CN116670288A

CN116670288A - Isolated anti-C5 a antibody, preparation and application thereof

Info

Publication number: CN116670288A
Application number: CN202280008248.5A
Authority: CN
Inventors: 沈丙权; 张雪瑾; 朱萍霞; 李忠
Original assignee: Staidson Beijing Biopharmaceutical Co Ltd
Current assignee: Staidson Beijing Biopharmaceutical Co Ltd
Priority date: 2021-12-22
Filing date: 2022-12-16
Publication date: 2023-08-29
Also published as: WO2023116574A1; TW202334230A

Abstract

An isolated anti-C5 a antibody is provided for use in the treatment of C5 a-mediated diseases and conditions. The preparation has strong stability under high temperature, illumination, oscillation, freeze thawing, acceleration and long-term conditions, can ensure that the preparation has good stability in the preparation, transportation and storage processes, and ensures the safety and quality controllability of clinical medication.

Description

Isolated anti-C5 a antibody, preparation and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an isolated anti-C5 a antibody, a preparation and application thereof.

Background

C5a is an active peptide in allergic and inflammatory processes that is formed by cleavage of complement component C5 by C5 convertase in the complement cascade. C5a stimulates mast cell degranulation, tumor necrosis factor-alpha (TNF-alpha) and histamine release, and recruits phagocytes to sites of infection and inflammation by increasing the expression of endothelial cell surface adhesion molecules (molnes, t.e. et al blood 2002,100,1869-1877;Riedemann,N.C.et al.Immunity 2003,19,193-202). In the case of some pathological stimuli, such as allograft rejection and asthma after transplantation, C5a also leads to increased vascular permeability (Gueler, f.et al j.am.soc.neprol.2008, 19,2302-2312;Krug,N.et al.Am.J.Respir.Crit.Care Med.2001,164,1841-1843;Khan,M.A.et al.Proc.Natl.Acad.Sci.USA 2013,110,6061-6066). C5a is a potent pro-inflammatory molecule that binds to a classical G-protein coupled receptor (GPCR) C5aRI (CD 88) and triggers activation of pro-inflammatory signaling pathways (Li, r.et al faseb j.2013,27, 855-864). C5aR is widely expressed on non-myeloid cells such as umbilical vascular endothelial cells (HUVEC), murine dermis, liver, lung and renal proximal tubules (Monsinjon, T.et al. FASEB J.2003,17,1003-1014;Gerard,C.et al.Annu.Rev.Immunol.1994,12,775-808;Haviland,D.L.et al.J.Immunol.1995,154,1861-1869). Furthermore, studies demonstrated that C5aR was expressed in glomerular endothelial cells, but not podocytes, suggesting that C5a may cause proteinuria primarily in renal endothelial cells (Tsai, i.j.et al cell. Mol. Life sci.2015,72, 3157-3171). Thus, blocking its binding to C5aR by neutralizing C5a becomes a method of treating C5a mediated diseases and conditions. The antibody INab308 (InflRx) against human C5a is disclosed in patent application WO2011063980, the C5a antibody MEDI-7814 (MedImmune) is disclosed in WO2012088247, and the C5a antibody BNJ383 (Alexion) is disclosed in US 10450370.

The present invention provides an isolated anti-C5 a antibody and its anti-C5 a antibody formulations having strong stability for the treatment of C5a mediated diseases and conditions.

Disclosure of Invention

In one aspect, the presentThe invention provides an isolated anti-C5 a antibody comprising V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO 9, LC-CDR2 comprising the amino acid sequence SEQ ID NO 10, and LC-CDR3 comprising the amino acid sequence SEQ ID NO 11; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO. 12, LC-CDR2 comprising the amino acid sequence SEQ ID NO. 10, and LC-CDR3 comprising the amino acid sequence SEQ ID NO. 11; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO. 4, HC-CDR2 comprising the amino acid sequence SEQ ID NO. 5, and HC-CDR3 comprising the amino acid sequence SEQ ID NO. 6; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO 13, LC-CDR2 comprising the amino acid sequence SEQ ID NO 14, and LC-CDR3 comprising the amino acid sequence SEQ ID NO 15; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO. 7, HC-CDR2 comprising the amino acid sequence SEQ ID NO. 8, and HC-CDR3 comprising the amino acid sequence SEQ ID NO. 3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO. 16, LC-CDR2 comprising the amino acid sequence SEQ ID NO. 17, and LC-CDR3 comprising the amino acid sequence SEQ ID NO. 11.

In some embodiments, the antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 18,v (V) _L Comprising the amino acid sequence SEQ ID NO. 22; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO 19, and V _L Comprising the amino acid sequence SEQ ID NO. 23; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 20, and V _L Comprising the amino acid sequence SEQ ID NO. 24; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 21, and V _L Which comprises the amino acid sequence SEQ ID NO. 25.

In some embodiments, any of the isolated anti-C5 a antibodies described above, comprising an Fc fragment. In some embodiments, the isolated anti-C5 a antibody is a full-length IgG antibody. In some embodiments, the isolated anti-C5 a antibody is a full length IgG1 or IgG4 antibody.

In some embodiments, the antibody comprises a heavy chain constant region comprising an amino acid sequence set forth in SEQ ID NO. 26 or 27 and a light chain constant region comprising an amino acid sequence set forth in SEQ ID NO. 28.

In some embodiments, the antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO. 18, said V _L Comprising the amino acid sequence SEQ ID NO. 22, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 26, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28; or (b)

The antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO 19, said V _L Comprising the amino acid sequence SEQ ID NO. 23, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 27, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28; or (b)

The antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO. 20, said V _L Comprising the amino acid sequence SEQ ID NO. 24, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 26, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28; or (b)

The antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO. 21, said V _L Comprising the amino acid sequence SEQ ID NO. 25, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 27, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28.

In another aspect, the invention provides an anti-C5 a antibody formulation comprising the isolated anti-C5 a antibody described above, a stabilizer, a surfactant, and a buffer.

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

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

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

In some embodiments, the buffer is any one of histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphate buffer, glutamate buffer.

In some embodiments, the buffer is at a concentration of 3mM-100mM; in some embodiments, the buffer is at a concentration of 5mM-80mM or 8mM-50mM; preferably, the concentration of the buffer is 10mM-30mM, in some embodiments, the concentration of the buffer is 10mM or 20mM or 30mM.

In some embodiments, the pH of the buffer is 4.8-8.0; in some embodiments, the pH of the buffer is 5.0-7.2; in some embodiments, the pH of the buffer is 5.0, 5.5, 5.8, 6, 6.2, 6.5, 6.8, 7.0, or 7.2; preferably, the pH of the buffer is between 5.5 and 7.0.

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.

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

In some embodiments, the formulation is any one of the following formulations:

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

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

(3) The concentration of the antibody is 10-200mg/ml; the stabilizer is arginine hydrochloride and/or sodium chloride with the concentration of 100mM-250mM, and/or sucrose, trehalose, mannitol and/or sorbitol with the concentration of 45mg/ml-100 mg/ml; the buffer solution is 10mM, 20mM, 30mM histidine-histidine hydrochloride buffer solution, citric acid-disodium hydrogen phosphate buffer solution, acetic acid-sodium acetate buffer solution and phosphate buffer solution; the pH value of the buffer solution is 5.5-7.0; the surfactant is tween-20 and/or tween-80 of 0.05mg/ml to 0.3 mg/ml;

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

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

In some embodiments, the formulation is any one of the following formulations:

(1) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer solution, 100mM-250mM of arginine hydrochloride, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(2) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer solution, 100mM-250mM of sodium chloride, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(3) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer solution, 45mg/ml-100mg/ml of sucrose, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(4) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM acetate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, and the pH value of the buffer is 5.5-7.0

(5) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM acetate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(6) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM acetate buffer, 45mg/ml-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(7) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM phosphate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(8) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM phosphate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(9) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of phosphate buffer solution, 45-100mg/ml of sucrose, 0.05mg/ml-0.3mg/ml of Tween 20 or Tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(10) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of citric acid-disodium hydrogen phosphate buffer solution, 100mM-250mM of arginine hydrochloride, 0.05mg/ml-0.3mg/ml of Tween 20 or Tween 80, and the pH value of the buffer solution is 5.5-7.0;

(11) The preparation comprises 10mg/ml-200mg/ml of an anti-C5 a antibody, 10mM-30mM of citric acid-disodium hydrogen phosphate buffer solution, 100mM-250mM of sodium chloride, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(12) The preparation comprises 10mg/ml-200mg/ml of an anti-C5 a antibody, 10mM-30mM of citric acid-disodium hydrogen phosphate buffer solution, 45-100mg/ml of sucrose, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(13) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer, 45mg/ml-100mg/ml of mannitol or sorbitol or trehalose, 0.05mg/ml-0.3mg/ml of Tween 80, and the pH value of the buffer is 5.5-7.0.

In some embodiments, the formulation is any one of the following formulations:

(1) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 150mM arginine hydrochloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.2;

(2) The preparation comprises 10mg/ml of anti-C5 a antibody, 10mM histidine-histidine hydrochloride buffer, 200mM arginine hydrochloride, 0.15mg/ml Tween 20, wherein the pH value of the buffer is 6.4;

(3) The preparation comprises 15mg/ml of anti-C5 a antibody, 30mM histidine-histidine hydrochloride buffer, 150mM arginine hydrochloride, 0.05mg/ml Tween 80, wherein the pH value of the buffer is 5.8;

(4) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 150mM sodium chloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(5) The preparation comprises 15mg/ml of anti-C5 a antibody, 10mM acetate buffer, 250mM arginine hydrochloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.2;

(6) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM phosphate buffer, 100mM arginine hydrochloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(7) The preparation comprises 15mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 100mg/ml sucrose, 0.2mg/ml Tween 80, wherein the pH value of the buffer is 6.8;

(8) The preparation comprises 15mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 50mg/ml mannitol, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(9) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 45mg/ml sorbitol, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 7.0;

(10) The preparation comprises 30mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 80mg/ml trehalose, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.2;

(11) The preparation comprises 50mg/ml of an anti-C5 a antibody, 30mM histidine-histidine hydrochloride buffer, 60mg/ml sucrose, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(12) The preparation comprises 100mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 50mg/ml sucrose, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(13) The preparation comprises 150mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 60mg/ml sucrose, 0.2mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(14) The preparation comprises 200mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 70mg/ml sucrose, 0.3mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(15) The preparation comprises 150mg/ml of anti-C5 a antibody, 20mM phosphate buffer, 150mM arginine hydrochloride, 0.2mg/ml Tween 80, wherein the pH value of the buffer is 5.5;

(16) The formulation comprises 150mg/ml of anti-C5 a antibody, 20mM citric acid-disodium hydrogen phosphate buffer, 100mM sodium chloride, 0.1mg/ml tween 20, the pH of the buffer being 5.8;

(17) The formulation contained 150mg/ml of anti-C5 a antibody, 10mM histidine-histidine hydrochloride buffer, 200mM sodium chloride, 0.2mg/ml Tween 80, the pH of the buffer being 6.8.

In some embodiments, the formulation may further comprise a preservative and/or an antioxidant. The preservative or antioxidant is a preservative or antioxidant commonly used in antibody preparations. In some embodiments, the preservative is ethylenediamine tetraacetic acid (EDTA), diethylenetriamine pentaacetic acid (DTPA), or a combination thereof; the antioxidant is methionine.

In some embodiments, the preservative is at a concentration of 0-0.5mg/ml, in some embodiments, 0mg/ml, 0.02mg/ml, 0.15mg/ml, 0.25mg/ml, 0.4ml/ml, or 0.5mg/ml. In some embodiments, the antioxidant is present at a concentration of 0-1.1mg/ml. In some embodiments, the concentration of antioxidant is 0mg/ml, 0.15mg/ml, 0.75mg/ml, or 0.11mg/ml.

In some embodiments, the antibody formulation described above is a liquid formulation or a powder for injection.

In another aspect, the invention provides the use of any of the anti-C5 a antibodies as described above, any of the anti-C5 a antibody formulations as described above, in the manufacture of a medicament for the treatment of a disease.

In another aspect, the invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of any one of the anti-C5 a antibodies or antibody formulations described above. The application mode comprises the following steps: intravenous, intraarterial, intraperitoneal, intrapulmonary, oral, inhalational, intravascular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, mucosal or transdermal. In some embodiments, the formulation is administered intravenously. In some embodiments, the formulation is administered subcutaneously. In some embodiments, the formulation is administered via muscle.

In some embodiments, the disease or disorder is an inflammatory, respiratory, or autoimmune disease or disorder; preferably, the disease is selected from any one or Several of Inflammatory Response Syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia/reperfusion-related injury, acute lung injury, pneumonia, acute and chronic graft rejection in transplanted patients, graft-versus-host reaction, glomerulonephritis, solid renal failure, rheumatoid arthritis, autoimmune disease, bechterew's disease, lupus disease, inflammatory bowel disease, crohn's disease, tumor growth and solid organ cancer.

The anti-C5 a antibody and the preparation thereof provided by the invention have the following excellent effects:

1. the anti-C5 a antibody can obviously reduce the expression of CD11b in endogenous C5a induced human neutrophils, does not inhibit the haemolytic activity of blood plasma, and can obviously reduce cytokine storm and inflammatory response caused by novel coronaviruses in vivo.

2. The anti-C5 a antibody preparation has strong stability under high temperature, illumination, oscillation, freeze thawing, acceleration and long-term conditions, can ensure that the preparation has good stability in the preparation, transportation and storage processes, and ensures the safety and quality controllability of clinical medication.

Drawings

FIGS. 1A-1B show the results of a CD11B blocking assay. The results in fig. 1A demonstrate that in human neutrophils, the anti-C5 a antibodies Cab42, cab44, cab45 can block human endogenous C5 a-induced up-regulation of CD11 b. The results in FIG. 1B show that the anti-C5 a antibody Cab42 blocks endogenous human C5 a-induced up-regulation of CD11B expression in human neutrophils even in the presence of more than 50-fold molar amounts of C5 in the reaction system as compared to the control antibody INab 308.

FIGS. 2A-2B show the plasma hemolytic activity of the C5a antibody. In the classical activation pathway, the anti-C5 a antibodies Cab35, cab42, cab44, cab45 (fig. 1A) did not inhibit plasma hemolytic activity compared to the control antibody ecllizumab. In the bypass-activated pathway, the anti-C5 a antibodies Cab35, cab42, cab44, cab45 (fig. 2B) did not inhibit plasma hemolytic activity compared to the control antibody ecllizumab.

Detailed Description

In order to make the technical problems to be solved, the technical scheme adopted and the advantages of the invention more clear, the invention will be described in detail with reference to the accompanying drawings and specific embodiments. The following examples are illustrative of the invention and are not intended to limit the scope of the invention.

The reagents used in the examples below, unless otherwise specified, were formulated in conventional manner or were commercially available; the experimental methods used, unless otherwise specified, are all conventional; the materials, instruments and the like used, unless otherwise specified, are commercially available.

Example 1: screening and preparation of antibodies

Blood RNA was extracted from 2000 human blood, and cDNA first strand was synthesized by reverse transcription, human V _H 、V _L Respectively amplifying the specific primers of V _H 、V _L The segment is recovered by glue, and V is recovered by a segment of linker _H 、V _L Splicing into scFv form, then inserting into yeast display plasmid PYD1, and electrotransferring yeast to obtain yeast display human antibody scFv library.

The pool of native yeast display human antibody scfvs was enriched by MACS magnetic bead sorting with biotinylated C5a, and the pool was transferred to phage display systems in order to allow rapid identification of the C5a antibody population. And continuing to perform 3 rounds of screening on phage by using C5a, and then selecting a monoclonal antibody to perform combination ELISA identification and in-vitro biological activity evaluation to obtain the lead antibody.

In order to reduce the potential immunogenicity risk of the candidate molecule, the human embryo gene with the closest relation with the maternal sequence is used as a template, and simultaneously, the structure prediction analysis is combined, and the amino acid sequence of the non-embryo gene in the maternal sequence framework region is restored and mutated into the amino acid sequence of the embryo gene, so that the humanization of the candidate molecule is improved. And then, after affinity maturation and isomerisation risk points are removed, antibodies of Cab35, cab42, cab44 and Cab45 are obtained after Fc modification. The 4 antibodies were purified for expression and used in subsequent experiments. The heavy chain constant region amino acid sequences of Cab35, cab42, cab44 and Cab45 antibodies are shown IN SEQ ID NO:26, the amino acid sequence of the light chain constant region is shown as SEQ ID NO:28, the amino acid sequences of specific antibodies are shown in the following table.

TABLE 1 CDR sequences of anti-C5 a antibodies

TABLE 2 anti-C5 a antibody sequences

Example 2: CD11b blocking assay in Whole blood

Upregulation of CD11b expression is a characteristic and sensitive marker of neutrophil activation, which is assessed by CD11b levels in neutrophils. The present invention uses a human whole blood model and uses INab308 (WO 2011063980A1, infllaRx) as a control to evaluate the blocking activity of antibodies Cab42, cab44 and Cab45 on endogenous human C5 a. Human whole blood was incubated with human C5a alone or in combination with various concentrations of each antibody, respectively. After incubation, the erythrocytes were lysed by staining with the detection antibody CD11b, FITC, and the CD11b MFI was analyzed by flow cytometry to reflect the level of neutrophil activation in the blood.

As shown in fig. 1A, the optimized antibodies all significantly reduced the expression of CD11b in endogenous C5 a-induced human neutrophils, even at an Ab: ag molar ratio of 0.5:1, achieving a comparable ability to control antibody INab308 to inhibit CD11b upregulation.

As shown in fig. 1B and table 3, since the binding of the antibody Cab42 to human C5 is weak, both the control antibody INab308 and the antibody Cab42 can inhibit the expression of CD11B in human neutrophils induced by endogenous C5a, even if more than 50 times of C5 is present in the reaction system, the antibody Cab42 reduces the up-regulation of CD11B in human neutrophils induced by endogenous C5a with higher potency than the control antibody INab 308.

TABLE 3 Table 3

Antibodies to	eC5a IC50(nM)	eC5a+50*C5 IC50(nM)
Cab42	2.05	31.04
INab308	1.95	42.53

Example 3: plasma hemolytic Activity of anti-c 5a antibodies

The complement system can be independently activated by three activation pathways, ultimately forming an tapping complex. Under specific experimental conditions, it can directly attack the cell membrane of erythrocytes, leading to erythrocyte lysis. Based on this mechanism, the present invention conducted experiments to assess whether the C5a antibodies of the present invention would affect the biological activity of C5 convertases to cleave C5 to C5 b.

The role of C5a antibodies in the complement-mediated classical activation pathway was examined: the 50% complement hemolysis assay is a method for determining total classical complement activity in serum. The assay is a lysis assay, which uses antibodies as activators of the classical complement pathway to sensitize erythrocytes and dilutions of test serum are performed at different concentrations to determine the amount required to achieve 50% lysis (CHSO). The rate of hemolysis can be determined by spectrophotometry. The 50% complement hemolysis assay provides an indirect measure of Terminal Complement Complex (TCC) formation, as TCC itself has a direct effect on the measured hemolysis. This experiment is well known and routine to those skilled in the art, such as, for example, limei Zhao et al front immunol 2017 May 31;8:636; zhao et al Parasites & vectors.2014 Feb 24; described in 7:80.

Briefly, guinea pig erythrocytes were prepared by centrifugation of fresh guinea pig whole blood and then sensitized with sheep anti-erythrocyte antibodies. This procedure activates the complement classical hemolysis pathway, leading to erythrolysis. The absorbance at 412nm was read. The C5 antibody Eculizumab was used as a control.

The role of C5a antibodies in complement-mediated alternative activation pathways was examined in short, rabbit erythrocytes could activate the alternative pathway to form an tapping complex, leading to lysis of rabbit erythrocytes without antibody sensitization. After adding ethylene glycol bisaminotetraacetic acid (EGTA) into the reaction system, the substance can be mixed with Ca in blood plasma ²⁺ Chelate with Mg ²⁺ The classical pathway is blocked because of its weak binding capacity. The above 50% complement hemolysis assay was used to measure activation of the alternative pathway. The C5 antibody Eculizumab was used as a control.

As shown in fig. 2A, the C5 antibody ecalizumab, when added, inhibits hemolytic reactions in a dose-dependent manner, whereas antibodies Cab35, cab42, cab44, cab45 do not inhibit total classical complement activity. As shown in FIG. 2B, the addition of the C5 antibody Eculizumab inhibited the hemolytic reaction, while antibodies Cab35, cab42, cab44, cab45 did not inhibit the alternative pathway activity.

In summary, the anti-C5 a antibodies of the invention do not affect the function of C5b in the complement-mediated classical activation pathway nor the function of C5b in the alternative activation pathway.

Example 4: in vivo effects of anti-C5 a antibodies in treating coronavirus induced ARDS

An ARDS animal disease model was constructed to evaluate the therapeutic effect of the anti-C5 a antibodies of the invention in vivo.

ARDS animal disease model and normal control group: 46C 5a humanized mice (purchased from Shanghai south mode biotechnology Co., ltd.) were used in total, and the raising conditions were room temperature 20-26℃and relative humidity 40-70%, and 12 hours of light and shade were alternated. At 4, 3 and 2 days prior to the experiment, 40 of the mice were injected with adenovirus carrying and expressing SARS-CoV-2N protein (see: ting Gao et al, highly pathogenic coronavirus N protein aggravates lung injury by MASP-2-mediated complement over-activation medRxiv 2020.03.29.20041962; https:// doi.org/10.1101/2020.03.29.20041962), 7.5X10 ⁸ PFU/100. Mu.L/time/day, the remaining 6 mice were injected with sodium chloride solution (as a normal control). Mice were injected with the corresponding doses of antibody or sodium chloride solution on day 0 (day of the experiment) as follows.

Dosing and animal grouping: mice were divided into the following groups and treated with the corresponding reagents: (1) Normal control group (n=6), 100 μl of 0.9% sodium chloride solution was injected; (2) Disease model control group (n=10), 100 μl of 9% sodium chloride solution was injected; (3) Low dose experimental group (n=10), injected with antibody, at a dose of 1mg/kg; (4) Medium dose experimental group (n=10), injected with antibody, at a dose of 3mg/kg; (5) High dose experimental group (n=10), antibody was injected at a dose of 10mg/kg. After 30min of administration as described above, the mice were injected with LPS-K235 (Sigma-Aldrich) at a concentration of 1mg/mL, 100. Mu.L/mouse for the disease model control group and each experimental group. For the normal control group, sodium chloride solution was injected. All reagents in this experiment were administered by tail vein injection. The experiment was performed under the approval of the ethical committee of the Beijing Biotechnology institute and met the relevant regulatory standards.

Survival rate detection: survival of each group of mice was observed and analyzed at 12h,24h,36h,48h,60h and 72h, respectively, after dosing.

White blood cell count: the mice were anesthetized and the orbit was collected 72 hours after dosing. By usingSerial hematology analyzers perform whole blood white blood cell counts and classifications, including white blood cell count (WBC), neutrophil (Neut), lymphocyte (Lymph), monocyte (Mono).

Survival rate results: within 72 hours after dosing, animals in both the normal control group and the high-dose experimental group administered different anti-C5 a antibodies (Cab 35, cab42, cab44, or Cab 45) survived. The overall mortality in the model control group was 30% (3/10). In the low dose experimental groups administered different anti-C5 a antibodies (Cab 35, cab42, cab44 or Cab 45), the overall mortality was 10-20% (1-2/10); in the medium dose experimental groups administered with different anti-C5 a antibodies (Cab 35, cab42, cab44 or Cab 45), the overall mortality was 10% (1/10). The results show that the anti-C5 a antibody can effectively reduce or prevent death of mice caused by coronaviruses and improve survival rate of the mice.

Whole blood white blood cell count results: the mice in the model control group had reduced levels of WBC, lymph and Mono compared to the normal control group, with statistically significant differences (P < 0.05). Three dose experimental groups administered with different anti-C5 a antibodies (Cab 35, cab42, cab44 or Cab 45) showed an increase in WBC and Lymph numbers compared to the model control group, and the differences between the model control group and the medium and high dose experimental groups were all statistically significant (P < 0.05). The above results indicate that the anti-C5 a antibodies of the application help restore the equilibrium state of immune cells in mice model for ARDS disease.

Inflammatory cytokine results: compared with the normal control group, the levels of GM-CSF, IL-1 beta, IL-6, TNF-alpha and MCP-1 are obviously increased in the animals of the model control group, and the difference between the two levels has statistical significance (P < 0.05). The levels of GM-CSF, IL-1β, IL-6, TNF- α, MCP-1, C5a were decreased in 3 dose experimental groups administered with different anti-C5 a antibodies (Cab 35, cab42, cab44, or Cab 45) as compared to the model control group. Moreover, the difference in the levels of most of these cytokines in the dose experimental and model groups was statistically significant (P < 0.05). The above results indicate that the anti-C5 a antibodies of the invention are capable of significantly reducing cytokine storm and inflammatory responses caused by novel coronaviruses in vivo.

EXAMPLE 5 preparation of anti-C5 a antibody formulations

The anti-C5 a antibody formulation was formulated as follows:

table 4 anti-C5 a antibody formulation recipe

Preparations containing respective C5a antibodies (Cab 35, cab42 (IgG 1 mutation), cab44, cab45 (IgG 1 mutation)) containing V were prepared separately with reference to the prescription of Table 4 _H 、V _L And heavy and light chain constant regions, the specific antibody sequences are as follows:

TABLE 5 antibody sequences

Antibody name	V _H	V _L	Heavy chain constant region	Light chain constant region
Cab35 antibody	SEQ ID NO:18	SEQ ID NO:22	SEQ ID NO:26	SEQ ID NO:28
Cab42 (IgG 1 mutant) antibody	SEQ ID NO:19	SEQ ID NO:23	SEQ ID NO:27	SEQ ID NO:28
Cab44 antibodies	SEQ ID NO:20	SEQ ID NO:24	SEQ ID NO:26	SEQ ID NO:28
Cab45 (IgG 1 mutant) antibodies	SEQ ID NO:21	SEQ ID NO:25	SEQ ID NO:27	SEQ ID NO:28

Example 6 stability test of anti-C5 a antibody preparation

Setting conditions such as high temperature, illumination, oscillation, freeze thawing, acceleration, long term and the like, and inspecting the stability of different prescriptions. The detection items include visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, insoluble particles, aggregation properties, degradation properties, and charge heterogeneity of the antibody preparation. Wherein, the polymer characteristic is detected by adopting a SEC method and an NR-CE-SDS method, and the charge heterogeneity is detected by adopting a CEX method.

1. Stability test under high temperature conditions

The stability of each formulation of each antibody was tested at high temperature (40 ℃ ± 2 ℃,75% ± 5% rh (relative humidity), 0 days, 1 week, 2 weeks, 3 weeks).

(1) The results of each formulation test for the exemplary Cab35 antibody are shown in table 6: the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, bioactivity, thermal stability, insoluble particles of each antibody formulation were substantially unchanged relative to day 0; the polymers and fragments increased slightly and the main peak decreased slightly; the variation amplitude of the SEC detection polymer is within 0.9 percent; the variation amplitude of the NR-CE-SDS detected polymer is within 0.8%, the variation amplitude of the main peak is within 2.7%, and the variation amplitude of the fragment is within 2.0%. The above results indicate that the formulation of Cab35 has good stability under high temperature conditions.

TABLE 6 results of high temperature test of Cab35 antibodies

(2) The respective formulations of Cab42 (IgG 1 mutation), cab44 and Cab45 (IgG 1 mutation) antibodies also show good stability under high temperature conditions: within 3 weeks of high temperature, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, bioactivity, thermostability, insoluble particles of the three antibody formulations were substantially unchanged, the aggregates and fragments were slightly increased, and the major peaks were slightly reduced, relative to day 0: the variation amplitude of the SEC detection polymer is within 1.0 percent; the variation amplitude of the NR-CE-SDS detected polymer is within 0.9%, the variation amplitude of the main peak is within 2.6%, and the variation amplitude of the fragment is within 2.0%.

The above results demonstrate that the aggregation and degradation properties of the anti-C5 a antibody formulations of the invention do not significantly change under high temperature conditions, and the formulations are relatively stable.

2. Stability test under illumination

Under the illumination condition (5+/-3 ℃, 4500+/-500 lx, 5W/m) ² Light for 0 day, 3 days, 5 days, 1 week, 2 weeks) to examine the stability of each antibody preparation.

(1) Table 7 shows the results of each formulation test for exemplary antibody Cab42 (IgG 1 mutation): within 2 weeks of light exposure, the antibody formulation showed substantially no change in visible foreign matter, concentration, pH, osmotic pressure, viscosity, bioactivity, thermal stability, insoluble particles relative to day 0; the polymers and fragments increased slightly and the main peak decreased slightly: the variation amplitude of the SEC detection polymer is within 3.10%; the variation range of the NR-CE-SDS detected polymer is within 1.9%, the variation range of the main peak is within 4.4%, and the variation range of the fragment is within 2.5%. The above results indicate that Cab42 (IgG 1 mutation) antibody preparations can remain substantially stable under light conditions.

TABLE 7 results of light test of Cab42 (IgG 1 mutant) antibody preparations

(2) The respective formulations of Cab35, cab44 and Cab45 (IgG 1 mutation) antibodies also showed good stability under light conditions: within 2 weeks of light exposure, the antibody formulation has substantially no change in visible foreign matter, concentration, pH, osmotic pressure, viscosity, bioactivity, thermal stability, insoluble particles over 2 weeks of light exposure relative to day 0; the polymers and fragments have an increasing tendency and the main peak has a slightly decreasing tendency: the variation amplitude of the SEC detection polymer is within 3.15 percent; the variation range of the NR-CE-SDS detected polymer is within 2.1%, the variation range of the main peak is within 4.8%, and the variation range of the fragment is within 2.3%.

The above results demonstrate that the aggregation and degradation properties of the anti-C5 a antibody formulations of the invention do not significantly change under light conditions and the formulations remain substantially stable.

3. Stability test under Oscillating conditions

The stability of each antibody preparation was examined under shaking conditions (5.+ -. 3 ℃ C., 100rpm shaking for 0 days, 1 week, 2 weeks).

(1) Table 8 shows the results of each formulation test for exemplary antibody Cab 44: within 2 weeks of shaking, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, bioactivity, thermal stability, insoluble particles of each antibody preparation were substantially unchanged relative to day 0; the variation amplitude of the SEC detection polymer is very small and is within 0.13 percent; the variation amplitude of the CEX detection acidic peak, the main peak and the alkaline peak is within 0.40 percent, and almost no variation exists; the amplitude of the variation of the NR-CE-SDS aggregate, the main peak and the fragment was within 0.42%, and almost no variation was observed. The above results demonstrate excellent stability of the Cab44 antibody formulation under shaking conditions.

TABLE 8 results of shaking test of anti-Cab 44 antibody formulations

(2) Each formulation of Cab35, cab42 (IgG 1 mutation) and Cab45 (IgG 1 mutation) antibodies also exhibited excellent stability under shaking conditions: within 2 weeks of shaking, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, bioactivity, thermal stability, insoluble particles of each antibody preparation were substantially unchanged relative to day 0; the variation amplitude of the SEC detection polymer is small, and the variation is within 0.15 percent; the variation amplitude of the CEX detection acidic peak, the main peak and the alkaline peak is within 0.38%, and almost no variation exists; the amplitude of the variation of the NR-CE-SDS aggregate, the main peak and the fragment was within 0.41%, and almost no variation was observed.

The results of each of the above shaking tests demonstrate that the anti-C5 a antibody formulations of the invention have excellent stability under shaking conditions.

4. Stability test under Freeze thawing conditions

The stability of each antibody preparation was tested under freeze-thawing conditions (freeze: -20 ℃ + -5 ℃ C.;. Thawing: room temperature, freeze-thawing 0, 1, 3, 5 times).

(1) Table 9 shows the results of each formulation test for exemplary antibody Cab45 (IgG 1 mutation): within 5 times of freeze thawing, the foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability and insoluble particles of each antibody preparation are not basically changed relative to the freeze thawing for 0 time; the variation of the SEC detection polymer is very small and is within 0.15 percent; CEX detects that the variation amplitude of the acid peak, the main peak and the alkaline peak is within 0.42 percent, and almost no variation exists; the variation amplitude of the detected polymer, main peak and fragment of NR-CE-SDS is within 0.39%, and almost no variation exists. The above results demonstrate the excellent stability of the formulations of Cab45 (IgG 1 mutant) antibodies under freeze-thawing conditions.

TABLE 9 freeze-thaw test results of Cap45 (IgG 1 mutant) antibody formulations

(2) Each formulation of Cab35, cab42 (IgG 1 mutation) and Cab44 antibodies also exhibited excellent stability under freeze-thawing conditions: within 5 times of freeze thawing, the foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability and insoluble particles of each antibody preparation are not basically changed relative to the freeze thawing for 0 time; the SEC detection polymer has little change, and the change is within 0.16 percent; CEX detects that the variation amplitude of the acid peak, the main peak and the alkaline peak is within 0.40 percent, and almost no variation exists; the variation amplitude of the detected polymer, main peak and fragment of NR-CE-SDS is within 0.42%, and almost no variation exists.

The results of each test item in the freeze-thawing test show that the anti-C5 a antibody preparation provided by the invention has excellent stability under the freeze-thawing condition.

5. Stability test under accelerated conditions

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, 2 months).

(1) Table 10 shows the results of each formulation test for exemplary antibody Cab42 (IgG 1 mutation): within 2 months of acceleration, the antibody formulation was substantially unchanged in visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particles relative to day 0; the SEC detection polymer has no change or little change, and the change range is within 0.31 percent; CEX detected the slight increase in acid and base peaks and the slight decrease in major peaks; the variation amplitude of the acid peak is within 3.10 percent, the variation amplitude of the alkali peak is within 0.9 percent, and the variation amplitude of the main peak is within 4 percent; the increase of the NR-CE-SDS detected polymer is within 0.5%, the decrease of the main peak is within 1%, and the increase of the fragment is within 0.5%. The above results indicate that the preparation of Cab42 (IgG 1 mutant) antibodies can remain substantially stable under accelerated conditions.

TABLE 10 accelerated test results of antibody preparations against Cab42 (IgG 1 mutation)

(2) Each formulation of Cab35, cab44 and Cab45 (IgG 1 mutation) antibodies also exhibited excellent stability under accelerated conditions: within 2 months of acceleration, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particles of each antibody formulation were substantially unchanged relative to day 0. The SEC detection polymer has no change or little change, and the change range is within 0.35 percent; CEX detected the slight increase in acid and base peaks and the slight decrease in major peaks; the variation amplitude of the acid peak is within 3.13 percent, the variation amplitude of the alkali peak is within 1.0 percent, and the variation amplitude of the main peak is within 4.2 percent; the increase of the NR-CE-SDS detected polymer is within 0.5%, the decrease of the main peak is within 1%, and the increase of the fragment is within 0.5%.

The acceleration test results show that the anti-C5 a antibody preparation provided by the invention has no obvious change under acceleration conditions and can basically keep stable.

6. Stability test under Long term conditions

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

(1) Table 11 shows the results of each formulation test for exemplary antibody Cab 44: the antibody formulation has substantially unchanged visible impurities, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles relative to day 0 over a period of 6 months under long term conditions; the variation amplitude of the SEC detection polymer is within 0.11 percent, and almost no variation exists; CEX detects that the acid peak, the main peak and the alkaline peak have almost no change, the change amplitude of the acid peak is within 0.44 percent, the change amplitude of the main peak is within 0.49 percent, and the change amplitude of the alkaline peak is within 0.13 percent; the NR-CE-SDS detects the polymer, main peak and fragment with almost no change, the increase of the polymer is within 0.5%, the decrease of the main peak is within 0.5%, and the increase of the fragment is within 0.3%. The above results demonstrate the excellent stability of the formulation of Cab44 antibodies under long term conditions.

TABLE 11 results of long-term stability of Cab44 antibody formulations

(2) Each formulation of Cab35, cab42 (IgG 1 mutation) and Cab45 (IgG 1 mutation) antibodies also exhibited excellent stability under long term conditions: the antibody formulation showed substantially no change in visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles over 6 months under long term conditions relative to day 0. The SEC detection polymer has little change, and the change range is within 0.13 percent; CEX detects that the acid peak, the main peak and the alkaline peak have almost no change, the change amplitude of the acid peak is within 0.46 percent, the change amplitude of the main peak is within 0.5 percent, and the change amplitude of the alkaline peak is within 0.15 percent; the NR-CE-SDS detects the polymer, main peak and fragment with almost no change, the increase of the polymer is within 0.51%, the decrease of the main peak is within 0.5%, and the increase of the fragment is within 0.4%.

The results of the above long-term tests demonstrate that the anti-C5 a antibody formulations of the invention have excellent stability under long-term conditions.

In conclusion, the anti-C5 a antibody preparation provided by the invention has strong stability under high temperature, illumination, oscillation, freeze thawing, acceleration and long-term conditions, and can ensure that the preparation has good stability in the preparation, transportation and storage processes, and ensure the safety and quality controllability of clinical medication.

Claims

An isolated anti-C5 a antibody comprising V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO 9, LC-CDR2, whichComprising the amino acid sequence SEQ ID NO. 10, and LC-CDR3 comprising the amino acid sequence SEQ ID NO. 11; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, HC-CDR2 comprising the amino acid sequence SEQ ID NO:2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO. 12, LC-CDR2 comprising the amino acid sequence SEQ ID NO. 10, and LC-CDR3 comprising the amino acid sequence SEQ ID NO. 11; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO. 4, HC-CDR2 comprising the amino acid sequence SEQ ID NO. 5, and HC-CDR3 comprising the amino acid sequence SEQ ID NO. 6; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO 13, LC-CDR2 comprising the amino acid sequence SEQ ID NO 14, and LC-CDR3 comprising the amino acid sequence SEQ ID NO 15; or (b)

The antibody comprises V _H The V is _H Comprising: HC-CDR1 comprising the amino acid sequence SEQ ID NO. 7, HC-CDR2 comprising the amino acid sequence SEQ ID NO. 8, and HC-CDR3 comprising the amino acid sequence SEQ ID NO. 3; v (V) _L The V is _L Comprising: LC-CDR1 comprising the amino acid sequence SEQ ID NO. 16, LC-CDR2 comprising the amino acid sequence SEQ ID NO. 17, and LC-CDR3 comprising the amino acid sequence SEQ ID NO. 11.
The antibody of claim 1, wherein the antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 18, and V _L Comprising the amino acid sequence SEQ ID NO. 22; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO 19, and V _L Comprising the amino acid sequence SEQ ID NO. 23; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 20, and V _L Comprising the amino acid sequence SEQ ID NO. 24; or (b)

The antibody comprises V _H Comprising the amino acid sequence SEQ ID NO. 21, and V _L Which comprises the amino acid sequence SEQ ID NO. 25.
The antibody of claim 2, wherein the antibody comprises a heavy chain constant region comprising amino acid sequence SEQ ID No. 26 or 27 and a light chain constant region comprising amino acid sequence SEQ ID No. 28.
The antibody of claim 2, wherein the antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO. 18, said V _L Comprising the amino acid sequence SEQ ID NO. 22, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 26, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28; or (b)

The antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO 19, said V _L Comprising the amino acid sequence SEQ ID NO. 23, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 27, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28; or (b)

The antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO. 20, said V _L Comprising the amino acid sequence SEQ ID NO. 24, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 26, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28; or (b)

The antibody comprises V _H 、V _L Heavy chain constant region and light chain constant region, the V _H Comprising the amino acid sequence SEQ ID NO. 21, said V _L Comprising the amino acid sequence SEQ ID NO. 25, the heavy chain constant region comprising the amino acid sequence SEQ ID NO. 27, and the light chain constant region comprising the amino acid sequence SEQ ID NO. 28.
An anti-C5 a antibody formulation comprising the isolated anti-C5 a antibody of any one of claims 1-4, a stabilizer, a surfactant, and a buffer.
The formulation of claim 5, wherein the antibody concentration is 1mg/ml to 300mg/ml; preferably, the antibody concentration is 10mg/ml to 200mg/ml.
The formulation of claim 5 or 6, wherein the stabilizer is any one or a combination of several of sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine hydrochloride, glycine, proline, lysine; preferably, the stabilizer is any one or a combination of a plurality of sucrose, arginine hydrochloride, sodium chloride, mannitol, trehalose and sorbitol.
The formulation of any one of claims 5-7, wherein the stabilizer is: sodium chloride, arginine hydrochloride, glycine, proline or lysine at a concentration of 50mM-300mM, preferably 100mM-250 mM; and/or sucrose, trehalose, maltose, sorbitol and/or mannitol at a concentration of 30mg/ml to 150mg/ml, preferably 45mg/ml to 100 mg/ml.
The formulation of any one of claims 5-8, wherein the buffer is any one of histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphate buffer, glutamate buffer.
The formulation of any one of claims 5-9, wherein the buffer is at a concentration of 3mM-100mM; preferably, the concentration of the buffer is 5mM-80mM or 8mM-50mM; more preferably, the concentration of the buffer is 10mM-30mM.
The formulation according to any one of claims 5 to 10, wherein the pH of the buffer is 4.8 to 8.0; preferably, the pH of the buffer is 5.0-7.2; more preferably, the pH of the buffer is between 5.5 and 7.0.
The formulation according to any one of claims 5 to 11, wherein the surfactant is a polysorbate and/or a poloxamer; preferably, the polysorbate is tween-20 or tween-80.
The formulation according to any one of claims 5 to 12, wherein the concentration of the surfactant is 0.01mgml to 2mg/ml; preferably, the concentration of the surfactant is 0.02mgml-1.5mg/ml or 0.03mg/ml-1mg/ml or 0.04mg/ml-0.5mg/ml; more preferably, the concentration of the surfactant is from 0.05mg/ml to 0.3mg/ml.
The formulation of claim 5, wherein the formulation is any one of the following:

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

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

(3) The concentration of the antibody is 10-200mg/ml; the stabilizer is 100mM-250mM arginine hydrochloride and/or sodium chloride, and/or 45mg/ml-100mg/ml sucrose, trehalose, mannitol and/or sorbitol; the buffer solution is 10mM, 20mM, 30mM histidine-histidine hydrochloride buffer solution, citric acid-disodium hydrogen phosphate buffer solution, acetic acid-sodium acetate buffer solution and phosphate buffer solution; the pH value of the buffer solution is 5.5-7.0; the surfactant is tween-20 and/or tween-80 of 0.05mg/ml to 0.3 mg/ml;

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

(5) The concentration of the antibody is 10mg/ml-200mg/ml; the stabilizer is 100mM-250mM arginine hydrochloride and/or sodium chloride, and/or 45mg/ml-100mg/ml sucrose, trehalose, mannitol and/or sorbitol; the buffer solution is 10mM-30mM histidine-histidine hydrochloride buffer solution, citric acid-disodium hydrogen phosphate buffer solution, acetic acid-sodium acetate buffer solution and phosphate buffer solution; the pH value of the buffer solution is 5.5-7.0; the surfactant is Tween-20 and/or Tween-80 at 0.05mg/ml, 0.10mg/ml, 0.15mg/ml, 0.2mg/ml, 0.25mg/ml, 0.3 mg/ml.
The formulation of claim 5, wherein the formulation is any one of the following:

(1) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer solution, 100mM-250mM of arginine hydrochloride, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(2) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer solution, 100mM-250mM of sodium chloride, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(3) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer solution, 45mg/ml-100mg/ml of sucrose, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(4) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM acetate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, and the pH value of the buffer is 5.5-7.0

(5) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM acetate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(6) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM acetate buffer, 45mg/ml-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(7) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM phosphate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(8) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM phosphate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml tween 20 or tween 80, wherein the pH value of the buffer is 5.5-7.0;

(9) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of phosphate buffer solution, 45-100mg/ml of sucrose, 0.05mg/ml-0.3mg/ml of Tween 20 or Tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(10) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of citric acid-disodium hydrogen phosphate buffer solution, 100mM-250mM of arginine hydrochloride, 0.05mg/ml-0.3mg/ml of Tween 20 or Tween 80, and the pH value of the buffer solution is 5.5-7.0;

(11) The preparation comprises 10mg/ml-200mg/ml of an anti-C5 a antibody, 10mM-30mM of citric acid-disodium hydrogen phosphate buffer solution, 100mM-250mM of sodium chloride, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(12) The preparation comprises 10mg/ml-200mg/ml of an anti-C5 a antibody, 10mM-30mM of citric acid-disodium hydrogen phosphate buffer solution, 45-100mg/ml of sucrose, 0.05mg/ml-0.3mg/ml of tween 20 or tween 80, wherein the pH value of the buffer solution is 5.5-7.0;

(13) The preparation comprises 10mg/ml-200mg/ml of anti-C5 a antibody, 10mM-30mM of histidine-histidine hydrochloride buffer, 45mg/ml-100mg/ml of mannitol or sorbitol or trehalose, 0.05mg/ml-0.3mg/ml of Tween 80, and the pH value of the buffer is 5.5-7.0.
The formulation of claim 5, wherein the formulation is any one of the following:

(1) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 150mM arginine hydrochloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.2;

(2) The preparation comprises 10mg/ml of anti-C5 a antibody, 10mM histidine-histidine hydrochloride buffer, 200mM arginine hydrochloride, 0.15mg/ml Tween 20, wherein the pH value of the buffer is 6.4;

(3) The preparation comprises 15mg/ml of anti-C5 a antibody, 30mM histidine-histidine hydrochloride buffer, 150mM arginine hydrochloride, 0.05mg/ml Tween 80, wherein the pH value of the buffer is 5.8;

(4) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 150mM sodium chloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(5) The preparation comprises 15mg/ml of anti-C5 a antibody, 10mM acetate buffer, 250mM arginine hydrochloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.2;

(6) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM phosphate buffer, 100mM arginine hydrochloride, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(7) The preparation comprises 15mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 100mg/ml sucrose, 0.2mg/ml Tween 80, wherein the pH value of the buffer is 6.8;

(8) The preparation comprises 15mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 50mg/ml mannitol, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(9) The preparation comprises 15mg/ml of anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 45mg/ml sorbitol, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 7.0;

(10) The preparation comprises 30mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 80mg/ml trehalose, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.2;

(11) The preparation comprises 50mg/ml of an anti-C5 a antibody, 30mM histidine-histidine hydrochloride buffer, 60mg/ml sucrose, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(12) The preparation comprises 100mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 50mg/ml sucrose, 0.1mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(13) The preparation comprises 150mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 60mg/ml sucrose, 0.2mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(14) The preparation comprises 200mg/ml of an anti-C5 a antibody, 20mM histidine-histidine hydrochloride buffer, 70mg/ml sucrose, 0.3mg/ml Tween 80, wherein the pH value of the buffer is 6.0;

(15) The preparation comprises 150mg/ml of anti-C5 a antibody, 20mM phosphate buffer, 150mM arginine hydrochloride, 0.2mg/ml Tween 80, wherein the pH value of the buffer is 5.5;

(16) The formulation comprises 150mg/ml of anti-C5 a antibody, 20mM citric acid-disodium hydrogen phosphate buffer, 100mM sodium chloride, 0.1mg/ml tween 20, the pH of the buffer being 5.8;

(17) The formulation contained 150mg/ml of anti-C5 a antibody, 10mM histidine-histidine hydrochloride buffer, 200mM sodium chloride, 0.2mg/ml Tween 80, the pH of the buffer being 6.8.
The formulation according to any one of claims 5 to 16, further comprising a preservative and/or an antioxidant.
The formulation of claim 17, wherein the preservative is ethylenediamine tetraacetic acid and/or diethylenetriamine pentaacetic acid; the antioxidant is methionine.
The formulation of claim 18, wherein the preservative is present at a concentration of 0-0.5mg/ml and the antioxidant is present at a concentration of 0-1.1mg/ml.
The formulation according to any one of claims 5 to 19, wherein the formulation is a liquid formulation or a powder for injection.
Use of an antibody according to any one of claims 1-4, a formulation according to any one of claims 5-20, in the manufacture of a medicament for the treatment of a disease.
The use according to claim 21, wherein the disease comprises an inflammatory, respiratory or autoimmune disease; preferably, the disease is selected from any one or more of inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, ischemia/reperfusion-related injury, acute lung injury, pneumonia, acute and chronic graft rejection in transplanted patients, graft-versus-host reaction, glomerulonephritis, solid renal failure, rheumatoid arthritis, autoimmune disease, bechterew's disease, lupus disease, inflammatory bowel disease, crohn's disease, tumor growth, and solid organ cancer.