An isolated anti-c5a antibody and formulations and uses thereof

Abstract

Provided is an isolated anti-C5a antibody for treating C5a-mediated diseases and disorders. Also provided is an anti-C5a antibody formulation that is stable under high temperature, light, shaking, freeze-thaw, accelerated, and long-term conditions, and that ensures good stability of the formulation during preparation, transportation, and storage, ensuring the safety and quality controllability of clinical medication.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an isolated anti-C5a antibody, its formulation, and its application. Background Technology

[0002] C5a is an active peptide involved in allergic reactions and inflammation. It is formed by the cleavage of complement component C5 by C5 convertase in the complement cascade. C5a stimulates mast cell degranulation, the release of tumor necrosis factor-α (TNF-α) and histamine, and also recruits phagocytes to sites of infection and inflammation by increasing the expression of adhesion molecules on the surface of endothelial cells (Mollnes, TE et al. Blood 2002, 100, 1869–1877; Riedemann, NC et al. Immunity 2003, 19, 193–202). In certain pathological conditions, such as post-transplant rejection and asthma, C5a also leads to increased vascular permeability (Gueler, F. et al. J. Am. Soc. Nephrol. 2008, 19, 2302–2312; Krug, N. et al. Am. J. Respir. Crit. Care Med. 2001, 164, 1841–1843; Khan, MA et al. Proc. Natl. Acad. Sci. USA 2013, 110, 6061–6066). C5a is a potent pro-inflammatory molecule that binds to a classic G protein-coupled receptor (GPCR) C5aRI (CD88) and triggers activation of pro-inflammatory signaling pathways (Li, R. et al. FASEB J. 2013, 27, 855–864). C5aR is widely expressed in non-myeloid cells such as umbilical vascular endothelial cells (HUVECs), mouse dermis, liver, lungs, and proximal tubules of the kidneys (Monsinjon, T. et al. FASEB J. 2003, 17, 1003–1014; Gerard, C. et al. Annu. Rev. Immunol. 1994, 12, 775–808; Haviland, D. Letal. J. Immunol. 1995, 154, 1861–1869). Furthermore, studies have shown that C5aR is expressed in glomerular endothelial cells rather than podocytes, suggesting that C5a may primarily cause proteinuria in renal endothelial cells (Tsai, IJ et al. Cell. Mol. Life Sci. 2015, 72, 3157–3171). Therefore, neutralizing C5a and blocking its binding to C5aR has become a method for treating C5a-mediated diseases and symptoms. Patent application WO2011063980 discloses the antibody INab308 (InflaRx) targeting human C5a, WO2012088247 discloses the C5a antibody MEDI-7814 (MedImmune), and US10450370 discloses the C5a antibody BNJ383 (Alexion).

[0003] This invention provides an isolated anti-C5a antibody and a highly stable anti-C5a antibody formulation for the treatment of C5a-mediated diseases and conditions. Summary of the Invention

[0004] In one aspect, the present invention provides an isolated anti-C5a antibody, said antibody comprising V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:9; LC-CDR2, which contains the amino acid sequence SEQ ID NO:10; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:11; or

[0005] The antibody contains V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:1; HC-CDR2, which contains the amino acid sequence SEQ ID NO:2; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:12; LC-CDR2, which contains the amino acid sequence SEQ ID NO:10; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:11; or

[0006] The antibody contains V H The V H Contains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:4; HC-CDR2, which contains the amino acid sequence SEQ ID NO:5; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:6; and V L The V L Contains: LC-CDR1, which contains the amino acid sequence SEQ ID NO:13; LC-CDR2, which contains the amino acid sequence SEQ ID NO:14; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:15; or

[0007] The antibody contains V H The V HContains: HC-CDR1, which contains the amino acid sequence SEQ ID NO:7; HC-CDR2, which contains the amino acid sequence SEQ ID NO:8; and HC-CDR3, which contains the amino acid sequence SEQ ID NO:3; and V L The V L It includes: LC-CDR1, which contains the amino acid sequence SEQ ID NO:16; LC-CDR2, which contains the amino acid sequence SEQ ID NO:17; and LC-CDR3, which contains the amino acid sequence SEQ ID NO:11.

[0008] In some embodiments, the antibody comprises V H It contains the amino acid sequence SEQ ID NO:18, and V L It contains the amino acid sequence SEQ ID NO:22; or

[0009] The antibody contains V H It contains the amino acid sequence SEQ ID NO:19, and V L It contains the amino acid sequence SEQ ID NO:23; or

[0010] The antibody contains V H It contains the amino acid sequence SEQ ID NO:20, and V L It contains the amino acid sequence SEQ ID NO:24; or

[0011] The antibody contains V H It contains the amino acid sequence SEQ ID NO:21, and V L It contains the amino acid sequence SEQ ID NO:25.

[0012] In some embodiments, the isolated anti-C5a antibody, as described above, comprises an Fc fragment. In some embodiments, the isolated anti-C5a antibody is a full-length IgG antibody. In some embodiments, the isolated anti-C5a antibody is a full-length IgG1 or IgG4 antibody.

[0013] In some embodiments, the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprising an amino acid sequence as shown in SEQ ID NO:26 or 27, and the light chain constant region comprising an amino acid sequence as shown in SEQ ID NO:28.

[0014] In some embodiments, the antibody comprises V H V L The heavy chain constant region and the light chain constant region, the V HContaining the amino acid sequence SEQ ID NO:18, the V L The heavy chain constant region contains the amino acid sequence SEQ ID NO:22, and the light chain constant region contains the amino acid sequence SEQ ID NO:26; or

[0015] The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO:19, the V L The heavy chain constant region contains the amino acid sequence SEQ ID NO:23, the light chain constant region contains the amino acid sequence SEQ ID NO:27, or the heavy chain constant region contains the amino acid sequence SEQ ID NO:28; or

[0016] The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO:20, the V L The heavy chain constant region contains the amino acid sequence SEQ ID NO:24, the light chain constant region contains the amino acid sequence SEQ ID NO:26, and the light chain constant region contains the amino acid sequence SEQ ID NO:28; or

[0017] The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO:21, the V L It contains the amino acid sequence SEQ ID NO:25, the heavy chain constant region contains the amino acid sequence SEQ ID NO:27, and the light chain constant region contains the amino acid sequence SEQ ID NO:28.

[0018] In another aspect, the present invention provides an anti-C5a antibody formulation comprising the isolated anti-C5a antibody, a stabilizer, a surfactant, and a buffer solution.

[0019] In some embodiments, the antibody concentration is 1 mg / ml to 300 mg / ml; preferably, the antibody concentration is 10 mg / ml to 200 mg / ml. In some specific embodiments, the antibody concentration is 10 mg / ml, 15 mg / ml, 25 mg / ml, 35 mg / ml, 50 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 175 mg / ml, or 200 mg / ml.

[0020] In some embodiments, the stabilizer is any one or a combination of several selected from sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine hydrochloride, glycine, proline, and lysine.

[0021] In some embodiments, the stabilizer is: sodium chloride, arginine hydrochloride, glycine, proline, or lysine at a concentration of 50 mM-300 mM, preferably 100 mM-250 mM (in some embodiments, concentrations are 100 mM, 150 mM, 200 mM, or 250 mM), or sucrose, trehalose, maltose, sorbitol, or mannitol at a concentration of 30 mg / ml-150 mg / ml, preferably 45 mg / ml-100 mg / ml (in some embodiments, concentrations are 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, or 100 mg / ml).

[0022] In some embodiments, the buffer solution is any one of histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer, and glutamate buffer.

[0023] In some embodiments, the concentration of the buffer solution is 3mM-100mM; in some embodiments, the concentration of the buffer solution is 5mM-80mM or 8mM-50mM; preferably, the concentration of the buffer solution is 10mM-30mM; and in some specific embodiments, the concentration of the buffer solution is 10mM, 20mM, or 30mM.

[0024] In some embodiments, the pH value of the buffer solution is 4.8-8.0; in some embodiments, the pH value of the buffer solution is 5.0-7.2; in some embodiments, the pH value of the buffer solution is 5.0, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7.0 or 7.2; preferably, the pH value of the buffer solution is 5.5-7.0.

[0025] In some embodiments, the surfactant is any one or a combination of two of polysorbate and / or poloxamer; preferably, the polysorbate is Tween-20 or Tween-80.

[0026] In some embodiments, the concentration of the surfactant is 0.01 mg / ml-2 mg / ml; preferably, the concentration of the surfactant is 0.02 mg / ml-1.5 mg / ml, 0.03 mg / ml-1 mg / ml, or 0.04 mg / ml-0.5 mg / ml; more preferably, the concentration of the surfactant is 0.05 mg / ml-0.3 mg / ml; in some specific embodiments, the concentration of the surfactant is 0.05 mg / ml, 0.10 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, or 0.3 mg / ml.

[0027] In some embodiments, the formulation is any one of the following:

[0028] (1) The antibody concentration is 10 mg / ml, 15 mg / ml, 25 mg / ml, 35 mg / ml, 50 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 175 mg / ml or 200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80;

[0029] (2) The antibody concentration is 10 mg / ml-200 mg / ml; the stabilizer is 100 mM, 150 mM, 200 mM or 250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml or 100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80;

[0030] (3) The antibody concentration is 10-200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM, 20 mM, 30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80;

[0031] (4) The antibody concentration is 10-200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5, 5.8, 6, 6.2, 6.5, 6.8 or 7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80;

[0032] (5) The antibody concentration is 10-200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml, 0.10 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml or 0.3 mg / ml Tween-20 and / or Tween-80.

[0033] In some embodiments, the formulation is any one of the following:

[0034] (1) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of histidine-histidine hydrochloride buffer, 100 mM-250 mM of arginine hydrochloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0035] (2) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of histidine-histidine hydrochloride buffer, 100 mM-250 mM of sodium chloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0036] (3) The formulation contains 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of histidine-histidine hydrochloride buffer, 45 mg / ml-100 mg / ml of sucrose, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0037] (4) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of acetate buffer, 100 mM-250 mM of arginine hydrochloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0.

[0038] (5) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of acetate buffer, 100 mM-250 mM of sodium chloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0.

[0039] (6) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of acetate buffer, 45 mg / ml-100 mg / ml of sucrose, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0040] (7) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of phosphate buffer, 100 mM-250 mM of arginine hydrochloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0041] (8) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of phosphate buffer, 100 mM-250 mM of sodium chloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0.

[0042] (9) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of phosphate buffer, 45-100 mg / ml of sucrose, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0.

[0043] (10) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of citrate-disodium hydrogen phosphate buffer, 100 mM-250 mM of arginine hydrochloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0044] (11) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of citrate-disodium hydrogen phosphate buffer, 100 mM-250 mM of sodium chloride, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0045] (12) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of citrate-disodium hydrogen phosphate buffer, 45-100 mg / ml of sucrose, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0;

[0046] (13) The formulation comprises 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of histidine-histidine hydrochloride buffer, 45 mg / ml-100 mg / ml of mannitol or sorbitol or trehalose, and 0.05 mg / ml-0.3 mg / ml of Tween 80, wherein the pH of the buffer is 5.5-7.0.

[0047] In some embodiments, the formulation is any one of the following:

[0048] (1) The formulation contains 15 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 150 mM of arginine hydrochloride, and 0.1 mg / ml of Tween 80, wherein the pH of the buffer is 6.2.

[0049] (2) The formulation contains 10 mg / ml of anti-C5a antibody, 10 mM of histidine-histidine hydrochloride buffer, 200 mM of arginine hydrochloride, and 0.15 mg / ml of Tween 20, wherein the pH of the buffer is 6.4.

[0050] (3) The formulation contains 15 mg / ml of anti-C5a antibody, 30 mM of histidine-histidine hydrochloride buffer, 150 mM of arginine hydrochloride, and 0.05 mg / ml of Tween 80, wherein the pH of the buffer is 5.8.

[0051] (4) The formulation contains 15 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 150 mM of sodium chloride, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.0.

[0052] (5) The formulation contains 15 mg / ml of anti-C5a antibody, 10 mM acetate buffer, 250 mM arginine hydrochloride, and 0.1 mg / ml of Tween 80, wherein the pH of the buffer is 6.2.

[0053] (6) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM of phosphate buffer, 100 mM of arginine hydrochloride, and 0.1 mg / ml of Tween 80, wherein the pH of the buffer is 6.0.

[0054] (7) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 100 mg / ml of sucrose, 0.2 mg / ml of Tween 80, and the pH of the buffer is 6.8.

[0055] (8) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM histidine-histidine hydrochloride buffer, 50 mg / ml mannitol, 0.1 mg / ml Tween 80, and the pH of the buffer is 6.0.

[0056] (9) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM histidine-histidine hydrochloride buffer, 45 mg / ml sorbitol, 0.1 mg / ml Tween 80, and the pH of the buffer is 7.0.

[0057] (10) The formulation comprises 30 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 80 mg / ml of trehalose, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.2.

[0058] (11) The formulation comprises 50 mg / ml of anti-C5a antibody, 30 mM of histidine-histidine hydrochloride buffer, 60 mg / ml of sucrose, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.0.

[0059] (12) The formulation comprises 100 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 50 mg / ml of sucrose, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.0.

[0060] (13) The formulation comprises 150 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 60 mg / ml of sucrose, 0.2 mg / ml of Tween 80, and the pH of the buffer is 6.0.

[0061] (14) The formulation comprises 200 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 70 mg / ml of sucrose, 0.3 mg / ml of Tween 80, and the pH of the buffer is 6.0.

[0062] (15) The formulation comprises 150 mg / ml of anti-C5a antibody, 20 mM of phosphate buffer, 150 mM of arginine hydrochloride, and 0.2 mg / ml of Tween 80, wherein the pH of the buffer is 5.5.

[0063] (16) The formulation comprises 150 mg / ml of anti-C5a antibody, 20 mM of citrate-disodium hydrogen phosphate buffer, 100 mM of sodium chloride, 0.1 mg / ml of Tween 20, and the pH of the buffer is 5.8.

[0064] (17) The formulation comprises 150 mg / ml of anti-C5a antibody, 10 mM of histidine-histidine hydrochloride buffer, 200 mM of sodium chloride, 0.2 mg / ml of Tween 80, and the pH of the buffer is 6.8.

[0065] In some embodiments, the formulation may further comprise a preservative and / or an antioxidant. The preservative or antioxidant is a commonly used preservative or antioxidant in antibody formulations. In some embodiments, the preservative is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), or a combination thereof; the antioxidant is methionine.

[0066] In some embodiments, the concentration of the preservative is 0-0.5 mg / ml. In some specific embodiments, the concentration of the preservative is 0 mg / ml, 0.02 mg / ml, 0.15 mg / ml, 0.25 mg / ml, 0.4 mg / ml, or 0.5 mg / ml. In some embodiments, the concentration of the antioxidant is 0-1.1 mg / ml. In some specific embodiments, the concentration of the antioxidant is 0 mg / ml, 0.15 mg / ml, 0.75 mg / ml, or 0.11 mg / ml.

[0067] In some embodiments, the antibody preparation described above is a liquid preparation or an injectable powder preparation.

[0068] In another aspect, the present invention provides the use of any of the anti-C5a antibodies described above, or any of the anti-C5a antibody preparations described above, in the preparation of medicaments for treating diseases.

[0069] In another aspect, the present invention provides a method for treating a desired individual disease or symptom, comprising administering to the individual an effective amount of any of the anti-C5a antibody or antibody preparations described above. The administration methods include: intravenous injection, intra-arterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration, or transdermal administration. In some embodiments, the preparation is administered intravenously. In some embodiments, the preparation is administered subcutaneously. In some embodiments, the preparation is administered intramuscularly.

[0070] In some embodiments, the disease or condition is an inflammatory, respiratory, or autoimmune disease or condition; preferably, the disease is selected from any one or more of the following: SIRS, sepsis, severe sepsis, septic shock, ischemia / reperfusion-related injury, acute lung injury, pneumonia, acute and chronic transplant rejection in transplant patients, graft-versus-host disease, glomerular disease, glomerulonephritis, solid kidney failure, rheumatoid arthritis, autoimmune diseases, Bechterew's disease, lupus-like diseases, inflammatory bowel disease, Crohn's disease, tumor growth, and solid organ cancer.

[0071] The anti-C5a antibody and its formulation provided by this invention have the following excellent effects:

[0072] 1. The anti-C5a antibody of the present invention can significantly reduce the expression of CD11b in endogenous C5a-induced human neutrophils without inhibiting plasma hemolytic activity, and can significantly reduce the cytokine storm and inflammatory response caused by the novel coronavirus in vivo.

[0073] 2. The anti-C5a antibody preparation of the present invention exhibits strong stability under conditions of high temperature, light, shaking, freeze-thaw, acceleration, and long-term use, ensuring good stability during preparation, transportation, and storage, and guaranteeing the safety and quality control of clinical use. Attached Figure Description

[0074] Figure 1A-1B The results of the CD11b blocking experiment are shown. Figure 1A The results showed that in human neutrophils, anti-C5a antibodies Cab42, Cab44, and Cab45 could block the upregulation of CD11b induced by endogenous C5a in humans. Figure 1BThe results showed that, compared with the control antibody INab308, even with more than 50 times the molar amount of C5 in the reaction system, the anti-C5a antibody Cab42 could still block the upregulation of CD11b expression induced by endogenous human C5a in human neutrophils.

[0075] Figure 2A-2B The figure shows the plasma hemolytic activity of the C5a antibody. In the classical activation pathway, compared to the control antibody Eculizumab, the anti-C5a antibodies Cab35, Cab42, Cab44, and Cab45 (…) exhibited… Figure 2A ) does not inhibit plasma hemolytic activity. In the bypass activation pathway, compared with the control antibody Eculizumab, anti-C5a antibodies Cab35, Cab42, Cab44, and Cab45 ( Figure 2B It does not inhibit plasma hemolytic activity. Detailed Implementation

[0076] To make the technical problem to be solved by the present invention, the technical solution adopted, and the advantages clearer, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0077] Unless otherwise specified, the reagents used in the following examples were prepared using conventional methods or obtained commercially; the experimental methods used were conventional methods; and the materials and instruments used were obtained commercially unless otherwise specified.

[0078] Example 1: Antibody Screening and Preparation

[0079] Blood RNA was extracted from 2000 blood samples, and the first strand of cDNA was synthesized via reverse transcription. Human V... H V L The specific primers amplified V respectively H V L The fragment, after glue recycling, is then transferred to a linker via V. H V L The cells were spliced ​​into scFv format and then inserted into the yeast display plasmid PYD1. Yeast cells were electroporated to obtain a yeast display human antibody scFv library.

[0080] A natural yeast-display human antibody scFv library was enriched using biotinylated C5a via MACS magnetic bead sorting. To facilitate rapid identification of the C5a antibody population, the yeast library was transferred to a phage display system. Phages were then screened using C5a for three rounds, and single clones were selected for binding ELISA identification and in vitro biological activity evaluation to obtain lead antibodies.

[0081] To reduce the potential immunogenicity risk of candidate molecules, the human germline gene closest in phylogenetic relationship to the maternal sequence was used as a template. Simultaneously, combined with structural prediction analysis, the non-germline gene amino acid sequences in the maternal sequence frame region were restored to germline gene amino acid sequences, thereby improving the humanization of the candidate molecules. After affinity maturation, removal of isomerization risk points, and Fc modification, Cab35, Cab42, Cab44, and Cab45 antibodies were obtained. These four antibodies were expressed and purified for subsequent experiments. The amino acid sequences of the heavy chain constant region of the Cab35, Cab42, Cab44, and Cab45 antibodies are shown in SEQ IN NO: 26, and the amino acid sequences of the light chain constant region are shown in SEQ IN NO: 28. The specific amino acid sequences of the antibodies are shown in the table below.

[0082] Table 1. CDR sequence of anti-C5a antibody

[0083]

[0084] Table 2 Anti-C5a antibody sequences

[0085]

[0086]

[0087] Example 2: CD11b blocking experiment in whole blood

[0088] Upregulation of CD11b expression is a characteristic and sensitive marker of neutrophil activation. This invention uses CD11b levels in neutrophils to evaluate neutrophil activation. Using a human whole blood model and INab308 (WO2011063980A1, InflaRx) as a control, this study evaluates the blocking activity of antibodies Cab42, Cab44, and Cab45 against endogenous human C5a. Human whole blood was incubated alone with human C5a or in combination with human C5a and different concentrations of the antibodies. After incubation, CD11b:FITC staining was performed, and after erythrocyte lysis, CD11b MFI was analyzed by flow cytometry to reflect the activation level of neutrophils in the blood.

[0089] like Figure 1A As shown, the optimized antibodies can significantly reduce the expression of CD11b in human neutrophils induced by endogenous C5a. Even when the Ab:Ag molar ratio is 0.5:1, they can achieve the same ability to inhibit CD11b upregulation as the control antibody INab308.

[0090] like Figure 1BAs shown in Table 3, due to the weak binding of antibody Cab42 to human C5, both the control antibody INab308 and antibody Cab42 can inhibit the expression of CD11b in human neutrophils induced by endogenous C5a. Even with more than 50-fold C5 in the reaction system, antibody Cab42 reduced the upregulation of CD11b expression in human neutrophils induced by endogenous C5a with greater efficacy compared to the control antibody INab308.

[0091] Table 3

[0092] Cab42 2.05 31.04 INab308 1.95 42.53

[0093] Example 3: Plasma hemolytic activity of anti-c5a antibody

[0094] The complement system can be activated independently via three pathways, ultimately forming a membrane attack complex. Under specific experimental conditions, this complex can directly attack the cell membrane of erythrocytes, leading to erythrocyte lysis. Based on this mechanism, this invention conducts experiments to evaluate whether the C5a antibody of this invention affects the biological activity of C5 convertase in cleaving C5 cells to generate C5b.

[0095] Detection of the role of C5a antibody in the complement-mediated classical activation pathway: The 50% complement hemolysis assay is a method for determining total classical complement activity in serum. This assay is a lysis test in which an antibody is used as an activator of the classical complement pathway, sensitizing erythrocytes and diluting the test serum at different concentrations to determine the amount required to achieve 50% lysis (CHSO). The hemolysis rate can be determined using a spectrophotometer. The 50% complement hemolysis assay provides an indirect method for measuring terminal complement complex (TCC) formation, as TCC itself has a direct effect on the hemolysis being measured. This assay is well-known and routinely performed by those skilled in the art, for example, as described in Limei Zhao et al. Front Immunol. 2017 May 31; 8:636; Zhao et al. Parasites & Vectors. 2014 Feb 24; 7:80.

[0096] In short, guinea pig erythrocytes were prepared by centrifuging fresh guinea pig whole blood, and then sensitized with sheep anti-erythrocyte antibodies. This process activates the classical complement hemolytic pathway, causing erythrocyte lysis. The absorbance was measured at 412 nm. The C5 antibody Eculizumab was used as a control.

[0097] Detection of the role of C5a antibody in the complement-mediated alternative pathway activation: In short, without antibody sensitization, rabbit erythrocytes can activate the alternative pathway to form the membrane attack complex, leading to erythrocyte lysis. The addition of ethylene glycol diaminotetraacetic acid (EGTA) to the reaction system allows this substance to react with Ca2+ in plasma. 2+Chelation, but with Mg 2+ The binding affinity for complement is very weak, thus blocking the classical pathway. The aforementioned 50% complement hemolysis assay was used to measure activation of the alternative pathway. The C5 antibody Eculizumab was used as a control.

[0098] like Figure 2A As shown, the addition of the C5 antibody Eculizumab inhibited hemolysis in a dose-dependent manner, while antibodies Cab35, Cab42, Cab44, and Cab45 did not inhibit total classical complement activity. Figure 2B As shown, the addition of the C5 antibody Eculizumab can inhibit the hemolytic reaction, while antibodies Cab35, Cab42, Cab44, and Cab45 do not inhibit the activity of the bypass pathway.

[0099] In summary, the anti-C5a antibody of the present invention does not affect the function of C5b in the complement-mediated classical activation pathway, nor does it affect the function of C5b in the alternative activation pathway.

[0100] Example 4: In vivo efficacy of anti-C5a antibody in treating coronavirus-induced ARDS

[0101] An animal model of ARDS was constructed to evaluate the therapeutic effect of the anti-C5a antibody of the present invention in vivo.

[0102] ARDS animal disease model and normal control group: A total of 46 C5a humanized mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) were used. The mice were housed at room temperature of 20℃-26℃, relative humidity of 40%-70%, and with 12-hour light-dark cycles. Four, three, and two days before the experiment, 40 of these mice were injected with adenovirus carrying and expressing the SARS-CoV-2 N protein (see: Ting Gao et al., Highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation medRxiv2020.03.29.20041962; https: / / doi.org / 10.1101 / 2020.03.29.20041962), 7.5×10⁻⁶. 8 PFU / 100μL / mouse / dose / day, with the remaining 6 mice injected with sodium chloride solution (as a normal control group). On day 0 (the day of the experiment), mice were injected with the corresponding dose of antibody or sodium chloride solution according to the following groups.

[0103] Dosage and Animal Grouping: Mice were divided into the following groups and treated with the corresponding reagents: (1) Normal control group (n=6), injected with 100 μL of 0.9% sodium chloride solution; (2) Disease model control group (n=10), injected with 100 μL of 9% sodium chloride solution; (3) Low-dose experimental group (n=10), injected with antibody at a dose of 1 mg / kg; (4) Medium-dose experimental group (n=10), injected with antibody at a dose of 3 mg / kg; (5) High-dose experimental group (n=10), injected with antibody at a dose of 10 mg / kg. 30 min after administration, LPS-K235 (Sigma-Aldrich) at a concentration of 1 mg / mL, 100 μL / mouse was injected into the disease model control group and each experimental group. Sodium chloride solution was injected into the normal control group. All reagents in this experiment were administered via tail vein injection. This experiment was conducted with the approval of the Ethics Committee of the Beijing Institute of Biotechnology and complied with relevant regulatory standards.

[0104] Survival rate detection: The survival of mice in each group was observed and analyzed at 12h, 24h, 36h, 48h, 60h and 72h after drug administration.

[0105] White blood cell count: 72 hours after administration, mice were anesthetized, and blood was collected from the orbital region. The 2120 series blood analyzer performs whole blood white blood cell count and differential, including white blood cell count (WBC), neutrophils (Neut), lymphocytes (Lymph), and monocytes (Mono).

[0106] Survival results: Within 72 hours after administration, animals in both the normal control group and the high-dose experimental groups treated with different anti-C5a antibodies (Cab35, Cab42, Cab44, or Cab45) survived. The overall mortality rate in the model control group was 30% (3 / 10). In the low-dose experimental groups treated with different anti-C5a antibodies (Cab35, Cab42, Cab44, or Cab45), the overall mortality rate was 10-20% (1-2 / 10); in the medium-dose experimental groups treated with different anti-C5a antibodies (Cab35, Cab42, Cab44, or Cab45), the overall mortality rate was 10% (1 / 10). These results indicate that the anti-C5a antibody of the present invention can effectively reduce or prevent coronavirus-induced death in mice and improve their survival rate.

[0107] Whole blood white blood cell count results: Compared with the normal control group, the levels of WBC, Lymph, and Mono in mice in the model control group were decreased, and the difference was statistically significant (P<0.05). Compared with the model control group, all three experimental groups administered different doses of anti-C5a antibody (Cab35, Cab42, Cab44, or Cab45) showed increased WBC and Lymph counts, and the differences between the model control group and the medium and high dose experimental groups were statistically significant (P<0.05). The above results indicate that the anti-C5a antibody of this application helps restore the balance of immune cells in ARDS disease model mice.

[0108] Inflammatory cytokine results: Compared with the normal control group, the levels of GM-CSF, IL-1β, IL-6, TNF-α, and MCP-1 were significantly increased in the model control group, with statistically significant differences (P<0.05). Compared with the model control group, the levels of GM-CSF, IL-1β, IL-6, TNF-α, MCP-1, and C5a in the three dose groups administered with different anti-C5a antibodies (Cab35, Cab42, Cab44, or Cab45) decreased in a dose-dependent manner. Moreover, the levels of most of these cytokines showed statistically significant differences between the dose groups and the model group (P<0.05). These results indicate that the anti-C5a antibody of the present invention can significantly reduce the cytokine storm and inflammatory response induced by the novel coronavirus in vivo.

[0109] Example 5: Preparation of anti-C5a antibody formulation

[0110] The formulation of the anti-C5a antibody preparation is shown below:

[0111] Table 4. Prescriptions for anti-C5a antibody preparations

[0112]

[0113] Preparations containing each C5a antibody (Cab35, Cab42 (IgG1 mutation), Cab44, Cab45 (IgG1 mutation)) were prepared according to the formulations in Table 4, wherein the antibody contains V H V L The antibody sequences include the heavy chain constant region and the light chain constant region, and are as follows:

[0114] Table 5 Antibody Sequences

[0115] Cab35 antibody SEQ ID NO:18 SEQ ID NO:22 SEQ ID NO:26 SEQ ID NO:28 Cab42 (IgG1 mutation) antibody SEQ ID NO:19 SEQ ID NO:23 SEQ ID NO:27 SEQ ID NO:28 Cab44 antibody SEQ ID NO:20 SEQ ID NO:24 SEQ ID NO:26 SEQ ID NO:28 Cab45 (IgG1 mutation) antibody SEQ ID NO:21 SEQ ID NO:25 SEQ ID NO:27 SEQ ID NO:28

[0116] Example 6: Stability test of anti-C5a antibody preparation

[0117] The stability of different formulations was investigated under various conditions, including high temperature, light exposure, shaking, freeze-thaw cycles, accelerated testing, and long-term exposure. The assays included the detection of visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, bioactivity, thermal stability, insoluble particles, aggregation characteristics, degradation characteristics, and charge heterogeneity. Aggregation characteristics were detected using the SEC method and the NR-CE-SDS method, while charge heterogeneity was detected using the CEX method.

[0118] 1. Stability test under high temperature conditions

[0119] The stability of each antibody formulation was tested under high temperature conditions (40℃±2℃, 75%±5%RH (relative humidity), 0 days, 1 week, 2 weeks, and 3 weeks).

[0120] (1) Table 6 shows the detection results of each formulation of the exemplary Cab35 antibody: Compared with day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody formulation remained essentially unchanged; aggregates and fragments increased slightly, and the main peak decreased slightly; the change in aggregates detected by SEC was within 0.9%; the change in aggregates detected by NR-CE-SDS was within 0.8%, the change in the main peak was within 2.7%, and the change in fragments was within 2.0%. The above results demonstrate that the Cab35 formulations have good stability under high temperature conditions.

[0121] Table 6 Results of the high-temperature test for Cab35 antibody

[0122]

[0123]

[0124] (2) The various formulations of Cab42 (IgG1 mutant), Cab44 and Cab45 (IgG1 mutant) antibodies also showed good stability under high temperature conditions: within 3 weeks at high temperature, compared with day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability and insoluble particles of the above three antibody formulations were basically unchanged, while the aggregates and fragments increased slightly and the main peak decreased slightly: the change in aggregates detected by SEC was within 1.0%; the change in aggregates detected by NR-CE-SDS was within 0.9%, the change in the main peak was within 2.6%, and the change in fragments was within 2.0%.

[0125] The above results indicate that the aggregation and degradation characteristics of the anti-C5a antibody formulation of the present invention do not change significantly under high temperature conditions, and the formulation is relatively stable.

[0126] 2. Stability test under light conditions

[0127] Under illumination conditions (5℃±3℃, 4500±500lx, 5W / m²) 2 The stability of each antibody preparation was tested under light exposure for 0 days, 3 days, 5 days, 1 week, and 2 weeks.

[0128] (1) Table 7 shows the detection results of various formulations of the exemplary antibody Cab42 (IgG1 mutant): Within 2 weeks of light exposure, compared to day 0, the visible foreign matter, concentration, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of the antibody formulation remained essentially unchanged; aggregates and fragments increased slightly, while the main peak decreased slightly: SEC detection showed aggregate changes within 3.10%; NR-CE-SDS detection showed aggregate changes within 1.9%, main peak changes within 4.4%, and fragment changes within 2.5%. These results indicate that the Cab42 (IgG1 mutant) antibody formulation remains essentially stable under light conditions.

[0129] Table 7. Results of light irradiation test of the Cab42 (IgG1 mutant) antibody preparation.

[0130]

[0131]

[0132] (2) The various formulations of Cab35, Cab44 and Cab45 (IgG1 mutant) antibodies also showed good stability under light conditions: within 2 weeks of light exposure, compared with day 0, the visible foreign matter, concentration, pH, osmotic pressure, viscosity, biological activity, thermal stability and insoluble particles of the antibody formulations remained basically unchanged within 2 weeks of light exposure; there was an increasing trend in aggregates and fragments, and a slight decreasing trend in the main peak: the change in aggregates detected by SEC was within 3.15%; the change in aggregates detected by NR-CE-SDS was within 2.1%, the change in the main peak was within 4.8%, and the change in fragments was within 2.3%.

[0133] The above results indicate that the aggregation and degradation characteristics of the anti-C5a antibody formulation of the present invention do not change significantly under light conditions, and the formulation can remain basically stable.

[0134] 3. Stability test under oscillation conditions

[0135] The stability of each antibody preparation was tested under oscillation conditions (5℃±3℃, 100rpm oscillation for 0 days, 1 week, and 2 weeks).

[0136] (1) Table 8 shows the detection results of various formulations of the exemplary antibody Cab44: Within two weeks of shaking, compared to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody formulation remained essentially unchanged; the SEC assay showed very small changes in aggregates, all within 0.13%; the CEX assay showed changes in the acidic peak, main peak, and basic peak all within 0.40%, indicating almost no change; the NR-CE-SDS assay showed changes in aggregates, main peak, and fragments within 0.42%, indicating almost no change. These results demonstrate the excellent stability of the Cab44 antibody formulations under shaking conditions.

[0137] Table 8 Results of the shaking test for the anti-Cab44 antibody preparation

[0138]

[0139] (2) The various formulations of Cab35, Cab42 (IgG1 mutant) and Cab45 (IgG1 mutant) antibodies also showed excellent stability under shaking conditions: within 2 weeks of shaking, compared with day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability and insoluble particles of each antibody formulation remained basically unchanged; the SEC detection showed very small changes in aggregates, with changes within 0.15%; the CEX detection showed changes in acidic peaks, main peaks and basic peaks within 0.38%, with almost no changes; the NR-CE-SDS aggregates, main peaks and fragments showed changes within 0.41%, with almost no changes.

[0140] The results of the above-mentioned oscillation test indicate that the anti-C5a antibody preparation of the present invention has excellent stability under oscillation conditions.

[0141] 4. Stability test under freeze-thaw conditions

[0142] The stability of each antibody preparation was tested under freeze-thaw conditions (freeze: -20℃±5℃; thaw: room temperature, freeze-thaw cycles of 0, 1, 3, and 5).

[0143] (1) Table 9 shows the test results of various formulations of the exemplary antibody Cab45 (IgG1 mutant): Within 5 freeze-thaw cycles, compared with 0 freeze-thaw cycles, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody formulation remained essentially unchanged; the SEC assay showed very small changes in aggregates, all within 0.15%; the CEX assay showed changes in acidic peaks, main peaks, and basic peaks within 0.42%, almost no change; the NR-CE-SDS assay showed changes in aggregates, main peaks, and fragments within 0.39%, almost no change. These results demonstrate the excellent stability of the Cab45 (IgG1 mutant) antibody formulations under freeze-thaw conditions.

[0144] Table 9. Freeze-thaw test results of the Cab45 (IgG1 mutant) antibody preparation.

[0145]

[0146] (2) The various formulations of Cab35, Cab42 (IgG1 mutation) and Cab44 antibodies also showed excellent stability under freeze-thaw conditions: within 5 freeze-thaw cycles, compared with 0 freeze-thaw cycles, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles of each antibody formulation remained basically unchanged; the SEC detection showed very small changes in aggregates, with changes within 0.16%; the CEX detection showed changes in acidic peaks, main peaks, and basic peaks within 0.40%, with almost no changes; the NR-CE-SDS detection showed changes in aggregates, main peaks, and fragments within 0.42%, with almost no changes.

[0147] The results of the above freeze-thaw test indicate that the anti-C5a antibody preparation of the present invention has excellent stability under freeze-thaw conditions.

[0148] 5. Stability test under accelerated conditions

[0149] The stability of each antibody preparation was tested under accelerated conditions (25℃±2℃, 60%±10%RH (relative humidity) for 0 days, 2 weeks, 1 month, and 2 months).

[0150] (1) Table 10 shows the test results of various formulations of the exemplary antibody Cab42 (IgG1 mutant): Within 2 months of accelerated testing, compared with day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particles of the antibody formulation remained essentially unchanged; SEC testing showed no change or very small change in aggregates, with a change range within 0.31%; CEX testing showed a slight increase in acid and base peaks and a slight decrease in the main peak; the change range of acid peak was within 3.10%, the change range of base peak was within 0.9%, and the change range of the main peak was within 4%; NR-CE-SDS testing showed an increase in aggregates within 0.5%, a decrease in the main peak within 1%, and an increase in fragments within 0.5%. The above results indicate that the formulations of the Cab42 (IgG1 mutant) antibody can remain essentially stable under accelerated conditions.

[0151] Table 10 Accelerated test results of the Cab42 (IgG1 mutant) antibody formulation

[0152]

[0153]

[0154]

[0155] (2) The various formulations of Cab35, Cab44, and Cab45 (IgG1 mutant) antibodies also exhibited excellent stability under accelerated conditions: within 2 months of acceleration, compared to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particles of each antibody formulation remained essentially unchanged. SEC assays showed no or minimal change in aggregates, with changes within 0.35%; CEX assays showed slight increases in acid and alkali peaks and a slight decrease in the main peak; the acid peak change was within 3.13%, the alkali peak change was within 1.0%, and the main peak change was within 4.2%; NR-CE-SDS assays showed an increase in aggregates within 0.5%, a decrease in the main peak within 1%, and an increase in fragments within 0.5%.

[0156] The accelerated test results show that the anti-C5a antibody formulation of the present invention does not change significantly under accelerated conditions and can remain basically stable.

[0157] 6. Stability test under long-term conditions

[0158] The stability of the antibody preparation was tested under long-term conditions (0 days, 2 weeks, 1 month, 2 months, 3 months, and 6 months at 5℃±3℃).

[0159] (1) Table 11 shows the test results of various formulations of the exemplary antibody Cab44: Under long-term conditions, over 6 months, compared to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particles of the antibody formulation remained essentially unchanged; SEC analysis showed almost no change in aggregate composition (less than 0.11%); CEX analysis showed almost no change in acidic peaks, main peak, and basic peaks, with changes in acidic peaks (less than 0.44%), main peaks (less than 0.49%), and basic peaks (less than 0.13%); NR-CE-SDS analysis showed almost no change in aggregate composition, main peak, and fragments, with increases in aggregate composition (less than 0.5%), decreases in main peak composition (less than 0.5%), and increases in fragments (less than 0.3%). These results demonstrate the excellent stability of the Cab44 antibody formulation under long-term conditions.

[0160] Table 11 Long-term stability results of the Cab44 antibody preparation

[0161]

[0162]

[0163]

[0164]

[0165] (2) The various formulations of Cab35, Cab42 (IgG1 mutant) and Cab45 (IgG1 mutant) antibodies also exhibited excellent stability under long-term conditions: within 6 months under long-term conditions, compared with day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particles of the antibody formulations remained essentially unchanged. SEC detection showed almost no change in aggregates, with a change range within 0.13%; CEX detection showed almost no change in acidic peaks, main peaks, and basic peaks, with changes in acidic peaks within 0.46%, main peaks within 0.5%, and basic peaks within 0.15%; NR-CE-SDS detection showed almost no change in aggregates, main peaks, and fragments, with an increase in aggregates within 0.51%, a decrease in main peaks within 0.5%, and an increase in fragments within 0.4%.

[0166] The results of the above long-term tests demonstrate that the anti-C5a antibody formulation of the present invention has excellent stability under long-term conditions.

[0167] In summary, the anti-C5a antibody formulation of the present invention exhibits strong stability under conditions of high temperature, light, shaking, freeze-thaw, acceleration, and long-term use, ensuring good stability during preparation, transportation, and storage, and guaranteeing the safety and quality control of clinical use.

Claims

1. An anti-C5a antibody preparation, characterized in that, The formulation comprises isolated anti-C5a antibody, stabilizer, surfactant, and buffer; the antibody concentration is 1 mg / ml-300 mg / ml; The antibody contains V H The V H Contains: HC-CDR1, with the amino acid sequence SEQ ID NO: 1; HC-CDR2, with the amino acid sequence SEQ ID NO: 2; and HC-CDR3, with the amino acid sequence SEQ ID NO: 3; and V L The V L Contains: LC-CDR1, with the amino acid sequence SEQ ID NO: 9; LC-CDR2, with the amino acid sequence SEQ ID NO: 10; and LC-CDR3, with the amino acid sequence SEQ ID NO: 11; or The antibody contains V H The V H Contains: HC-CDR1, with the amino acid sequence SEQ ID NO: 1; HC-CDR2, with the amino acid sequence SEQ ID NO: 2; and HC-CDR3, with the amino acid sequence SEQ ID NO: 3; and V L The V L Contains: LC-CDR1, with the amino acid sequence SEQ ID NO: 12; LC-CDR2, with the amino acid sequence SEQ ID NO: 10; and LC-CDR3, with the amino acid sequence SEQ ID NO: 11; or The antibody contains V H The V H Contains: HC-CDR1, with the amino acid sequence SEQ ID NO: 4; HC-CDR2, with the amino acid sequence SEQ ID NO: 5; and HC-CDR3, with the amino acid sequence SEQ ID NO: 6; and V L The V L Contains: LC-CDR1, with the amino acid sequence SEQ ID NO: 13; LC-CDR2, with the amino acid sequence SEQ ID NO: 14; and LC-CDR3, with the amino acid sequence SEQ ID NO: 15; or The antibody contains V H The V H Contains: HC-CDR1, with the amino acid sequence SEQ ID NO: 7; HC-CDR2, with the amino acid sequence SEQ ID NO: 8; and HC-CDR3, with the amino acid sequence SEQ ID NO: 3; and V L The V L It includes: LC-CDR1, whose amino acid sequence is SEQ ID NO: 16; LC-CDR2, whose amino acid sequence is SEQ ID NO: 17; and LC-CDR3, whose amino acid sequence is SEQ ID NO:

11.

2. The formulation according to claim 1, characterized in that, The antibody contains V H It contains the amino acid sequence SEQ ID NO: 18, and V. L It contains the amino acid sequence SEQ ID NO: 22; or The antibody contains V H It contains the amino acid sequence SEQ ID NO: 19, and V. L It contains the amino acid sequence SEQ ID NO: 23; or The antibody contains V H It contains the amino acid sequence SEQ ID NO: 20, and V L It contains the amino acid sequence SEQ ID NO: 24; or The antibody contains V H It contains the amino acid sequence SEQ ID NO: 21, and V L It contains the amino acid sequence SEQ ID NO:

25.

3. The formulation according to claim 2, characterized in that, The antibody comprises a heavy chain constant region and a light chain constant region, wherein the heavy chain constant region comprises the amino acid sequence SEQ ID NO: 26 or 27, and the light chain constant region comprises the amino acid sequence SEQ ID NO:

28.

4. The formulation according to claim 2, characterized in that, The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO: 18, the V L Containing the amino acid sequence SEQ ID NO: 22, the heavy chain constant region containing the amino acid sequence SEQ ID NO: 26, and the light chain constant region containing the amino acid sequence SEQ ID NO: 28; or The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO:19, the V L Containing the amino acid sequence SEQ ID NO: 23, the heavy chain constant region containing the amino acid sequence SEQ ID NO: 27, and the light chain constant region containing the amino acid sequence SEQ ID NO: 28; or The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO:20, the V L Containing the amino acid sequence SEQ ID NO: 24, the heavy chain constant region containing the amino acid sequence SEQ ID NO: 26, and the light chain constant region containing the amino acid sequence SEQ ID NO: 28; or The antibody contains V H V L The heavy chain constant region and the light chain constant region, the V H Containing the amino acid sequence SEQ ID NO:21, the V L It contains the amino acid sequence SEQ ID NO: 25, the heavy chain constant region contains the amino acid sequence SEQ ID NO: 27, and the light chain constant region contains the amino acid sequence SEQ ID NO:

28.

5. The formulation according to claim 1, characterized in that, The antibody concentration is 10 mg / ml-200 mg / ml.

6. The formulation according to claim 1, characterized in that, The stabilizer is any one or a combination of several of the following: sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine hydrochloride, glycine, proline, and lysine.

7. The formulation according to claim 6, characterized in that, The stabilizer is any one or a combination of several of the following: sucrose, arginine hydrochloride, sodium chloride, mannitol, trehalose, and sorbitol.

8. The formulation according to claim 1, characterized in that, The stabilizer is: sodium chloride, arginine hydrochloride, glycine, proline or lysine at a concentration of 50 mM-300 mM; and / or sucrose, trehalose, maltose, sorbitol and / or mannitol at a concentration of 30 mg / ml-150 mg / ml.

9. The formulation according to claim 8, characterized in that, The stabilizer is: sodium chloride, arginine hydrochloride, glycine, proline or lysine at a concentration of 100mM-250mM; and / or sucrose, trehalose, maltose, sorbitol and / or mannitol at a concentration of 45mg / ml-100mg / ml.

10. The formulation according to claim 1, characterized in that, The buffer solution is any one of histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer, and glutamate buffer.

11. The formulation according to claim 1, characterized in that, The concentration of the buffer solution is 3mM-100mM.

12. The formulation according to claim 11, characterized in that, The concentration of the buffer solution is 5mM-80mM.

13. The formulation according to claim 11, characterized in that, The concentration of the buffer solution is 8mM-50mM.

14. The formulation according to claim 11, characterized in that, The concentration of the buffer solution is 10mM-30mM.

15. The formulation according to claim 1, characterized in that, The pH value of the buffer solution is 4.8-8.

0.

16. The formulation according to claim 15, characterized in that, The pH value of the buffer solution is 5.0-7.

2.

17. The formulation according to claim 15, characterized in that, The pH value of the buffer solution is 5.5-7.

0.

18. The formulation according to claim 1, characterized in that, The surfactant is polysorbate and / or poloxamer.

19. The formulation according to claim 18, characterized in that, The polysorbate is Tween-20 or Tween-80.

20. The formulation according to claim 1, characterized in that, The concentration of the surfactant is 0.01 mg / ml to 2 mg / ml.

21. The formulation according to claim 20, characterized in that, The concentration of the surfactant is 0.02 mg / ml to 1.5 mg / ml.

22. The formulation according to claim 20, characterized in that, The concentration of the surfactant is 0.03 mg / ml to 1 mg / ml.

23. The formulation according to claim 20, characterized in that, The concentration of the surfactant is 0.04 mg / ml to 0.5 mg / ml.

24. The formulation according to claim 20, characterized in that, The concentration of the surfactant is 0.05 mg / ml to 0.3 mg / ml.

25. The formulation according to claim 1, characterized in that, The formulation is any one of the following: (1) The antibody concentration is 10 mg / ml, 15 mg / ml, 25 mg / ml, 35 mg / ml, 50 mg / ml, 75 mg / ml, 100 mg / ml, 125 mg / ml, 150 mg / ml, 175 mg / ml or 200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80; (2) The antibody concentration is 10 mg / ml - 200 mg / ml; the stabilizer is 100 mM, 150 mM, 200 mM or 250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml or 100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM - 30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml - 0.3 mg / ml Tween-20 and / or Tween-80; (3) The antibody concentration is 10-200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM, 20 mM, 30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80; (4) The antibody concentration is 10-200 mg / ml; the stabilizer is 100 mM-250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml-100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM-30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5, 5.8, 6, 6.2, 6.5, 6.8 or 7.0; the surfactant is 0.05 mg / ml-0.3 mg / ml Tween-20 and / or Tween-80; (5) The antibody concentration is 10 mg / ml - 200 mg / ml; the stabilizer is 100 mM - 250 mM arginine hydrochloride and / or sodium chloride, and / or 45 mg / ml - 100 mg / ml sucrose, trehalose, mannitol and / or sorbitol; the buffer is 10 mM - 30 mM histidine-histidine hydrochloride buffer, citrate-disodium hydrogen phosphate buffer, acetate-sodium acetate buffer, phosphate buffer; the pH of the buffer is 5.5-7.0; the surfactant is 0.05 mg / ml, 0.10 mg / ml, 0.15 mg / ml, 0.2 mg / ml, 0.25 mg / ml, 0.3 mg / ml Tween-20 and / or Tween-80.

26. The formulation according to claim 1, characterized in that, The formulation is any one of the following: (1) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of histidine-histidine hydrochloride buffer, 100 mM - 250 mM of arginine hydrochloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (2) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of histidine-histidine hydrochloride buffer, 100 mM - 250 mM of sodium chloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (3) The formulation contains 10 mg / ml-200 mg / ml of anti-C5a antibody, 10 mM-30 mM of histidine-histidine hydrochloride buffer, 45 mg / ml-100 mg / ml of sucrose, and 0.05 mg / ml-0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (4) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of acetate buffer, 100 mM - 250 mM of arginine hydrochloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.

0. (5) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of acetate buffer, 100 mM - 250 mM of sodium chloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (6) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of acetate buffer, 45 mg / ml - 100 mg / ml of sucrose, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (7) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of phosphate buffer, 100 mM - 250 mM of arginine hydrochloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (8) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of phosphate buffer, 100 mM - 250 mM of sodium chloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (9) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of phosphate buffer, 45-100 mg / ml of sucrose, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (10) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of citrate-disodium hydrogen phosphate buffer, 100 mM - 250 mM of arginine hydrochloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (11) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of citrate-disodium hydrogen phosphate buffer, 100 mM - 250 mM of sodium chloride, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (12) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of citrate-disodium hydrogen phosphate buffer, 45-100 mg / ml of sucrose, and 0.05 mg / ml - 0.3 mg / ml of Tween 20 or Tween 80, wherein the pH of the buffer is 5.5-7.0; (13) The formulation comprises 10 mg / ml - 200 mg / ml of anti-C5a antibody, 10 mM - 30 mM of histidine-histidine hydrochloride buffer, 45 mg / ml - 100 mg / ml of mannitol or sorbitol or trehalose, 0.05 mg / ml - 0.3 mg / ml of Tween 80, and the pH of the buffer is 5.5-7.

0.

27. The formulation according to claim 1, characterized in that, The formulation is any one of the following: (1) The formulation contains 15 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 150 mM of arginine hydrochloride, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.

2. (2) The formulation contains 10 mg / ml of anti-C5a antibody, 10 mM of histidine-histidine hydrochloride buffer, 200 mM of arginine hydrochloride, and 0.15 mg / ml of Tween 20, wherein the pH of the buffer is 6.

4. (3) The formulation contains 15 mg / ml of anti-C5a antibody, 30 mM of histidine-histidine hydrochloride buffer, 150 mM of arginine hydrochloride, and 0.05 mg / ml of Tween 80, wherein the pH of the buffer is 5.

8. (4) The formulation contains 15 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 150 mM of sodium chloride, and 0.1 mg / ml of Tween 80, wherein the pH of the buffer is 6.0; (5) The formulation contains 15 mg / ml of anti-C5a antibody, 10 mM acetate buffer, 250 mM arginine hydrochloride, and 0.1 mg / ml of Tween 80, wherein the pH of the buffer is 6.2; (6) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM of phosphate buffer, 100 mM of arginine hydrochloride, and 0.1 mg / ml of Tween 80, wherein the pH of the buffer is 6.0; (7) The formulation contains 15 mg / ml of anti-C5a antibody, 20 mM histidine-histidine hydrochloride buffer, 100 mg / ml sucrose, 0.2 mg / ml Tween 80, and the pH of the buffer is 6.8; (8) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM histidine-histidine hydrochloride buffer, 50 mg / ml mannitol, 0.1 mg / ml Tween 80, and the pH of the buffer is 6.0; (9) The formulation comprises 15 mg / ml of anti-C5a antibody, 20 mM histidine-histidine hydrochloride buffer, 45 mg / ml sorbitol, 0.1 mg / ml Tween 80, and the pH of the buffer is 7.0; (10) The formulation comprises 30 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 80 mg / ml of trehalose, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.2; (11) The formulation comprises 50 mg / ml of anti-C5a antibody, 30 mM of histidine-histidine hydrochloride buffer, 60 mg / ml of sucrose, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.0; (12) The formulation comprises 100 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 50 mg / ml of sucrose, 0.1 mg / ml of Tween 80, and the pH of the buffer is 6.0; (13) The formulation comprises 150 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 60 mg / ml of sucrose, 0.2 mg / ml of Tween 80, and the pH of the buffer is 6.0; (14) The formulation comprises 200 mg / ml of anti-C5a antibody, 20 mM of histidine-histidine hydrochloride buffer, 70 mg / ml of sucrose, 0.3 mg / ml of Tween 80, and the pH of the buffer is 6.0; (15) The formulation comprises 150 mg / ml of anti-C5a antibody, 20 mM of phosphate buffer, 150 mM of arginine hydrochloride, and 0.2 mg / ml of Tween 80, wherein the pH of the buffer is 5.5; (16) The formulation comprises 150 mg / ml of anti-C5a antibody, 20 mM of citrate-disodium hydrogen phosphate buffer, 100 mM of sodium chloride, 0.1 mg / ml of Tween 20, wherein the pH of the buffer is 5.8; (17) The formulation comprises 150 mg / ml of anti-C5a antibody, 10 mM of histidine-histidine hydrochloride buffer, 200 mM of sodium chloride, 0.2 mg / ml of Tween 80, and the pH of the buffer is 6.

8.

28. The formulation according to claim 1, characterized in that, The formulation also contains preservatives and / or antioxidants.

29. The formulation according to claim 28, characterized in that, The preservative is ethylenediaminetetraacetic acid and / or diethylenetriaminepentaacetic acid; the antioxidant is methionine.

30. The formulation according to claim 29, characterized in that, The concentration of the preservative is 0-0.5 mg / ml, and the concentration of the antioxidant is 0-1.1 mg / ml.

31. The formulation according to claim 1, characterized in that, The preparation is a liquid preparation or a powder for injection.

32. Use of the formulation according to any one of claims 1-31 in the preparation of a medicament for treating diseases. The disease is selected from any one or more of the following: SIRS, ischemia / reperfusion-related injury, acute lung injury, pneumonia, inflammatory bowel disease, and ARDS caused by coronavirus.

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