WO2023116574A1 - Isolated anti-c5a antibody, and preparation and use thereof - Google Patents

Abstract

Provided is an isolated C5a antibody for treating C5a-mediated diseases and disorders. Further provided is an anti-C5a antibody preparation. The preparation has a strong stability under high temperature, light, oscillation, freeze-thaw, acceleration and long-term conditions, which can ensure that the preparation maintains a good stability during preparation, transportation and storage, and ensures the safety and quality controllability of clinical medication.

Description

A kind of isolated anti-C5a antibody and its preparation and application technical field

The invention belongs to the technical field of biomedicine, and in particular relates to an isolated anti-C5a antibody and its preparation and application.

Background technique

C5a is an active peptide in allergic reactions and inflammatory processes, which is formed by the cleavage of complement component C5 by C5 convertase in the complement cascade. C5a stimulates mast cell degranulation, release of tumor necrosis factor-α (TNF-α) and histamine, and also recruits phagocytes to sites of infection and inflammation by increasing expression of endothelial cell surface adhesion molecules (Mollnes, 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 post-transplant allograft 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, 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 (CD88) 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). In addition, studies have demonstrated that C5aR is expressed in glomerular endothelial cells but not podocytes, suggesting that C5a may mainly cause proteinuria in renal endothelial cells (Tsai, I.J. et al. Cell. Mol. Life Sci. 2015, 72, 3157 –3171). Therefore, blocking its binding to C5aR by neutralizing C5a becomes an approach to treat C5a-mediated diseases and disorders. Patent application WO2011063980 disclosed the antibody INab308 (InflaRx) against human C5a, WO2012088247 disclosed the C5a antibody MEDI-7814 (MedImmune), and US10450370 disclosed the C5a antibody BNJ383 (Alexion).

The present invention provides an isolated anti-C5a antibody and anti-C5a antibody preparation with strong stability for treating C5a-mediated diseases and disorders.

Contents of the invention

In one aspect, the invention provides an isolated anti-C5a antibody comprising a _VH comprising: HC- _CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid Sequence SEQ ID NO:2, and HC-CDR3, which comprises the amino acid sequence of SEQ ID NO:3; and V _L , which comprises: LC- _CDR1 , which comprises the amino acid sequence of SEQ ID NO:9, LC-CDR2, It comprises the amino acid sequence of SEQ ID NO: 10, and LC-CDR3, which comprises the amino acid sequence of SEQ ID NO: 11; or

The antibody comprises _{a VH} comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; and V _L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, LC-CDR2 comprising the amino acid sequence of _SEQ ID NO:10, and LC-CDR3 comprising comprising the amino acid sequence of SEQ ID NO: 11; or

The antibody comprises _{a VH} comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and HC-CDR3 comprising the amino acid sequence SEQ ID NO: 6; and V _L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of _SEQ ID NO: 14, and LC-CDR3 comprising comprising the amino acid sequence of SEQ ID NO: 15; or

The antibody comprises _{a VH} comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; and V _L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:16, LC-CDR2 comprising _the amino acid sequence of SEQ ID NO:17, and LC-CDR3 comprising Contains the amino acid sequence of SEQ ID NO:11.

In some embodiments, the antibody comprises a _VH comprising the amino acid sequence of SEQ ID NO: 18, and a _VL comprising the amino acid sequence of SEQ ID NO: 22; or

The antibody comprises a V _H comprising the amino acid sequence of SEQ ID NO: 19, and a V _L comprising the amino acid sequence of SEQ ID NO: 23; or

The antibody comprises a V _H comprising the amino acid sequence of SEQ ID NO: 20, and a V _L comprising the amino acid sequence of SEQ ID NO: 24; or

The antibody comprises a _VH comprising the amino acid sequence of SEQ ID NO:21, and a _VL comprising the amino acid sequence of SEQ ID NO:25.

In some embodiments, any of the isolated anti-C5a antibodies described above, the isolated anti-C5a antibody comprising 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 IgGl or IgG4 antibody.

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

In some embodiments, the antibody comprises a _VH , a _VL , a heavy chain constant region and a light chain constant region, the _VH comprising the amino acid sequence of SEQ ID NO: 18, and the _VL comprising the amino acid sequence of SEQ ID NO: 22. The heavy chain constant region comprises the amino acid sequence of SEQ ID NO:26, and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28; or

The antibody comprises V _H , V _L , a heavy chain constant region and a light chain constant region, the V _H comprising the amino acid sequence of SEQ ID NO: 19, the V _L comprising the amino acid sequence of SEQ ID NO: 23, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO:27 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28; or

The antibody comprises V _H , V _L , a heavy chain constant region and a light chain constant region, the V _H comprising the amino acid sequence of SEQ ID NO: 20, the V _L comprising the amino acid sequence of SEQ ID NO: 24, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO:26 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28; or

The antibody comprises V _H , V _L , a heavy chain constant region and a light chain constant region, the V _H comprising the amino acid sequence of SEQ ID NO: 21, the V _L comprising the amino acid sequence of SEQ ID NO: 25, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO:27 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28.

In another aspect, the present invention provides an anti-C5a antibody preparation, which comprises the above-mentioned isolated anti-C5a antibody, a stabilizer, a surfactant, and a buffer.

In some embodiments, the antibody concentration is 1 mg/ml-300 mg/ml; preferably, the antibody concentration is 10 mg/ml-200 mg/ml. In some specific 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 of sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine hydrochloride, glycine, proline, lysine or Several combinations. In some embodiments, the stabilizer is any one or a combination of sucrose, arginine hydrochloride, sodium chloride, mannitol, trehalose, and sorbitol.

In some embodiments, the stabilizing agent is: sodium chloride, arginine hydrochloride, glycine at a concentration of 50mM-300mM, preferably 100mM-250mM (in some embodiments, at a concentration of 100mM, 150mM, 200mM or 250mM) , proline or lysine, and or a concentration of 30mg/ml-150mg/ml, preferably 45mg/ml-100mg/ml (in some embodiments, a concentration of 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) of sucrose, trehalose, maltose, sorbitol, mannitol.

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

In some embodiments, the concentration of the buffer is 3mM-100mM; in some embodiments, the concentration of the buffer is 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 buffer solution has a pH value of 5.5-7.0.

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

In some embodiments, the concentration of the surfactant is 0.01mg/ml-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 0.05mg/ml-0.3mg/ml; in some specific embodiments, the concentration of the 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 of the following formulations:

(1) The antibody concentration is 10mg/ml, 15mg/ml, 25mg/ml, 35mg/ml, 50mg/ml, 75mg/ml, 100mg/ml, 125mg/ml, 150mg/ml, 175mg/ml or 200mg/ml 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 Liquid is 10mM-30mM histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphate buffer; the pH value of the buffer is 5.5 -7.0; The surfactant is Tween-20 and/or Tween-80 of 0.05mg/ml-0.3mg/ml;

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

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

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

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

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

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

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

(3) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM histidine-histidine hydrochloride buffer, 45mg/ml-100mg/ml sucrose, 0.05mg/ml - 0.3mg/ml of Tween 20 or Tween 80, the pH of the buffer is 5.5-7.0;

(4) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM acetate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer is 5.5-7.0

(5) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM acetate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml Tween 20 Or Tween 80, the pH value of the buffer is 5.5-7.0;

(6) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM acetate buffer, 45mg/ml-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml spit Wen 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

(7) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM phosphate buffer saline, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml Wen 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

(8) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM phosphate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

(9) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM phosphate buffer, 45-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

(10) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM citric acid-disodium hydrogen phosphate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3 mg/ml of Tween 20 or Tween 80, the pH of the buffer is 5.5-7.0;

(11) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM citric acid-disodium hydrogen phosphate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer is 5.5-7.0;

(12) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM citric acid-disodium hydrogen phosphate buffer, 45-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer is 5.5-7.0;

(13) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM histidine-histidine hydrochloride buffer, 45mg/ml-100mg/ml mannitol or sorbitol or Trehalose, 0.05mg/ml-0.3mg/ml Tween 80, the pH value of the buffer solution is 5.5-7.0.

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

(1) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer of 20mM, the arginine hydrochloride of 150mM, the Tween 80 of 0.1mg/ml, the described The pH of the buffer is 6.2;

(2) the preparation comprises the anti-C5a antibody of 10mg/ml, the histidine-histidine hydrochloride buffer of 10mM, the arginine hydrochloride of 200mM, the Tween 20 of 0.15mg/ml, the described The pH of the buffer is 6.4;

(3) the preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer of 30mM, the arginine hydrochloride of 150mM, the Tween 80 of 0.05mg/ml, the described The pH of the buffer is 5.8;

(4) the preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sodium chloride of 150mM, the Tween 80 of 0.1mg/ml, the Tween 80 of described buffer solution The pH value is 6.0;

(5) The preparation comprises 15 mg/ml of anti-C5a antibody, 10 mM acetate buffer, 250 mM arginine hydrochloride, 0.1 mg/ml Tween 80, and the pH value of the buffer is 6.2 ;

(6) The preparation comprises 15 mg/ml of anti-C5a antibody, 20 mM phosphate buffer, 100 mM arginine hydrochloride, 0.1 mg/ml Tween 80, and the pH of the buffer is 6.0;

(7) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 100mg/ml, the Tween 80 of 0.2mg/ml, the pH of the buffer solution The value is 6.8;

(8) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the mannitol of 50mg/ml, the Tween 80 of 0.1mg/ml, the Tween 80 of the buffer solution The pH value is 6.0;

(9) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sorbitol of 45mg/ml, the Tween 80 of 0.1mg/ml, the Tween 80 of described buffer solution The pH value is 7.0;

(10) The preparation comprises the anti-C5a antibody of 30mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the trehalose of 80mg/ml, the Tween 80 of 0.1mg/ml, the Tween 80 of described buffer solution The pH value is 6.2;

(11) The preparation comprises the anti-C5a antibody of 50mg/ml, the histidine-histidine hydrochloride buffer solution of 30mM, the sucrose of 60mg/ml, the Tween 80 of 0.1mg/ml, the pH of the buffer solution Value is 6.0;

(12) The preparation comprises the anti-C5a antibody of 100mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 50mg/ml, the Tween 80 of 0.1mg/ml, the pH of the buffer solution Value is 6.0;

(13) The preparation comprises the anti-C5a antibody of 150mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 60mg/ml, the Tween 80 of 0.2mg/ml, the pH of the buffer solution Value is 6.0;

(14) The preparation comprises the anti-C5a antibody of 200mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 70mg/ml, the Tween 80 of 0.3mg/ml, the pH of the buffer solution Value is 6.0;

(15) The preparation comprises 150 mg/ml of anti-C5a antibody, 20 mM phosphate buffer, 150 mM arginine hydrochloride, 0.2 mg/ml Tween 80, and the pH of the buffer is 5.5;

(16) The preparation comprises 150 mg/ml of anti-C5a antibody, 20 mM citric acid-disodium hydrogen phosphate buffer, 100 mM sodium chloride, 0.1 mg/ml Tween 20, and the pH of the buffer is 5.8;

(17) The preparation comprises the anti-C5a antibody of 150mg/ml, the histidine-histidine hydrochloride buffer solution of 10mM, the sodium chloride of 200mM, the Tween 80 of 0.2mg/ml, the Tween 80 of described buffer solution The pH is 6.8.

In some embodiments, the formulations may also include preservatives and/or antioxidants. The preservative or antioxidant is a preservative or antioxidant commonly used in antibody preparations. In some embodiments, the preservative is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) or a combination thereof; the antioxidant is methionine.

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 , 0.4ml/ml or 0.5mg/ml. In some embodiments, the concentration of the antioxidant is 0-1.1 mg/ml. In some embodiments, the concentration of the antioxidant is 0 mg/ml, 0.15 mg/ml, 0.75 mg/ml or 0.11 mg/ml.

In some embodiments, the above-mentioned antibody preparation is a liquid preparation or a powder for injection.

In another aspect, the present invention provides the use of any anti-C5a antibody and any anti-C5a antibody preparation described above in the preparation of a drug for treating diseases.

In another aspect, the invention provides a method of treating a disease or condition in an individual in need thereof, comprising administering to said individual an effective amount of any one of the anti-C5a antibodies or antibody preparations described above. The administration methods include: intravenous injection, intraarterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, intramuscular injection, intratracheal administration, subcutaneous injection, intraocular administration , intrathecal, mucosal or transdermal administration. In some embodiments, the formulation is administered intravenously. In some embodiments, the formulation is administered subcutaneously. In some embodiments, the formulation is administered intramuscularly.

In some embodiments, the disease or condition is an inflammatory, respiratory or autoimmune disease or condition; preferably, the disease is selected from the group consisting of inflammatory response syndrome (SIRS), sepsis, severe sepsis, septic shock, Blood/reperfusion-related injury, acute lung injury, pneumonia, acute and chronic graft rejection in transplant patients, graft-versus-host reaction, glomerular disease, glomerulonephritis, solid renal failure, rheumatoid arthritis, autoimmunity disease, Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, tumor growth, and solid organ cancer.

The anti-C5a antibody and its preparation provided by the present invention have the following excellent effects:

1. The anti-C5a antibody of the present invention can significantly reduce the expression of CD11b in human neutrophils induced by endogenous C5a, does not inhibit plasma hemolytic activity, and can significantly reduce the cytokine storm and inflammatory response caused by the new coronavirus in vivo .

2. The anti-C5a antibody preparation of the present invention has strong stability under high temperature, light, vibration, freeze-thaw, acceleration and long-term conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, and ensures the safety of clinical medication and quality controllability.

Description of drawings

Figures 1A-1B show the results of CD11b blocking experiments. The results in Figure 1A show that in human neutrophils, anti-C5a antibodies Cab42, Cab44, and Cab45 can block the upregulation of CD11b induced by human endogenous C5a. The results in Figure 1B show that compared with the control antibody INab308, the anti-C5a antibody Cab42 can block the up-regulation of CD11b expression induced by endogenous human C5a in human neutrophils even if there is more than 50 times the molar amount of C5 in the reaction system.

Figures 2A-2B show plasma hemolytic activity of C5a antibody. In the classical activation pathway, anti-C5a antibodies Cab35, Cab42, Cab44, Cab45 (Fig. 1A) did not inhibit plasma hemolytic activity compared with the control antibody Eculizumab. In the alternative activation pathway, anti-C5a antibodies Cab35, Cab42, Cab44, Cab45 (Fig. 2B) did not inhibit plasma hemolytic activity compared to the control antibody Eculizumab.

Detailed ways

In order to make the technical problems to be solved, the technical solutions adopted and the advantages of the present invention clearer, the present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

The reagents used in the following examples, if not specified, are prepared by conventional methods or obtained from commercial sources; the experimental methods used, if not specified, are conventional methods; the materials, instruments, etc. used, Unless otherwise specified, all were obtained from commercial sources.

Example 1: Screening and preparation of antibodies

Blood RNA was extracted from the blood of 2,000 people, and the first strand of cDNA was synthesized by reverse transcription. V _H and V _L fragments were respectively amplified with specific primers for human V _H and V _L. After being recovered by gel, V _H was synthesized by a linker , V _L were spliced into scFv form, and then inserted into the yeast display plasmid PYD1, and the yeast was electroporated to obtain the yeast display human antibody scFv library.

The natural yeast display human antibody scFv library was enriched by MACS magnetic bead sorting with biotinylated C5a. In order to quickly identify the C5a antibody population, the yeast library was transferred to a phage display system. Continue to use C5a to screen the phage for three rounds, and then select a single clone for binding ELISA identification and in vitro biological activity evaluation to obtain the lead antibody.

In order to reduce the potential immunogenicity risk of candidate molecules, the human germline gene with the closest relationship to the maternal sequence was used as a template, and combined with structure prediction analysis, the amino acid sequence of the non-germline gene in the framework region of the maternal sequence was restored to mutation It is the amino acid sequence of the germline gene, thereby improving the human origin of the candidate molecule. After affinity maturation, isomerization risk points are removed, and Fc is modified to obtain Cab35, Cab42, Cab44, and Cab45 antibodies. The above four antibodies were expressed and purified for subsequent experiments. The amino acid sequence of the heavy chain constant region of Cab35, Cab42, Cab44, and Cab45 antibodies is shown in SEQ IN NO: 26, and the amino acid sequence of the light chain constant region is shown in SEQ IN NO: 28. The specific amino acid sequence of the antibody is shown in the table below.

Table 1 Anti-C5a antibody CDR sequence

Table 2 Anti-C5a antibody sequence

Example 2: CD11b blocking experiment in whole blood

Upregulation of CD11b expression is a characteristic and sensitive marker of neutrophil activation, and the level of CD11b in neutrophils is used to evaluate the activation of neutrophils. The present invention uses a human whole blood model and uses INab308 (WO2011063980A1, InflaRx) as a control to evaluate the blocking activity of antibodies Cab42, Cab44 and Cab45 on endogenous human C5a. Human whole blood was incubated with human C5a alone or in combination with human C5a and different concentrations of each antibody. After incubation, stain with detection antibody CD11b:FITC, and after lysing red blood cells, analyze CD11b MFI by flow cytometry to reflect the activation level of neutrophils in the blood.

As shown in Figure 1A, all the optimized antibodies could significantly reduce the expression of CD11b in human neutrophils induced by endogenous C5a, even when the molar ratio of Ab:Ag was 0.5:1, it could also achieve the same level as that of the control antibody INab308. Comparable ability to inhibit CD11b upregulation.

As shown in Figure 1B and 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 in the reaction system In the presence of 50-fold more C5, antibody Cab42 reduced endogenous C5a-induced upregulation of CD11b expression in human neutrophils with greater potency than control antibody INab308.

table 3

Antibody	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-c5a antibodies

The complement system can be activated independently by three activation pathways, which eventually form the membrane attack complex. Under certain experimental conditions, it can directly attack the cell membrane of red blood cells, resulting in lysis of red blood cells. Based on this mechanism, the present invention conducted experiments to evaluate whether the C5a antibody of the present invention would affect the biological activity of C5 convertase to cleave C5 to generate C5b.

Detection of the role of C5a antibodies in the complement-mediated classical activation pathway: 50% complement hemolysis assay is a method to measure the total classical complement activity in serum. The assay is a lysis assay in which antibodies are used as activators of the classical complement pathway to sensitize red blood cells and the test serum is diluted to different concentrations to determine the amount required to achieve 50% lysis (CHSO). The hemolysis rate can be measured with a spectrophotometer. The 50% complement hemolysis assay provides an indirect measure of terminal complement complex (TCC) formation, since the TCC itself has a direct effect on the hemolysis measured. This experiment 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 .

Briefly, guinea pig erythrocytes were prepared by centrifugation from fresh guinea pig whole blood and then sensitized with sheep anti-erythrocyte antibody. The above operations can activate the classical hemolytic pathway of complement, causing red blood cell lysis. Absorbance was read at 412nm. The C5 antibody Eculizumab was used as a control.

Examination of the role of C5a antibody in the complement-mediated alternative pathway activation: Briefly, in the absence of antibody sensitization, rabbit erythrocytes can activate the alternative pathway to form membrane attack complexes, leading to lysis of rabbit erythrocytes. After adding ethylene glycol diaminotetraacetic acid (EGTA) to the reaction system, the substance can chelate Ca ²⁺ in plasma, but has a weak binding ability to Mg ²⁺ , so the classical pathway is blocked. The 50% complement hemolysis assay described above was used to measure activation of the alternative pathway. The C5 antibody Eculizumab was used as a control.

As shown in Figure 2A, after adding the C5 antibody Eculizumab, it can inhibit the hemolytic reaction in a dose-dependent manner, while the antibodies Cab35, Cab42, Cab44, and Cab45 did not inhibit the total classical complement activity. As shown in Figure 2B, the addition of the C5 antibody Eculizumab can inhibit the hemolytic reaction, while the antibodies Cab35, Cab42, Cab44, and Cab45 do not inhibit the activity of the alternative pathway.

In summary, the anti-C5a antibody of the present invention neither affects the function of C5b in the classical complement-mediated activation pathway nor affects the function of C5b in the alternative activation pathway.

Example 4: In vivo effect of anti-C5a antibody in treating ARDS caused by coronavirus

An ARDS animal disease model is constructed to evaluate the therapeutic effect of the anti-C5a antibody of the present invention in vivo.

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, and the feeding conditions were room temperature 20°C-26°C, relative humidity 40%-70% , 12 hours alternating light and dark. 4 days, 3 days and 2 days before the experiment, 40 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), 7.5×10 ⁸ PFU/100μL/mouse/time/day, and the remaining 6 mice were injected with Sodium chloride solution (as a normal control group). On day 0 (the day of the experiment), mice were injected with corresponding doses of antibody or sodium chloride solution in groups as follows.

Dosage and animal grouping: the mice were divided into the following groups and treated with 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), inject 100 μ L 9% sodium chloride solution; (3) low-dose experimental group (n=10), inject antibody, and administration dosage is 1mg/kg; (4) medium-dose experimental group (n=10 ), the antibody was injected at a dosage of 3 mg/kg; (5) the high-dose experimental group (n=10), the antibody was injected at a dosage of 10 mg/kg. After 30 minutes of administration as above, for the disease model control group and each experimental group, mice were injected with LPS-K235 (Sigma-Aldrich) at a concentration of 1 mg/mL, 100 μL/mouse. For the normal control group, sodium chloride solution was injected. All reagents in this experiment were administered by tail vein injection. This experiment was performed under the approval of the Ethics Committee of the Beijing Institute of Biotechnology and complied with relevant regulatory standards.

Detection of survival rate: observe and analyze the survival of mice in each group at 12h, 24h, 36h, 48h, 60h and 72h after administration.

White blood cell count: 72 hours after administration, the mice were anesthetized, and blood was collected from the orbit. use

A series of blood analyzers can count and classify white blood cells, including white blood cell count (WBC), neutrophils (Neut), lymphocytes (Lymph), and monocytes (Mono).

Survival rate results: within 72 hours after administration, animals in the normal control group and the high-dose experimental groups administered 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 administered with different anti-C5a antibodies (Cab35, Cab42, Cab44 or Cab45), the overall mortality rate was 10-20% (1-2/10); or Cab45) in the middle-dose experimental group, the overall mortality rate was 10% (1/10). The above results show that the anti-C5a antibody of the present invention can effectively reduce or prevent the death of mice caused by coronavirus and improve the survival rate of mice.

Complete blood white blood cell count results: Compared with the normal control group, the levels of WBC, Lymph and Mono in the model control group decreased, and there was a statistically significant difference between the two (P<0.05). Compared with the model control group, the three doses of experimental groups administered with different anti-C5a antibodies (Cab35, Cab42, Cab44 or Cab45) all showed an increase in the number of WBC and Lymph, and the difference between the model control group and the middle and high dose experimental groups The difference between them was statistically significant (P<0.05). The above results show that the anti-C5a antibody of the present application helps to restore the balance of immune cells in ARDS disease model mice.

Inflammatory cytokine results: Compared with the normal control group, the levels of GM-CSF, IL-1β, IL-6, TNF-α, and MCP-1 in the model control group were significantly increased, and the differences between the two were statistically significant. Scientific significance (P<0.05). Compared with the model control group, GM-CSF, IL-1β, IL-6, TNF-α, MCP-1, C5a in the experimental group administered with 3 doses of different anti-C5a antibodies (Cab35, Cab42, Cab44 or Cab45) The levels were decreased in a dose-dependent manner. Moreover, the levels of most of these cytokines had statistically significant differences between the dosage experiment group and the model group (P<0.05). The above results show that the anti-C5a antibody of the present invention can significantly reduce the cytokine storm and inflammatory response caused by the novel coronavirus in vivo.

Embodiment 5, preparation of anti-C5a antibody preparation

The anti-C5a antibody preparation prescription is as follows:

Table 4 Prescription of anti-C5a antibody preparation

Preparations (Cab35, Cab42 (IgG1 mutation), Cab44, Cab45 (IgG1 mutation)) containing each C5a antibody were prepared with reference to the prescription in Table 4, and the antibodies included V _H , V _L , and the constant region of the heavy chain and the constant region of the light chain , the specific antibody sequence is as follows:

Table 5 Antibody Sequence

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 (IgG1 mutant) 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 mutant) antibody	SEQ ID NO:21	SEQ ID NO:25	SEQ ID NO:27	SEQ ID NO:28

Embodiment 6, anti-C5a antibody preparation stability test

Set conditions such as high temperature, light, vibration, freeze-thaw, acceleration, and long-term conditions to investigate the stability of different formulations. Test items include visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, insoluble particles, aggregation characteristics, degradation characteristics and charge heterogeneity of antibody preparations. Among them, the characteristics of the polymer were detected by the SEC method and the NR-CE-SDS method, and the charge heterogeneity was detected by the CEX method.

1. Stability test under high temperature conditions

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

(1) Table 6 shows the test results of each preparation of the exemplary Cab35 antibody: relative to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, The insoluble particles basically did not change; aggregates and fragments increased slightly, and the main peak decreased slightly; the range of aggregates detected by SEC was within 0.9%; the range of aggregates detected by NR-CE-SDS was within 0.8%, and the range of changes of the main peak was 2.7% Within , the range of fragment variation is within 2.0%. The above results indicate that the preparation of Cab35 has good stability under high temperature conditions.

Table 6 Cab35 antibody high temperature test results

(2) The preparations of Cab42 (IgG1 mutation), Cab44 and Cab45 (IgG1 mutation) antibodies also showed good stability under high temperature conditions: within 3 weeks at high temperature, relative to the 0th day, the visible Foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles are basically unchanged, aggregates and fragments are slightly increased, and the main peak is slightly decreased: the change range of aggregates detected by SEC is within 1.0%; NR-CE-SDS detected that the change range of aggregates was all within 0.9%, the change range of main peak was all within 2.6%, and the change range of fragments was all within 2.0%.

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

2. Stability test under light conditions

The stability of each antibody preparation was tested under light conditions (5°C±3°C, 4500±500lx, 5W/m ² , 0 day, 3 days, 5 days, 1 week, 2 weeks of light).

(1) Table 7 shows the test results of each preparation of the exemplary antibody Cab42 (IgG1 mutation): within 2 weeks of light, relative to day 0, the visible foreign matter, concentration, pH, osmotic pressure, viscosity, and biological activity of the antibody preparation , thermal stability, and insoluble particles basically did not change; aggregates and fragments increased slightly, and the main peak decreased slightly: the range of aggregates detected by SEC was within 3.10%; the range of aggregates detected by NR-CE-SDS was within 1.9%, and the main peak The range of variation is within 4.4%, and the range of fragment variation is within 2.5%. The above results show that the Cab42 (IgG1 mutation) antibody preparation can basically remain stable under light conditions.

The light test result of table 7Cab42 (IgG1 mutation) antibody preparation

(2) The preparations of Cab35, Cab44 and Cab45 (IgG1 mutation) antibodies also showed good stability under light conditions: within 2 weeks of light, compared with day 0, the visible foreign matter, concentration, pH, penetration of antibody preparations Pressure, viscosity, biological activity, thermal stability, and insoluble particles basically did not change within 2 weeks of light; aggregates and fragments tended to increase, and the main peak showed a slight decrease trend: the change range of aggregates detected by SEC was within 3.15%; NR - CE-SDS detects that the variation range of the 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 show that the aggregation and degradation characteristics of the anti-C5a antibody preparation of the present invention do not change significantly under light conditions, and the preparation can basically remain stable.

3. Stability test under oscillating conditions

The stability of each antibody preparation was tested under shaking conditions (5°C±3°C, 100rpm shaking for 0 days, 1 week, and 2 weeks).

(1) Table 8 shows the test results of each preparation of the exemplary antibody Cab44: within 2 weeks of shaking, relative to day 0, the visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, biological activity, Thermal stability and insoluble particles basically did not change; SEC detection aggregates had a small change range, all within 0.13%; CEX detection acid peak, main peak and basic peak change range were all within 0.40%, almost no change; NR- CE-SDS aggregates, main peaks and fragments vary within 0.42%, almost no change. The above results demonstrate the excellent stability of the formulation of the Cab44 antibody under shaking conditions.

The shaking test result of table 8 anti-Cab44 antibody preparation

(2) The preparations of Cab35, Cab42 (IgG1 mutation) and Cab45 (IgG1 mutation) 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 basically do not change; SEC detects a small change in aggregates, within 0.15%; CEX detects acidic peaks, main peaks and basic peaks The variation ranges of NR-CE-SDS aggregates, main peaks and fragments were all within 0.38%, almost unchanged; the variation ranges of NR-CE-SDS aggregates, main peaks and fragments were within 0.41%, almost unchanged.

The results of each item of the shaking test above indicate that the anti-C5a antibody preparation of the present invention has excellent stability under shaking conditions.

4. Stability test under freeze-thaw conditions

The stability of each antibody preparation was tested under freeze-thaw conditions (freeze: -20°C ± 5°C; thaw: room temperature, freeze- thaw 0, 1, 3, 5 times).

(1) Table 9 shows the test results of each preparation of the exemplary antibody Cab45 (IgG1 mutation): within 5 times of freezing and thawing, compared with 0 times of freezing and thawing, foreign matter, concentration, turbidity, pH, osmotic pressure can be seen in each antibody preparation , viscosity, biological activity, thermal stability, and insoluble particles basically did not change; the aggregates detected by SEC changed very little, all within 0.15%; Changes; NR-CE-SDS detected aggregates, main peaks and fragments within 0.39%, almost no change. The above results demonstrate the excellent stability of the formulation of the Cab45 (IgG1 mutant) antibody under freeze-thaw conditions.

The freeze-thaw test result of table 9 Cab45 (IgG1 mutation) antibody preparation

(2) The preparations of Cab35, Cab42 (IgG1 mutation) and Cab44 antibodies also showed excellent stability under freeze-thaw conditions: within 5 times of freeze-thaw, compared with 0 times of freeze-thaw, foreign matter, concentration , turbidity, pH, osmotic pressure, viscosity, biological activity, thermal stability, and insoluble particles are basically unchanged; SEC detects little change in aggregates, within 0.16%; CEX detects changes in acidic peaks, main peaks and basic peaks The range is within 0.40%, almost no change; NR-CE-SDS detects that the change range of aggregates, main peaks and fragments is within 0.42%, almost no change.

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

5. Stability test under accelerated conditions

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

(1) Table 10 shows the test results of each preparation of the exemplary antibody Cab42 (IgG1 mutation): within 2 months of acceleration, relative to day 0, visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity of the antibody preparation , activity, thermal stability, and insoluble particles basically did not change; SEC detected polymers did not change or changed very little, and the range of change was within 0.31%; CEX detected acid peaks and base peaks increased slightly, and the main peak decreased slightly; Within 3.10%, the change range of the base peak is within 0.9%, and the change range of the main peak is within 4%; the increase range of the aggregate detected by NR-CE-SDS is within 0.5%, the decrease range of the main peak is within 1%, and the increase range of the fragment is within 0.5%. . The above results indicate that the preparation of the Cab42 (IgG1 mutant) antibody can be basically stable under accelerated conditions.

The accelerated test result of table 10 Cab42 (IgG1 mutation) antibody preparation

(2) The preparations of Cab35, Cab44 and Cab45 (IgG1 mutation) antibodies also showed excellent stability under accelerated conditions: within 2 months of acceleration, relative to day 0, the visible foreign matter, concentration, turbidity and The temperature, pH, osmotic pressure, viscosity, activity, thermal stability and insoluble particles are basically unchanged. SEC detects no change or little change in the polymer, and the change range is within 0.35%. CEX detects that the acid peak and base peak slightly increase, and the main peak decreases slightly; the change range of the acid peak is within 3.13%, and the change range of the base peak is within 1.0%. The change range of the main peak was within 4.2%; the increase range of the aggregate detected by NR-CE-SDS was within 0.5%, the decrease range of the main peak was within 1%, and the increase range of fragments was within 0.5%.

The results of the accelerated test show that the anti-C5a antibody preparation of the present invention has no significant change under accelerated conditions and can basically remain stable.

6. Stability test under long-term conditions

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

(1) Table 11 shows the test results of each preparation of the exemplary antibody Cab44: within 6 months under long-term conditions, relative to day 0, visible foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity of the antibody preparation , thermal stability and insoluble particles are basically unchanged; SEC detects that the change range of aggregates is within 0.11%, almost no change; The range of change is within 0.49%, and the range of change of the basic peak is within 0.13%. NR-CE-SDS detects that there is almost no change in the aggregate, main peak, and fragment. The increase of the aggregate is within 0.5%, and the decrease of the main peak is within 0.5%. The increase is within 0.3%. The above results demonstrate the excellent stability of the formulation of the Cab44 antibody under long-term conditions.

The long-term stability result of table 11 Cab44 antibody preparation

(2) The respective formulations of Cab35, Cab42 (IgG1 mutation) and Cab45 (IgG1 mutation) antibodies also showed excellent stability under long-term conditions: within 6 months under long-term conditions, relative to day 0, the visible Foreign matter, concentration, turbidity, pH, osmotic pressure, viscosity, activity, thermal stability, and insoluble particulates were essentially unchanged. SEC detects that there is almost no change in the polymer, and the change range is within 0.13%. CEX detects that the acidic peak, main peak and basic peak have almost no change. The amplitude was within 0.15%; NR-CE-SDS detected aggregates, main peaks, and fragments had almost no changes, aggregates increased within 0.51%, main peaks decreased within 0.5%, and fragments increased within 0.4%.

The results of each item of the long-term test above indicate that the anti-C5a antibody preparation of the present invention has excellent stability under long-term conditions.

In summary, the anti-C5a antibody preparation of the present invention has strong stability under high temperature, light, shaking, freeze-thaw, acceleration and long-term conditions, which can ensure that the preparation maintains good stability during preparation, transportation and storage, and ensures Clinical drug safety and quality controllability.

Claims (22)

1. An isolated anti-C5a antibody, characterized in that the antibody comprises _VH , and the _VH comprises: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO :2, and HC-CDR3, which comprises the amino acid sequence of SEQ _ID NO:3; and _VL , which comprises: LC-CDR1, which comprises the amino acid sequence of SEQ ID NO:9, LC-CDR2, which comprises the amino acid sequence SEQ ID NO: 10, and LC-CDR3 comprising the amino acid sequence of SEQ ID NO: 11; or

   The antibody comprises _{a VH} comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 1, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; and V _L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:12, LC-CDR2 comprising the amino acid sequence of _SEQ ID NO:10, and LC-CDR3 comprising comprising the amino acid sequence of SEQ ID NO: 11; or

   The antibody comprises _{a VH} comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO:4, HC-CDR2 comprising the amino acid sequence of SEQ ID NO:5, and HC-CDR3 comprising the amino acid sequence SEQ ID NO: 6; and V _L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO: 13, LC-CDR2 comprising the amino acid sequence of _SEQ ID NO: 14, and LC-CDR3 comprising comprising the amino acid sequence of SEQ ID NO: 15; or

   The antibody comprises _{a VH} comprising: HC-CDR1 comprising the amino acid sequence of SEQ ID NO: 7, HC-CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and HC-CDR3 comprising the amino acid sequence SEQ ID NO:3; and V _L comprising: LC-CDR1 comprising the amino acid sequence of SEQ ID NO:16, LC-CDR2 comprising _the amino acid sequence of SEQ ID NO:17, and LC-CDR3 comprising Contains the amino acid sequence of SEQ ID NO:11.
2. The antibody according to claim 1, wherein the antibody comprises V _H comprising the amino acid sequence of SEQ ID NO: 18, and V _L comprising the amino acid sequence of SEQ ID NO: 22; or

   The antibody comprises a V _H comprising the amino acid sequence of SEQ ID NO: 19, and a V _L comprising the amino acid sequence of SEQ ID NO: 23; or

   The antibody comprises a V _H comprising the amino acid sequence of SEQ ID NO: 20, and a V _L comprising the amino acid sequence of SEQ ID NO: 24; or

   The antibody comprises a _VH comprising the amino acid sequence of SEQ ID NO:21, and a _VL comprising the amino acid sequence of SEQ ID NO:25.
3. The antibody according to claim 2, wherein the antibody comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region comprises the amino acid sequence SEQ ID NO: 26 or 27, and the light chain constant region Comprising the amino acid sequence of SEQ ID NO:28.
4. The antibody according to claim 2, wherein the antibody comprises _VH , _VL , a heavy chain constant region and a light chain constant region, the _VH comprises the amino acid sequence of SEQ ID NO: 18, and the _VL comprising the amino acid sequence of SEQ ID NO:22, the heavy chain constant region comprising the amino acid sequence of SEQ ID NO:26, and the light chain constant region comprising the amino acid sequence of SEQ ID NO:28; or

   The antibody comprises V _H , V _L , a heavy chain constant region and a light chain constant region, the V _H comprising the amino acid sequence of SEQ ID NO: 19, the V _L comprising the amino acid sequence of SEQ ID NO: 23, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO:27 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28; or

   The antibody comprises V _H , V _L , a heavy chain constant region and a light chain constant region, the V _H comprising the amino acid sequence of SEQ ID NO: 20, the V _L comprising the amino acid sequence of SEQ ID NO: 24, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO:26 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28; or

   The antibody comprises V _H , V _L , a heavy chain constant region and a light chain constant region, the V _H comprising the amino acid sequence of SEQ ID NO: 21, the V _L comprising the amino acid sequence of SEQ ID NO: 25, the heavy chain The constant region comprises the amino acid sequence of SEQ ID NO:27 and the light chain constant region comprises the amino acid sequence of SEQ ID NO:28.
5. An anti-C5a antibody preparation, characterized in that the preparation comprises the isolated anti-C5a antibody according to any one of claims 1-4, a stabilizer, a surfactant, and a buffer.
6. The preparation according to claim 5, wherein the antibody concentration is 1 mg/ml-300 mg/ml; preferably, the antibody concentration is 10 mg/ml-200 mg/ml.
7. The preparation according to claim 5 or 6, wherein the stabilizer is sucrose, trehalose, maltose, sorbitol, mannitol, sodium chloride, arginine hydrochloride, glycine, proline, Any one or several combinations of lysine; preferably, the stabilizer is any one or several of sucrose, arginine hydrochloride, sodium chloride, mannitol, trehalose, sorbitol combination of species.
8. According to the preparation according to any one of claims 5-7, it is characterized in that the stabilizer is: sodium chloride, arginine hydrochloride, glycine, proline with a concentration of 50mM-300mM, preferably 100mM-250mM amino acid or lysine; and/or sucrose, trehalose, maltose, sorbitol and/or mannitol at a concentration of 30mg/ml-150mg/ml, preferably 45mg/ml-100mg/ml.
9. The preparation according to any one of claims 5-8, wherein the buffer is histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-acetic acid Any of sodium buffer, phosphate buffer, and glutamate buffer.
10. The preparation according to any one of claims 5-9, wherein the buffer has a concentration of 3mM-100mM; preferably, the buffer has a concentration of 5mM-80mM or 8mM-50mM; more preferably Preferably, the concentration of the buffer is 10mM-30mM.
11. The preparation according to any one of claims 5-10, wherein the buffer has a pH value of 4.8-8.0; preferably, the buffer has a pH value of 5.0-7.2; more preferably, The pH value of the buffer is 5.5-7.0.
12. The preparation according to any one of claims 5-11, wherein the surfactant is polysorbate and/or poloxamer; preferably, the polysorbate is Tween- 20 or Tween-80.
13. The preparation according to any one of claims 5-12, wherein the concentration of the surfactant is 0.01mgml-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 0.05mg/ml-0.3mg/ml.
14. The preparation according to claim 5, wherein the preparation is any one of the following preparations:

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

    (2) The antibody concentration 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 sucrose, trehalose, mannitol and/or Or sorbitol; The buffer is 10mM-30mM histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphate buffer; the The pH value of the buffer solution is 5.5-7.0; the surfactant is 0.05mg/ml-0.3mg/ml Tween-20 and/or Tween-80;

    (3) The antibody concentration 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 is 10mM, 20mM, 30mM histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphoric acid Salt buffer solution; the pH value of the buffer solution is 5.5-7.0; the surfactant is 0.05mg/ml-0.3mg/ml Tween-20 and/or Tween-80;

    (4) The antibody concentration 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 is 10mM-30mM histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphate buffer solution; the pH value of the buffer is 5.5, 5.8, 6, 6.2, 6.5, 6.8 or 7.0; the surfactant is Tween-20 and/or Tween-20 of 0.05mg/ml-0.3mg/ml 80;

    (5) The antibody concentration 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 is 10mM-30mM histidine-histidine hydrochloride buffer, citric acid-disodium hydrogen phosphate buffer, acetic acid-sodium acetate buffer, phosphoric acid Salt buffer; the pH value of the buffer is 5.5-7.0; the surfactant is 0.05mg/ml, 0.10mg/ml, 0.15mg/ml, 0.2mg/ml, 0.25mg/ml, 0.3mg/ml ml of Tween-20 and/or Tween-80.
15. The preparation according to claim 5, wherein the preparation is any one of the following preparations:

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

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

    (3) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM histidine-histidine hydrochloride buffer, 45mg/ml-100mg/ml sucrose, 0.05mg/ml - 0.3mg/ml of Tween 20 or Tween 80, the pH of the buffer is 5.5-7.0;

    (4) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM acetate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer is 5.5-7.0

    (5) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM acetate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml Tween 20 Or Tween 80, the pH value of the buffer is 5.5-7.0;

    (6) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM acetate buffer, 45mg/ml-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml spit Wen 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

    (7) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM phosphate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3mg/ml Wen 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

    (8) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM phosphate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

    (9) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM phosphate buffer, 45-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer solution is 5.5-7.0;

    (10) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM citric acid-disodium hydrogen phosphate buffer, 100mM-250mM arginine hydrochloride, 0.05mg/ml-0.3 mg/ml of Tween 20 or Tween 80, the pH of the buffer is 5.5-7.0;

    (11) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM citric acid-disodium hydrogen phosphate buffer, 100mM-250mM sodium chloride, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer is 5.5-7.0;

    (12) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM citric acid-disodium hydrogen phosphate buffer, 45-100mg/ml sucrose, 0.05mg/ml-0.3mg/ml Tween 20 or Tween 80, the pH value of the buffer is 5.5-7.0;

    (13) The preparation comprises 10mg/ml-200mg/ml anti-C5a antibody, 10mM-30mM histidine-histidine hydrochloride buffer, 45mg/ml-100mg/ml mannitol or sorbitol or Trehalose, 0.05mg/ml-0.3mg/ml Tween 80, the pH value of the buffer solution is 5.5-7.0.
16. The preparation according to claim 5, wherein the preparation is any one of the following preparations:

    (1) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer of 20mM, the arginine hydrochloride of 150mM, the Tween 80 of 0.1mg/ml, the described The pH of the buffer is 6.2;

    (2) the preparation comprises the anti-C5a antibody of 10mg/ml, the histidine-histidine hydrochloride buffer of 10mM, the arginine hydrochloride of 200mM, the Tween 20 of 0.15mg/ml, the described The pH of the buffer is 6.4;

    (3) the preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer of 30mM, the arginine hydrochloride of 150mM, the Tween 80 of 0.05mg/ml, the described The pH of the buffer is 5.8;

    (4) the preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sodium chloride of 150mM, the Tween 80 of 0.1mg/ml, the Tween 80 of described buffer solution The pH value is 6.0;

    (5) The preparation comprises 15 mg/ml of anti-C5a antibody, 10 mM acetate buffer, 250 mM arginine hydrochloride, 0.1 mg/ml Tween 80, and the pH value of the buffer is 6.2 ;

    (6) The preparation comprises 15 mg/ml of anti-C5a antibody, 20 mM phosphate buffer, 100 mM arginine hydrochloride, 0.1 mg/ml Tween 80, and the pH of the buffer is 6.0;

    (7) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 100mg/ml, the Tween 80 of 0.2mg/ml, the pH of the buffer solution The value is 6.8;

    (8) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the mannitol of 50mg/ml, the Tween 80 of 0.1mg/ml, the Tween 80 of the buffer solution The pH value is 6.0;

    (9) The preparation comprises the anti-C5a antibody of 15mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sorbitol of 45mg/ml, the Tween 80 of 0.1mg/ml, the Tween 80 of described buffer solution The pH value is 7.0;

    (10) The preparation comprises the anti-C5a antibody of 30mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the trehalose of 80mg/ml, the Tween 80 of 0.1mg/ml, the Tween 80 of described buffer solution The pH value is 6.2;

    (11) The preparation comprises the anti-C5a antibody of 50mg/ml, the histidine-histidine hydrochloride buffer solution of 30mM, the sucrose of 60mg/ml, the Tween 80 of 0.1mg/ml, the pH of the buffer solution Value is 6.0;

    (12) The preparation comprises the anti-C5a antibody of 100mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 50mg/ml, the Tween 80 of 0.1mg/ml, the pH of the buffer solution Value is 6.0;

    (13) The preparation comprises the anti-C5a antibody of 150mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 60mg/ml, the Tween 80 of 0.2mg/ml, the pH of the buffer solution Value is 6.0;

    (14) The preparation comprises the anti-C5a antibody of 200mg/ml, the histidine-histidine hydrochloride buffer solution of 20mM, the sucrose of 70mg/ml, the Tween 80 of 0.3mg/ml, the pH of the buffer solution Value is 6.0;

    (15) The preparation comprises 150 mg/ml of anti-C5a antibody, 20 mM phosphate buffer, 150 mM arginine hydrochloride, 0.2 mg/ml Tween 80, and the pH of the buffer is 5.5;

    (16) The preparation comprises 150 mg/ml of anti-C5a antibody, 20 mM citric acid-disodium hydrogen phosphate buffer, 100 mM sodium chloride, 0.1 mg/ml Tween 20, and the pH of the buffer is 5.8;

    (17) The preparation comprises the anti-C5a antibody of 150mg/ml, the histidine-histidine hydrochloride buffer solution of 10mM, the sodium chloride of 200mM, the Tween 80 of 0.2mg/ml, the Tween 80 of described buffer solution The pH is 6.8.
17. The preparation according to any one of claims 5-16, characterized in that, the preparation may further comprise a preservative and/or an antioxidant.
18. The preparation according to claim 17, wherein the preservative is ethylenediaminetetraacetic acid and/or diethylenetriaminepentaacetic acid; the antioxidant is methionine.
19. The preparation according to claim 18, 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.
20. The preparation according to any one of claims 5-19, characterized in that the preparation is a liquid preparation or a powder for injection.
21. Use of the antibody according to any one of claims 1-4 and the preparation according to any one of claims 5-20 in the preparation of medicines for treating diseases.
22. The use according to claim 21, wherein the disease comprises inflammatory, respiratory or autoimmune disease; preferably, the disease is selected from the group consisting of inflammatory response syndrome (SIRS), sepsis, severe sepsis, infectious Shock, ischemia/reperfusion-related injury, acute lung injury, pneumonia, acute and chronic graft rejection in transplant patients, graft-versus-host reaction, glomerular disease, glomerulonephritis, solid renal failure, rheumatoid arthritis , autoimmune disease, Bechterew's disease, lupus-like disease, inflammatory bowel disease, Crohn's disease, tumor growth, and solid organ cancer.

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