL, 0.04-0.3 mg / mL, 0.04-0.2 mg / mL, 0.04-0.1 mg / mL, 0.1-1 mg / mL, 0.1-0.5 mg / mL, 0.1-0.4 mg / mL, 0.1-0.3 mg / mL, 0.1-0.2 mg / mL, 0.2-1 mg / mL, 0.2-0.5 mg / mL, 0.2-0.4 mg / mL, 0.2-0.3 mg / mL, 0.3-1 mg / mL, 0.3-0.5 mg / mL, 0.3-0.4 mg / mL, 0.4-1 mg / mL, and 0.4-0.5 mg / mL. In a more preferred embodiment, the concentration of the surfactant is about 0.1-0.4 mg / mL. In a more preferred embodiment, the concentration of the surfactant is about 0.2-0.4 mg / mL. In the most preferred embodiment, the concentration of the surfactant is about 0.3 or 0.4 mg / mL.

[0052] In some embodiments, the pH of the IgM antibody formulation is 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, the pH of the formulation is approximately 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, or 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, 5.4-5.9, 5.4-5.8, 5.4-5.7, 5.4-5.6, 5.4-5.5 5.5-5.6, 5.6-6.6, 5.6-6.5, 5.6-6.4, 5.6-6.3, 5.6-6.2, 5.6-6.1, 5.6-6.0, 5.6-5.9, 5.6-5.8, 5.6-5.7, 5.7-5.8, 5.8-6.6, 5.8-6.5, 5.8-6.4, 5.8-6.3, 5.8-6.2, 5.8-6.1, 5.8-6.0, 5.8-5.9, 5.9-6.6, 5.9-6.5, 5.9-6.4, 5.9-6.3 pH values ​​are 5.9-6.2, 5.9-6.1, 5.9-6.0, 6.0-6.6, 6.0-6.5, 6.0-6.4, 6.0-6.3, 6.0-6.2, 6.0-6.1, 6.1-6.6, 6.1-6.5, 6.1-6.4, 6.1-6.3, 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 about 5.3-6.8. In a more preferred embodiment, the pH of the formulation is about 5.4-6.6. In a more preferred embodiment, the pH of the formulation is about 5.6-6.4.In a more preferred embodiment, the pH of the formulation is about 5.8-6.2. In a more preferred embodiment, the pH of the formulation is about 6.0-6.4. In the most preferred embodiment, the pH of the formulation is about 6.0.

[0053] In some implementations, the IgM antibody formulation comprises:

[0054] IgM antibody levels are approximately 0.1-20 mg / mL;

[0055] The citrate buffer system is approximately 10-30 mM;

[0056] Sorbitol is approximately 20-35 mg / mL;

[0057] NaCl is approximately 40-65 mM;

[0058] Polysorbate 20 is approximately 0.1-0.4 mg / mL;

[0059] and water; and

[0060] The pH of the formulation is approximately 5.6-6.4.

[0061] In some implementations, the IgM antibody formulation comprises:

[0062] IgM antibody concentration is approximately 1-20 mg / mL;

[0063] The citrate buffer system is approximately 10-20 mM;

[0064] Sorbitol is approximately 20-35 mg / mL;

[0065] NaCl is approximately 40-60 mM;

[0066] Polysorbate 20 is approximately 0.2-0.4 mg / mL;

[0067] and water; and

[0068] The pH of the formulation is approximately 5.6-6.4.

[0069] In some implementations, the IgM antibody formulation comprises:

[0070] IgM antibody is approximately 1 mg / mL or approximately 5 mg / mL;

[0071] The citrate buffer system is approximately 20 mM.

[0072] Sorbitol is approximately 30 mg / mL;

[0073] NaCl approximately 60 mM (3.5 mg / mL);

[0074] Polysorbate 20 is approximately 0.3 or 0.4 mg / mL;

[0075] and water; and

[0076] The pH of the formulation is approximately 6.0.

[0077] IgM antibody

[0078] In some embodiments, the IgM antibody is an IgM antibody formed from a nanobody fusion protein, for example, an IgM antibody formed from a nanobody fusion protein and a J chain, wherein the nanobody fusion protein is a fusion protein comprising a nanobody and an Fc fragment, wherein the nanobody is a nanobody that specifically binds to SARS-CoV-2 RBD, and the Fc fragment is selected from the Fc fragment of human IgM, wherein the nanobody and Fc fragment are optionally linked by a linker.

[0079] In a preferred embodiment, the structure of the nanobody fusion protein from the N-terminus to the C-terminus is shown in formula (I):

[0080] ALB(I)

[0081] in,

[0082] A represents a nanobody, for example, a nanobody that specifically binds to SARS-CoV-2 RBD;

[0083] B is the Fc fragment of human IgM;

[0084] L is (GGGGS)m, where m = 0, 1, 2, 3 or 4.

[0085] In a preferred embodiment, the nanobody (e.g., a nanobody that specifically binds to SARS-CoV-2 RBD) comprises the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO:1, CDR2 with the amino acid sequence shown in SEQ ID NO:2, and CDR3 with the amino acid sequence shown in SEQ ID NO:3.

[0086] In a preferred embodiment, the nanobody (e.g., a nanobody that specifically binds to SARS-CoV-2 RBD) further includes four framework regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence; more preferably, the FR1-4 are as shown in SEQ ID NO:4, 5, 6 and 7, respectively.

[0087] In a preferred embodiment, the nanobody (e.g., a nanobody that specifically binds to SARS-CoV-2RBD) has an amino acid sequence as shown in SEQ ID NO:8.

[0088] In a preferred embodiment, the Fc fragment of human IgM has an amino acid sequence as shown in SEQ ID NO:10.

[0089] In a preferred embodiment, the nanobody fusion protein has an amino acid sequence as shown in SEQ ID NO:9.

[0090] In a preferred embodiment, the IgM antibody comprises a J chain having an amino acid sequence as shown in SEQ ID NO:11.

[0091] In some implementations, the IgM antibody is a recombinant IgM pentamer produced via a mammalian expression system. The IgM structure is as follows: Figure 1 As shown, its antibody variable region differs from the traditional H-chain and L-chain combination, instead employing the SARS-CoV-2 alpaca-derived nanobody R14. This nanobody was obtained by simultaneously immunizing alpacas with SARS-CoV-2 RBD and SARS-CoV-2 NTD proteins, constructing an antibody library, and screening using phage display technology. Its amino acid sequence is shown in SEQ ID NO:8 (i.e., QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGST SYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAG GMDYWGQGTQVTVSS).

[0092] The amino acid sequence R14 as shown in SEQ ID NO:8 is combined with the amino acid sequence as shown in SEQ ID NO:8. NO:10 (i.e., VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY) shows a human IgM Fc fragment that, when fused via homologous recombination, yields the nanobody fusion protein R14-Fc (SEQ ID). As shown in NO:9, that is QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPA GGMDYWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNA SSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTTCTVTHTDLPSPLKQTISRPKGVALHRPDVY LLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY).

[0093] The nanobody fusion protein R14-Fc and the J chain with the amino acid sequence shown in SEQ ID NO:11 (i.e., QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFV YHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYG GETKMVETALTPDACYPD) can self-assemble in cells to form a recombinant IgM pentameric antibody.

[0094] IgM antibody formulation dosage form

[0095] In some embodiments, the formulation is in the form of a nasal spray, an oral formulation, a suppository, or a parenteral formulation.

[0096] In a preferred embodiment, the nasal spray is selected from aerosols, sprays, and powders.

[0097] In a preferred embodiment, the oral formulation is selected from tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, and ointments.

[0098] In a preferred embodiment, the parenteral preparation is a transdermal preparation, ointment, plaster, topical liquid, injectable or bolus-applied preparation.

[0099] Uses of IgM antibody preparations

[0100] This invention also relates to the use of the formulations of this invention in the preparation of products or medicines for the prevention or treatment of COVID-19 infection, wherein the COVID-19 virus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

[0101] In a preferred embodiment, the SARS-CoV-2 variant strain is an 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, or BA.2 strain.

[0102] Abbreviation Description

[0103] AA Acetic acid CA Citric acid His Histidine HCl Hydrochloric acid NaCl Sodium chloride PS20 Polysorbate 20 SEC Size exclusion chromatography nrCE-SDS Non-reduced sodium dodecyl sulfate capillary gel electrophoresis LMW low molecular weight HMW High molecular weight Main Protein main peak

[0104] Pharmaceutical formulation testing

[0105] In some examples of formulation screening for liquid IgM antibody preparations, the opalescence phenomenon mentioned in the observation of visible particles refers to the diffuse reflection effect of colloidal dispersed phases or ultra-microscopic particles. Slight opalescence refers to a colorless, clear, and transparent solution under normal lighting conditions, but a transparent bluish-green state when placed under a clarity instrument; severe opalescence refers to opalescence visible even under natural light.

[0106] The detection items and detection methods in the following embodiments:

[0107] (1) Observation of visible particles: Place the sample in a visible light and clarity meter and observe the state of the solution with the naked eye.

[0108] (2) Purity: Detected by size exclusion chromatography (SEC) and non-reduced sodium dodecyl sulfate capillary gel electrophoresis (nrCE-SDS).

[0109] (3) Visible foreign objects: detected by light inspection instrument.

[0110] (4) Protein concentration: detected by ultraviolet spectrophotometer at a wavelength of 280 nm.

[0111] (5) Osmotic pressure detection: Osmotic pressure molar concentration was measured by an osmotic pressure molar concentration meter.

[0112] The IgM antibody used in the following examples is MR14 (an IgM in pentameric form formed by the nanobody fusion protein R14-Fc (amino acid sequence as shown in SEQ ID NO:9) and the J chain (amino acid sequence as shown in SEQ ID NO:11), with the structure shown in [image description missing]. Figure 1 (As shown). R14-Fc and J chains are expressed in mammalian host cell CHO K1, and the two self-assemble within the cell to form a pentamer form of IgM antibody.

[0113] The expressed cell supernatant proteins were purified and then identified by SDS-PAGE. The Native-Page results are as follows: Figure 2 As shown, Sample 1 is the cell pool supernatant, and Sample 2 is the purified IgM antibody.

[0114] Example 1: Screening of IgM antibody formulation basic buffer system

[0115] After expression, the IgM antibody of this invention was purified and ultrafiltered to obtain a stock solution (concentration of approximately 52 mg / mL) that was replaced in PBS buffer. Although no precipitation occurred during the buffer replacement, the solution became turbid. The applicant selected four buffer systems and prepared six formulations after pH adjustment. The suitable buffer system was screened based on the observation of visible particles during the buffer replacement.

[0116] As shown in Table 1, IgM antibody molecules are sensitive to buffer systems. When one drop of the stock solution is added to approximately 5 mL of buffer, precipitation occurs in all systems except the citrate buffer system (F6), the acetate buffer system (F1), the histidine-acetate buffer systems (F2 and F3), and the histidine-hydrochloric acid buffer systems (F4 and F5). Further investigation of F6 revealed that the buffer was difficult to centrifuge during ultrafiltration, indicating that IgM antibody molecules are sensitive to shear forces. The applicant attempted to use molecular sieves for buffer changes and added different stabilizers to protect the molecules from shear forces. As shown in F7-F11, the citrate buffer system formulations with different stabilizers allowed for normal buffer changes via the molecular sieve system, resulting in a clear stock solution after the change; however, the acetate buffer system formulations clogged the column during molecular sieve changes.

[0117] Based on the observations of the above F1-F11 buffer exchange process, the citrate buffer system was selected as the basic buffer system for IgM antibody.

[0118] Table 1

[0119]

[0120]

[0121] Example 2: Screening of IgM antibody formulation stabilizers

[0122] 3.5 mg / mL (60 mM) NaCl was added to the 20 mM citrate buffer system formulation to increase ionic strength and regulate osmotic pressure, achieving an isotonic state in the human body. Mannitol, trehalose, sorbitol, sucrose, arginine hydrochloride, proline, glycine, and methionine were selected as stabilizers, and 0.4 mg / mL PS20 was added as a surfactant to prepare the F12-F20 formulation with a pH of 5.3 ± 0.1 and a protein concentration of 50 mg / mL. Accelerated stability studies were conducted on the F12-F20 formulation samples at 40°C. Sampling times were Day 0, Day 3, week 1 (Day 7), week 2 (Day 14), and week 3 (Day 21), with visible particle detection and size exclusion chromatography (SEC) analysis performed. As shown in Table 2, after being stored at 40℃ for 3 weeks, visible particle observation revealed that formulations F12 (containing 30 mg / mL sorbitol), F15 (containing 60 mg / mL sucrose), and F16 (containing 60 mg / mL trehalose) were relatively stable. Samples of formulations F12, F15, and F16 were taken at Day 0, one week at 40℃, and two weeks at 40℃, respectively. Protein purity was determined by SEC, and the results are shown in Table 3. In formulations F12, F15, and F16, the main peak area of ​​the protein decreased by 31.5%, 33.4%, and 29.8% respectively compared to Day 0, mainly due to an increase in high molecular weight aggregates (HMW).

[0123] The applicant also investigated the stabilizing effects of 40 mg / mL mannitol, trehalose, and sorbitol as stabilizers on 39 mg / mL of the IgM antibody protein of this invention at pH 5.0 (see formulations F21-F23). As shown in Table 2, through a one-week accelerated stability study at 40°C, it was found that formulation F21, with mannitol as a stabilizer, and formulation F22, with trehalose as a stabilizer, became turbid after overnight incubation at 40°C, while formulation F23, with sorbitol as a stabilizer, only showed opalescence after overnight incubation at 40°C. Therefore, sorbitol has a better stabilizing effect on IgM molecules than mannitol and trehalose.

[0124] Formulations F24, F25, and F26 use 30 mg / mL sorbitol as a stabilizer. The formulations are similar to F12, except for the pH and protein concentration. Visible particle observation results after one week of accelerated treatment at 40°C show that sorbitol has a good stabilizing effect on IgM molecules.

[0125] Evaluation results of multiple stabilizers indicate that sorbitol has the best stabilizing effect on IgM antibodies.

[0126] Table 2

[0127]

[0128]

[0129] Table 3

[0130]

[0131] Example 3: Screening of IgM antibody formulation stabilizer concentration

[0132] The effects of stabilizer concentration and salt concentration on the stability of IgM molecules were investigated. Sorbitol stabilizer concentrations were set at 30, 35, and 40 mg / mL, and NaCl osmotic pressure regulator concentrations were set at 40, 50, and 60 mM. Formulations F27, F28, and F29 were prepared according to Table 4. Accelerated stability studies were conducted by placing formulations F27, F28, and F29 at 40°C for 2 weeks. Visible particle count, protein concentration, osmotic pressure, size exclusion chromatography (SEC), and non-reduced sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS) were used for detection.

[0133] Based on the results of visible particle observation and protein concentration retesting (Table 4), the three formulations F27, F28, and F29 showed similar effects on the stability of IgM molecules, with no significant differences in protein concentration retesting results at different time points, and an osmotic pressure range of 333-356 mOsm. Based on the purity detection results of nr CE-SDS and SEC (Table 5), changing the concentration of the stabilizer had no significant effect on the stability of IgM protein molecules.

[0134] Table 4

[0135]

[0136]

[0137] Table 5

[0138]

[0139] Example 4: pH optimization of IgM antibody formulation

[0140] In Example 2, the visible particle observation results of formulations F24-F26 after accelerated stability testing at 40°C for one week showed that IgM molecules were relatively stable at pH 5.3, with no precipitation. The applicant optimized the pH based on pH 5.3. The basic formulations for F30-F34 were 20 mM CA + 3.5 mg / mL (60 mM) NaCl + 30 mg / mL sorbitol + 0.4 mg / mL PS20, with a protein concentration of 50 mg / mL and a pH range of 5.2-6.0. Accelerated stability studies were conducted on each formulation solution at 40°C for three weeks, evaluating aspects such as visible particle appearance, protein concentration, osmotic pressure, size exclusion chromatography (SEC), and non-reduced sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS).

[0141] As shown in Table 6, visible particle observation results indicate that the stability of IgM molecules varies under different pH conditions. Formulations with pH = 5.6-6.0 did not exhibit severe opalescence or turbidity. Combined with the accelerated observation of each formulation at 40℃ for 3 weeks in Table 7, the SEC purity detection showed a 27.0% decrease in the main peak area ratio for formulation F32 at pH = 5.6, a 22.7% decrease for formulation F33 at pH = 5.8, and a 19.0% decrease for formulation F34 at pH = 6.0, indicating that IgM antibody molecules are more stable in formulations with pH = 6.0.

[0142] The applicant further optimized the pH based on pH=6.0. The basic formulation of F35-F39 is still 20mM CA + 3.5mg / mL (60mM) NaCl + 30mg / mL sorbitol + 0.4mg / mL PS20, with a protein concentration of 20mg / mL, a pH range of 6.0-6.8, and an osmotic pressure range of 320-339.

[0143] Based on the visible particle observation results, protein concentration retest results, and nrCE-SDS and SEC purity test results of the F35-F39 formulations in Tables 8 and 9, only the F39 formulation with pH=6.8 showed moderate particle production (5 < visible foreign matter count < 20) after two weeks of accelerated testing at 40℃. As the pH gradually increased from 6.0 to 6.8, the percentage decrease in the proportion of the main protein peak in each of the F35-F39 formulations was 7.1%, 8.4%, 10.1%, 11.8%, and 13.6%, respectively, indicating a gradual increase in the instability of the IgM antibody. The results show that the decrease in the proportion of the main protein peak is less than 10% within the pH range of 6.0-6.4, with the F35 formulation at pH=6.0 being superior.

[0144] It can be seen that IgM antibodies have good solubility and stability in the pH range of 5.3-6.8, with the pH being better at 6.0.

[0145] Table 6

[0146]

[0147] Table 7

[0148]

[0149]

[0150] Table 8

[0151]

[0152] Table 9

[0153]

[0154]

[0155] Example 5: IgM antibody prescription antibody concentration

[0156] Select IgM antibody concentrations of 5 mg / mL, 10 mg / mL, 15 mg / mL and 20 mg / mL, and prepare prescriptions F40, F41, F42, F43, F44 and F45 according to Table 10.

[0157] Table 10 shows the visible particle observation results and concentration retest results, indicating that formulations F40, F41, F42, F43, F44, and F45 remained clear after two weeks of accelerated treatment at 40℃, with normal slight opalescence visible in the clarity instrument. No significant differences were observed in the concentration retest results at different time points, indicating that the existing formulations can better guarantee the good solubility of IgM at different antibody concentrations. Table 11 shows the purity test results of nr CE-SDS and SEC, indicating that the instability of IgM molecules increases with increasing protein concentration, but the overall decrease in the main peak is within 10%, indicating that the existing formulations can guarantee good stability of IgM. There was no significant difference in the stabilizing effect of surfactants and their different concentrations on IgM antibodies.

[0158] Table 10

[0159]

[0160]

[0161] Table 11

[0162]

[0163] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

[0164] sequence list

[0165] CDR1 sequence of the VHH chain of nanobody R14 (SEQ ID NO:1)

[0166] GFTLDYYAIG

[0167] CDR2 sequence of the VHH chain of SEQ ID NO:2 nanobody R14

[0168] CISSSDGSTSYADSVKG

[0169] CDR3 sequence of the VHH chain of SEQ ID NO:3 nanobody R14

[0170] TPATYYSGRYYYQCPAGGMDY

[0171] FR1 sequence of the VHH chain of SEQ ID NO:4 nanobody R14

[0172] QVQLQESGGGLVQPGGSLRLSCAVS

[0173] FR2 sequence of the VHH chain of SEQ ID NO:5 nanobody R14

[0174] WFRQAPGKEREGVS

[0175] FR3 sequence of the VHH chain of SEQ ID NO:6 nanobody R14

[0176] RFTISRDNAKNTVYLQMNSLKPEDTALYYCAA

[0177] FR4 sequence of the VHH chain of SEQ ID NO:7 nanobody R14

[0178] WGQGTQVTVSS

[0179] Amino acid sequence of the VHH chain of nanobody R14 (SEQ ID NO:8)

[0180] QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQVTVSS

[0181] The amino acid sequence of SEQ ID NO:9 nanobody fusion protein R14-Fc

[0182] QVQLQESGGGLVQPGGSLRLSCAVSGFTLDYYAIGWFRQAPGKEREGVSCISSSDGSTSYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTALYYCAATPATYYSGRYYYQCPAGGMDYWGQGTQVTVSSVIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0183] Fc sequence of human IgM antibody of SEQ ID NO:10

[0184] VIAELPPKVSVFVPPRDGFFGNPRKSKLICQATGFSPRQIQVSWLREGKQVGSGVTTDQVQAEAKESGPTTYKVTSTLTIKESDWLGQSMFTCRVDHRGLTFQQNASSMCVPDQDTAIRVFAIPPSFASIFLTKSTKLTCLVTDLTTYDSVTISWTRQNGEAVKTHTNISESHPNATFSAVGEASICEDDWNSGERFTCTVTHTDLPSPLKQTISRPKGVALHRPDVYLLPPAREQLNLRESATITCLVTGFSPADVFVQWMQRGQPLSPEKYVTSAPMPEPQAPGRYFAHSILTVSEEEWNTGETYTCVVAHEALPNRVTERTVDKSTGKPTLYNVSLVMSDTAGTCY

[0185] J chain sequence of human IgM antibody of SEQ ID NO:11

[0186] QEDERIVLVDNKCKCARITSRIIRSSEDPNEDIVERNIRIIVPLNNRENISDPTSPLRTRFVYHLSDLCKKCDPTEVELDNQIVTATQSNICDEDSATETCYTYDRNKCYTAVVPLVYGGETKMVETALTPDACYPD.

Claims

1. An IgM antibody formulation, said formulation comprising; IgM antibody: 0.1-50 mg / mL; Citrate buffer system: 10-30 mM; Sorbitol: 20-35 mg / mL; NaCl: 40-65 mM; Polysorbate 20: 0.1-0.4 mg / mL; and water; and The pH of the formulation is 5.6-6.4; in, The IgM antibody is an IgM antibody formed by a nanobody fusion protein and a J chain. The nanobody fusion protein is a fusion protein containing a nanobody and an Fc fragment, wherein the nanobody and the Fc fragment are directly linked. The nanobody includes the following CDRs: CDR1 with an amino acid sequence as shown in SEQ ID NO:1, CDR2 with an amino acid sequence as shown in SEQ ID NO:2, and CDR3 with an amino acid sequence as shown in SEQ ID NO:

3. The nanobody is a nanobody that specifically binds to SARS-CoV-2 RBD.

2. The formulation of claim 1, wherein the structure of the nanobody fusion protein from the N-terminus to the C-terminus is as shown in formula (I): ALB (I) in, A represents a nanobody; B is the Fc fragment of human IgM; L is (GGGGS)m, where m = 0, 1, 2, 3 or 4; The nanobody includes the following CDRs: CDR1 with the amino acid sequence shown in SEQ ID NO:1, CDR2 with the amino acid sequence shown in SEQ ID NO:2, and CDR3 with the amino acid sequence shown in SEQ ID NO:

3. The nanobody also includes four framework regions FR1-4, which are arranged alternately with CDR1, CDR2 and CDR3 in sequence; FR1-4 are shown as SEQ ID NO:4, 5, 6 and 7 respectively.

3. The formulation of claim 2, wherein the nanobody has the amino acid sequence shown in SEQ ID NO:8; the Fc fragment of the human IgM has the amino acid sequence shown in SEQ ID NO:10; the nanobody fusion protein has the amino acid sequence shown in SEQ ID NO:9; and the J chain has the amino acid sequence shown in SEQ ID NO:

11.

4. 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.

5. Use of the formulation according to any one of claims 1-4 in the preparation of a medicament for the prevention or treatment of COVID-19 infection, wherein the COVID-19 virus is the original SARS-CoV-2 strain and / or a SARS-CoV-2 variant strain.

Images