CN113667015B - Antibodies targeting PSGL-1 protein and uses thereof - Google Patents

Abstract

The embodiment of the specification discloses an antibody targeting PSGL-1 protein and application thereof. The antibody may comprise: an antibody targeting human PSGL-1 comprising a light chain and a heavy chain, wherein: the light chain comprises the amino acid sequence of SEQ ID NO. 1 and the heavy chain comprises the amino acid sequence of SEQ ID NO. 5; alternatively, the light chain comprises the amino acid sequence of SEQ ID NO. 9 and the heavy chain comprises the amino acid sequence of SEQ ID NO. 13. The antibody provided by the invention can inhibit inflammatory reaction, and is applied to medicines for treating or preventing inflammatory diseases.

Description

Antibodies targeting PSGL-1 protein and uses thereof

Technical Field

The application relates to the technical field of immunology, in particular to an antibody targeting PSGL-1 protein and application thereof.

Background

P-selectin (P-selectin) is a glycoprotein with a molecular mass of 140kD, and is present on the Weibe-l Palade microsomal membrane and the platelet alpha-granule membrane of vascular endothelial cells. The stimulation of histamine, thrombin, phorbol ester and calcium ion carrier can quickly regulate the expression of membrane P-selectin, and the expression can be induced by hypoxia/reoxidation or oxygen free radical. P-selectin and E-selectin are expressed in low to zero amounts on the vascular endothelium under normal physiological conditions, and are rapidly up-regulated within minutes under inflammatory conditions.

P-Selectin Glycoprotein Ligand 1 (P selecting Glycoprotein Ligand 1, PSGL-1 for short) is also called CD162, the corresponding amino acid is SELPLG, and the P-Selectin Glycoprotein Ligand is formed by connecting two 120kD homologous sialylated mucins through a disulfide bond. After the PSGL-1 subunit is synthesized, the total length is 412 amino acids, after the synthesis, the 1-17 Signal peptide (Signal peptide) sequence is cut off, and then the 18-41 Propeptide (Propeptide) sequence is cut off through processing, so that the mature protein is finally formed. PSGL-1 was found to bind to all selectins including P-Selectin, E-Selectin (E-Selectin) and L-Selectin (L-Selectin) to recruit chemokines and activate integrins to regulate T cells. The glycosylation of PSGL-1 subunit is complex and various, and can be combined with P-selectin and the like in Ca2+ dependency after sialylation, fucosylation and sulfation modification. The differences in N-terminal glycosylation of PSGL-1 form multiple PSGL-1 isoforms that exhibit different affinities for L-or E-selectin.

PSGL-1, a cell surface adhesion molecule, is expressed on neutrophils, natural killer cells, monocytes, dendritic cells, T and B lymphocytes, and some CD34+ stem cells, and is also abundantly expressed on the platelet surface in the early stages of differentiation. PSGL-1 expression is rapidly increased under inflammatory conditions, induces leukocyte rolling and tethering on vascular endothelial cells by binding to L-/E-selectin on the surface of the vascular endothelial cells, and mediates infiltration or migration of leukocytes into inflammatory tissues. Since PSGL-1 mediates the pathological recruitment of leukocytes at the site of inflammation, PSGL-1 antibodies or repressors are potential anti-inflammatory drug candidates.

At present, no clinically available PSGL-1 antibody drug is available. Antibody drugs against PSGL-1 in clinical phase II trials: neihulizumab and SelK-2, and the corresponding indications are type I diabetes, graft-versus-host disease, psoriasis, psoriatic arthritis, ulcerative colitis, venous thromboembolism, asthma, chronic obstructive pulmonary disease and Crohn's disease. These conditions are all linked to PSGL-1 mediated intravascular rolling or tethering of leukocytes and recruitment to sites of inflammation. Clinical proof of concept and safety has been validated in 170 patients, who are currently being studied in multi-dose trials.

In addition, by targeting the highly expressed adhesion molecule PSGL-1 on tumor-associated macrophages, PSGL-1 antibody-VTX-0811 developed by sansheng drugs in combination with Verseau Therapeutics, inc, macrophages can be reprogrammed to a pro-inflammatory state, activating T cells and attracting other immune cells to synergistically produce potent anti-tumor effects. In both tumors that respond and do not respond to PD-1, VTX-0811 exhibits a stronger inflammatory response than current immunotherapy.

Therefore, aiming at the important target point PSGL-1, the development of candidate antibodies with stronger blocking effect and capable of inhibiting inflammatory reaction is urgently needed.

Disclosure of Invention

The embodiments of the present disclosure provide a method, apparatus, device and computer readable medium for targeting PSGL-1 protein to provide a candidate antibody with enhanced blocking effect for inhibiting inflammatory response.

In order to solve the above technical problem, the embodiments of the present specification are implemented as follows:

the embodiments of the present specification provide an antibody targeting human PSGL-1 comprising a light chain and a heavy chain, wherein: the light chain comprises the amino acid sequence of SEQ ID NO. 1 and the heavy chain comprises the amino acid sequence of SEQ ID NO. 5; alternatively, the light chain comprises the amino acid sequence of SEQ ID NO. 9 and the heavy chain comprises the amino acid sequence of SEQ ID NO. 13.

The embodiments of the present specification provide an antibody targeting human PSGL-1 comprising a light chain variable region and a heavy chain comprising a heavy chain variable region, wherein: the light chain variable region comprises CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 3 and CDR3 of SEQ ID NO. 4; and the heavy chain variable region comprises CDR1 of SEQ ID NO. 6, CDR2 of SEQ ID NO. 7 and CDR3 of SEQ ID NO. 8; or, a light chain comprising a light chain variable region comprising CDR1 of SEQ ID NO. 10, CDR2 of SEQ ID NO. 11 and CDR3 of SEQ ID NO. 12; and the heavy chain variable region comprises CDR1 of SEQ ID NO. 14, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 16.

Optionally, the antibody is a whole antibody or a Fab fragment.

Optionally, the antibody is a monoclonal antibody.

Optionally, the antibody further comprises a heavy chain constant region of an IgG1 subtype.

Optionally, the antibody further comprises a light chain constant region of the kappa subtype.

In alternative embodiments, the antibodies can inhibit endothelial cell adhesion to leukocytes.

In alternative embodiments, the antibodies block binding of THP-1 cells to recombinant human P-selectin/CD62P Fc chimeras.

The embodiments of the present disclosure also provide a pharmaceutical composition comprising an antibody targeting human PSGL-1 and a pharmaceutically acceptable excipient, diluent, or carrier. Optionally, the antibody targeting human PSGL-1 specifically comprises: a light chain comprising the amino acid sequence of SEQ ID NO. 1 and a heavy chain comprising the amino acid sequence of SEQ ID NO. 5; alternatively, a light chain comprising the amino acid sequence of SEQ ID NO 9 and a heavy chain comprising the amino acid sequence of SEQ ID NO 13. Optionally, the antibody targeting human PSGL-1 specifically comprises: a light chain variable region comprising CDR1 of SEQ ID NO. 2, CDR2 of SEQ ID NO. 3 and CDR3 of SEQ ID NO. 4 and a heavy chain variable region comprising CDR1 of SEQ ID NO. 6, CDR2 of SEQ ID NO. 7 and CDR3 of SEQ ID NO. 8; alternatively, the light chain variable region comprising CDR1 of SEQ ID NO 10, CDR2 of SEQ ID NO 11 and CDR3 of SEQ ID NO 12 and the heavy chain variable region comprising CDR1 of SEQ ID NO 14, CDR2 of SEQ ID NO 15 and CDR3 of SEQ ID NO 16.

The embodiments also provide a polynucleotide encoding an antibody or antigen-binding portion that targets human PSGL-1 as provided in the embodiments.

The present specification also provides a vector comprising the aforementioned polynucleotide encoding an antibody or antigen-binding portion targeting human PSGL-1 as provided by the embodiments of the specification.

In embodiments of the present specification, there is provided the use of the aforementioned antibody or a corresponding pharmaceutical composition in a medicament for the treatment or prophylaxis of an inflammatory disease.

Alternatively, the inflammatory disease may comprise dextran sodium sulfate induced chronic colitis. Specifically, the treatment or prevention may include: relieving colitis clinical symptoms, relieving colonic involvement, inhibiting inflammatory infiltration, relieving edema, and reducing surface necrosis.

Alternatively, the inflammatory disease may include acute pancreatitis induced by rana peptides. Specifically, the treatment or prevention may include: inhibiting inflammatory infiltration, improving pancreatic interstitial edema, and reducing acinar cell necrosis.

One embodiment of the present description can achieve at least the following advantages: the provided PSGL-1-targeting antibody can bind to PSGL-1 protein with high affinity, thereby playing a role in inhibiting inflammation and being used for preventing or treating inflammation.

The practice of the present application employs, unless otherwise indicated, conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques.

Unless otherwise indicated, terms used in the present application have meanings commonly understood by those skilled in the art.

Human PSGL-1 can also be referred to in the art as a selectin P ligand, SELPG, CLA, CD162, or PSGL1.

The term "antibody", as used herein, refers to an immunoglobulin molecule capable of specifically binding to a target via at least one antigen recognition site located in a variable region of the immunoglobulin molecule from a different group of mice. Targets include, but are not limited to, carbohydrates, multiple different groups of mouse polynucleotides, lipids, polypeptides, and the like. As used herein, "antibody" includes not only intact (i.e., full-length) antibodies, but also polypeptides comprising fragments thereof (such as Fab, fab ', F (ab') 2, fv); single chain variable fragment (scFv), single chain diabody (scDb), tandem single chain variable fragment (scFv) units (tandem scFv known as taFv) and mutants or other configurations thereof, fusion proteins comprising an antibody portion, any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site.

Typically, a complete or full-length antibody comprises two heavy chains and two light chains. Each heavy chain contains a heavy chain variable region (VH) and a constant region (CH). Each light chain contains a light chain variable region (VL) and a constant region (CL). Full-length antibodies can be of any class, such as IgD, igE, igG, igA, or IgM (or subclasses thereof), but the antibodies need not belong to any particular class. Depending on the antibody amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to different classes. Generally, there are five main classes of immunoglobulins: igA, igD, igE, igG and IgM, and several of these classes can be further classified into subclasses (isotypes), such as IgG1, igG2, igG3, igG4, igA1 and IgA2. The heavy chain constant domains corresponding to different immunoglobulin classes are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional structures of different classes of immunoglobulins are well known.

The term "antigen-binding fragment or antigen-binding portion" as used herein refers to a portion or region of an intact antibody molecule that is responsible for binding an antigen. The antigen-binding domain may comprise a heavy chain variable region (VH), a light chain variable region (VL), or both. Each of VH and VL typically contains three complementarity determining regions CDR1, CDR2, and CDR3. It is well known to those skilled in the art that the complementarity determining regions (CDRs, usually CDR1, CDR2 and CDR 3) are the regions of the variable region that have the greatest impact on the affinity and specificity of an antibody. There are two common definitions of CDR sequences for VH or VL, namely the Chothia definition and the Kabat definition. For a given antibody variable region sequence, according to Chothia definition or Kabat definition to determine VH and VL sequences in CDR sequence. For a given antibody variable region sequence, the variable region sequence of the middle CDR sequence can be analyzed in a variety of ways.

In describing the structure of the antibodies herein, the description relating to the numbering of amino acid positions refers to the EUnum be ring definition of the human IgG1 antibody, which is well known and readily ascertainable by one skilled in the art.

The term "specific binding" as used herein refers to a non-random binding reaction between two molecules, e.g. binding of an antibody to an epitope of an antigen.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in a small number of individuals. Monoclonal antibodies are typically highly specific for a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

As used herein, "chimeric antibody" refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. A complete chimeric antibody comprises two copies of a chimeric light chain and two copies of a chimeric heavy chain. The production of chimeric antibodies is known in the art. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic those of an antibody derived from one mammalian species, while the constant portions are homologous to sequences in an antibody derived from another species. One clear advantage of such chimeric forms is that the variable regions can be conveniently derived from currently known sources using, for example, readily available hybridomas or B cells from non-human host organisms in combination with constant regions derived from, for example, human cell preparations. While the variable region has the advantage of being easy to prepare and the specificity is not affected by its source, the constant region from a human is less likely to elicit an immune response in a human subject when injected with an antibody than the constant region from a non-human source. However, the definition is not limited to this particular example. In some embodiments, amino acid modifications are made in the variable and/or constant regions.

As used herein, "humanized antibody" refers to a form of a non-human (e.g., murine) antibody that is a specific chimeric immunoglobulin, immunoglobulin chain, or fragment thereof, such as the Fv, fab ', F (ab') 2 or other antigen binding subsequences of an antibody, that contains minimal sequence derived from a non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a recipient's Complementarity Determining Regions (CDRs) are replaced by residues from a CDR (donor antibody) of a non-human species, such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some cases, fv Framework Region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies may contain residues that are not in the recipient antibody nor in the imported CDR or framework sequences, but are included to further improve and optimize antibody performance. Typically, a humanized antibody will comprise substantially at least one and typically two variable domains in which all or substantially all of the CDR regions correspond to all or substantially all of the FR regions of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also comprises at least a portion of an immunoglobulin constant region or domain (e.g., an Fc domain), typically a portion of a human immunoglobulin.

"polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and/or RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any matrix that can be incorporated into the polymer by DNA or RNA polymerase (substrate). Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs.

As used herein, "vector" means a construct capable of delivery and, ideally, expression of one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells such as producer cells.

The term "inflammatory disease" as used herein refers to the general term for diseases in which inflammation is the main damaging factor. Inflammation is the biological response of tissue to noxious stimuli, a condition that is accompanied by three events, tissue degeneration, circulatory disturbance and fluid exudation, and hypertrophy. Examples of inflammatory diseases include acute and chronic diseases including, but not limited to, allergic pneumonia, atopic dermatitis, asthma, rhinitis, crohn's disease, ankylosing spondylitis, rheumatic fever, fibromyalgia, psoriatic arthritis, chronic nephritis, sjogren's syndrome, and multiple sclerosis, among others.

Drawings

In order to more clearly illustrate the embodiments of the present specification or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the description below are only some embodiments described in the present application, and for those skilled in the art, other drawings may be obtained according to the drawings without creative efforts.

FIG. 1 shows the sorting results of a flow cytometric sorter for antigen-specific memory B cells;

FIG. 2 shows the results of ELISA assays of cell culture supernatants;

FIG. 3 shows a eukaryotic expression vector used in the examples of the present specification;

FIG. 4A shows fluorescence microscopy observations of endothelial cell adhesion to leukocytes; FIGS. 4B and 4C show flow cytometry measurements reflecting the binding of various groups of THP-1 cells to recombinant human P-selectin/CD62P Fc chimeras;

figures 5A to 5D show the results of disease activity index analysis of different groups of mice in the DSS-induced mouse chronic colitis model; figures 5E and 5F show colon shortening and engorgement in different groups of mice in the DSS-induced mouse chronic colitis model;

figure 6A shows flow cytometry analyses of whole blood cells, neutrophil and monocyte percentage of leukocytes, from different groups of mice in a DSS-induced mouse model of chronic colitis; figure 6B shows the results of staining colon tissue of different groups of mice in the DSS-induced mouse model of chronic colitis; FIG. 6C shows the results of the measurement of MPO activity in plasma and colon tissues of different groups of mice in a DSS-induced mouse chronic colitis model; figure 6D shows the results of an analysis of the levels of inflammatory cytokines in the plasma of different groups of mice in the DSS-induced mouse model of chronic colitis;

fig. 7A shows the results of staining of pancreatic tissue of different groups of mice in a rana peptide-induced mouse acute pancreatitis model; fig. 7B shows the results of analysis of plasma amylase levels in different groups of mice in the rana peptides-induced mouse acute pancreatitis model; fig. 7C shows the results of analysis of inflammatory cytokines in plasma of different groups of mice in the rana peptide-induced mouse acute pancreatitis model; FIG. 7D shows the analysis results of the percentage of neutrophils and monocytes in whole blood cells of different groups of mice in the Rana dybowskii peptide-induced mouse acute pancreatitis model;

FIG. 8A shows stained sections of organs of different groups of mice; fig. 8B is a graph showing the analysis results of the cardiotoxicity test, the liver function test, and the kidney function test for different groups of mice.

Description of the sequence:

SEQ ID NO:1 the amino acid sequence of the light chain variable region (VL) of antibody Rh 001-6;

the amino acid sequence of SEQ ID NO: amino acid sequences of VL CDR1 to CDR3 of 2-4 antibody Rh 001-6;

SEQ ID NO: amino acid sequence of heavy chain variable region (VH) of antibody Rh 001-6;

SEQ ID NO: amino acid sequences of VH CDR1 to CDR3 of 6-8 antibody Rh 001-6;

SEQ ID NO:9 the amino acid sequence of the light chain variable region (VL) of antibody Rh 001-22;

SEQ ID NO: amino acid sequences of VL CDR1 to CDR3 of 10-12 antibody Rh 001-22;

SEQ ID NO:13 the amino acid sequence of the heavy chain variable region (VH) of antibody Rh 001-22;

the amino acid sequence of SEQ ID NO: amino acid sequences of VH CDR1 to CDR3 of 14-16 antibody Rh001-22.

Detailed Description

In the examples of the present specification, antigen vaccines of different PSGL-1 were designed to immunize rhesus monkeys three times, with higher abundance of antigen-specific memory B cells 2 weeks after the third immunization. Then, memory B cells are harvested by using PSGL-1 specific antigen, cultured in vitro, and cultured in vitro for 2 weeks under the action of CD40L, interleukin 2 (IL 2) and interleukin 21 (IL 21), and the memory B cells proliferate and differentiate into plasma cells to secrete antibody. And then ELISA is used for detecting the antibody specificity in the supernatant, and antibody gene cloning is carried out subsequently to obtain the novel monkey-derived monoclonal antibody targeting PSGL-1. Finally, expression purification of the obtained novel antibodies (e.g., antibodies Rh001-6 and Rh 001-22) can be performed. Furthermore, the biological function of newly obtained antibodies targeting PSGL-1 (e.g., rh001-6 and Rh 001-22) can be identified by in vivo and in vitro experiments.

Based on the scheme of the embodiment of the specification, the main effective component of the obtained product is monkey-derived monoclonal antibody, the molecular weight is 150kD, the solvent is PBS, the pH value is 7.4, and the purity is more than 95%. The basic structure of the obtained monoclonal antibody is composed of four peptide chains, i.e., two identical smaller molecular weight peptide chains (light chains) and two identical larger molecular weight peptide chains (heavy chains), wherein the light chains are of the Kappa (. Kappa.) subtype and the heavy chains are of the gamma chain (i.e., igG subtype).

The PSGL-1-targeting antibody provided by the embodiment of the invention can be combined with PSGL-1 protein with high affinity, plays a role in inhibiting inflammation, can be used for preventing or treating inflammation, and has a wide application prospect.

The following is a detailed description of specific embodiments.

The first embodiment is as follows: in vitro culture of monkey memory B cells and antibody gene cloning construction

1.1 in vitro culture of monkey memory B cells

1.1.1 preparation of feeder cells

The artificially constructed NIH/3T3 cell line over expressing human CD40L and IL21 is recovered from liquid nitrogen, and the cell state is kept after passage to about 5 generations, and the cell density is 80%. The medium was discarded, replaced with a complete medium containing 50. Mu.g/mL mitomycin, and placed in an incubator at 37 ℃ for 2 hours. The culture medium was discarded, washed 7 times with DMEM medium and 3 times with PBS, and mitomycin was completely removed. The cells were then digested with pancreatin, blown off, centrifuged, resuspended in 3T3 medium and counted.

1.1.2 preparation of B cell Medium

45mL of IMDM medium was taken Plus 5mL of LFBS, 100. Mu.l of the antibiotic complex MycoZap Plus-PR, IL-2 (final concentration 100U/mL) and IL-21 (final concentration 100 ng/mL).

1.1.3 resuspend 3T3 cells in B cell culture media and add 200. Mu.l to a 96-well U-bottom plate (50000 cells/well).

The day 1.1.4, 10mL to 20mL of fresh monkey blood was drawn to isolate PBMCs, and antigen-specific memory B cells (CD 3-CD14-CD20+ CD27+ IgG +) were sorted into 5 cells per well using a flow cytometric sorter. The results of sorting the antigen-specific memory B cells by flow cytometric sorting are shown in fig. 1.

1.1.5A 96-well plate containing B cells and feeder cells was incubated at 37 ℃ in an 8% carbon dioxide incubator for 14 days.

1.1.6 two weeks after incubation 50. Mu.l of cell culture supernatant was taken for ELISA to detect antibody specificity.

1.2ELISA screening of clone supernatants bound to the antigen hPSGL-1-ECD

Coating hPSGL-1-ECD antigen protein 1 mu g/mL and 100 mu l/hole overnight at 4 ℃; discard the liquid, PBST wash 2 times, 200 u l/hole; sealing 5% skimmed milk 200 μ l/hole at normal temperature for 2 hr; discarding the liquid, and washing for 2 times by PBST; adding 50. Mu.l or 100. Mu.l of B cell culture supernatant, and incubating at room temperature for 2 hours; discarding the liquid, PBST washing 4 times; mu.l of HRP-labeled goat anti-human Fab antibody was added per well as a secondary antibody, 1:1000 to 1:3000 dilution, and incubation for 1 hour at normal temperature; discarding the liquid, PBST washing 4 times; adding 100 mul of TMB color development solution into each hole, and incubating for 2-5 minutes in a dark place; add 50. Mu.l of stop buffer (2N dilute sulfuric acid) and read the OD 450.

FIG. 2 shows the results of ELISA detection of cell culture supernatants. As shown in FIG. 2, the positive clones were determined to be more than 2 times higher than the background value, and 10 positive clones were selected in total.

1.3 Mixed B cell antibody Gene cloning after in vitro culture

1.3.1 discard B cell culture supernatant, add 200. Mu.l TRIZOL and lyse for 10 minutes at room temperature to extract RNA.

1.3.2 reverse transcription cDNA synthesis of the extracted RNA. Table 1 shows the reaction system for reverse transcription of RNA for cDNA synthesis.

TABLE 1 cDNA Synthesis reaction System

1.4 nested PCR amplification of variable regions of heavy and light chains of antibody genes

1.4.1 materials and methods

Primers for the first round of PCR were made to follow exactly the literature sequence (literature references: weixu Meng, leike Li, wei Xiong, et al (2015) Efficient generation of monoclonal antibodies from single species bacillus macaque cloning cells, mAbs,7, 4,707-718, DOI: 10.1080/19420862.2015.1051440), and primers for the second round of PCR were made to add a 15bp cloning vector homologous sequence to the 5' end of the literature sequence. Each sequence is prepared into 100 mu mol concentration, and the equal volumes of the gene sequence primers of different subtypes are mixed together, so that the final concentration of each primer is 10 mu mol. As the DNA polymerase, rrimeSTAR GXL DNA polymerase was used. Nested PCR systems are shown in tables 2 to 5 below:

TABLE 2 first round PCR reaction System

TABLE 3 first round PCR reaction conditions

TABLE 4 second round PCR reaction System

TABLE 5 second round PCR reaction System

1.4.2 results

The nested PCR products were identified by 2% agarose electrophoresis, the size of the heavy chain variable region was about 400bp, and the light chain was 350bp. Cutting the positive PCR target strip, recovering antibody gene fragments by using a gel recovery kit, and cloning antibody genes by using a homologous recombination kit. The eukaryotic expression vector plasmids AbVec-hIgkappa or AbVec-hIgG1 containing light and heavy chain constant region fragments are respectively constructed to constitute the complete antibody light and heavy chain full-length genes, for example, containing the sequences encoding SEQ ID NO:1,5,9, 13, or a pharmaceutically acceptable salt thereof. FIG. 3 illustrates a eukaryotic expression vector used in the examples of the present specification.

And respectively transforming the constructed vectors containing the full-length genes of the light and heavy chains of the antibody into escherichia coli TOP10, culturing overnight at 37 ℃, and extracting the plasmids of the light and heavy chains of the antibody by using an endotoxin-free plasmid extraction kit for sequencing and eukaryotic expression.

Extracting plasmid for second generation sequencing. The antibody sequence was known to be completely novel.

Example two: expression and purification of full-length antibodies

In this example, 2 Fab antibodies with better blocking activity for binding hPSGL-1-ECD obtained in the first example were constructed as human IgG1 subtype, wherein all light chains are kappa type and the antibody type is fully human antibody.

Candidate antibodies Rh001-6 and Rh001-22 were expressed by an Expi293 transient expression system. The specific method comprises the following steps: on the day of transfection when Expi293 cells were cultured in 250mL culture flasks, the cell density was confirmed to be about 7X 106 viable cells/mL, and the cell viability was confirmed to be around 7X 106 viable cells/mL>98% cells were adjusted to a final concentration of 6X 106 cells/mL (final volume 75 mL) using 37 ℃ pre-warmed Expi293 expression medium. 1mL of Expi293 solution precooled at 4 DEG C ^TM Expression mediumThe plasmid of interest (50. Mu.g) was diluted and used in the presence of 1mL of Expi293 ^TM The expression medium dilutes the transfection reagent 75 mu L FectoPro, then 1mL of the two are mixed in equal volume and are gently mixed to prepare the Expi293 ^TM The expression medium/plasmid DNA mixture, incubated at room temperature for 15 minutes, slowly added to the prepared cell suspension, placed in a cell culture shaker, and cultured at 37 deg.C, 5% CO2. At 24 hours after transfection, fectoPRO boroster was added to the culture system (final concentration of 0.6. Mu.L/mL), and the culture was continued in a shaker at 37 ℃ and in 5% CO2. On day 5 post-transfection, the same volume of Expi293 was added slowly ^TM The medium was expressed into the culture system, and after collecting cell culture supernatant after 10 days of transfection, centrifugation was carried out for 10 minutes at 4000G, affinity purification was carried out using a Protein G agarose column, the objective Protein was eluted with 100mM sodium acetate (pH 3.0), and after neutralization with 1M Tris-HCl, the resultant Protein was replaced into PBS buffer by a 30kD ultrafiltration concentration tube.

The relative molecular weight of two candidate antibodies Rh001-6 and Rh001-22 is determined to be 150kD, and the purity is more than 90 percent. Specifically, the protein concentration of the purified antibody was measured by an ultramicrospectrophotometer, and the value obtained by dividing the measured a280 value by the theoretical extinction coefficient of the antibody was defined as the antibody concentration value. Candidate antibodies were then dispensed and stored at-80 ℃.

Example three: detection of adhesion capacity of endothelial cells and leukocytes mediated by antibody Rh001-6 and Rh001-22 of targeting PSGL-1

3.1 fluorescent microscope observation of Rh001-6 and Rh001-22 antibodies inhibiting the adhesion of endothelial cells and leukocytes

3.1.1 endothelial cell culture: HUVEC cells of human umbilical vein endothelial cells in good growth state were cultured in an ECM culture chamber at 37 ℃ and 5% CO2.

3.1.2 Take 96-well cell culture plates, resuspend HUVEC cells (1X 105/mL) with ECM medium, add 100. Mu.L HUVEC cells per well.

3.1.3 morning, 1. Mu.g/mL, 5. Mu.g/mL, 10. Mu.g/mL of Rh001-6 and Rh001-22 antibodies, respectively, were added to HUVEC cells and cultured for 48 hours.

3.1.4 preparation of human peripheral blood mononuclear cells THP-1 cells, culture of THP-1 cells in RPMI-1640 medium containing 10% FBS +1% P/S, culture environment 37 ℃ 5% CO2. THP-1 cells in good growth state were collected, the leukocyte concentration was adjusted to 1X 109/mL, and the THP-1 cells were stained with BCEC-F-AM for 30 minutes, followed by washing twice with 1mL PBS, centrifugation at 1000rpm at 4 ℃ for 5 minutes, and finally re-suspension of the THP-1 cells with PBS.

3.1.5 incubation of stained THP-1 cells with treated endothelial cells in a cell incubator at 37 ℃ C. And 5% CO2 for 1 hour. The cells were then washed three times with PBS to remove non-adherent cells. The adhesion of endothelial cells to leukocytes was observed using a fluorescence microscope.

As shown in fig. 4A, the fluorescence microscope observation results of the adhesion of endothelial cells to leukocytes are shown. The results show that the number of fluorescing cells is significantly reduced after treatment with the Rh001-6 and Rh001-22 antibodies compared to the positive control, i.e., the Rh001-6 and Rh001-22 antibodies can significantly inhibit the adhesion between endothelial cells and leukocytes, and have a significant concentration-dependent effect.

3.2Rh001-6 and Rh001-22 antibodies block the ability of THP-1 cells to bind to Recombinant Human P-selectin/CD62P Fc Chimera (Recombinant Human P-selectin/CD62P Fc Chimera)

3.2.1 THP-1 cells resuspended in HBSS were added separately to flow tubes (100. Mu.l/tube).

3.2.2 blocking peripheral blood mononuclear cell Fc fragment antigen: purified murine anti-human CD16/CD32 (murine BD Fc Block reagent) was added to the THP-1 suspension and incubated for 30 minutes at room temperature.

3.2.31mL PBS stops the antigen-antibody combination reaction, centrifuges for 5 minutes at the temperature of 4 ℃ and at the rpm of 2000, and discards and washes the excessive antibody; the washing was repeated once to wash out the remaining antibody.

After 3.2.4100 μ l PBS resuspended THP-1 cells, the cells were divided into four groups: a blank control group, a recombinant human P-selectin/CD62P Fc chimera treatment group, and a recombinant human P-selectin/CD62P Fc chimera added with Rh001-6 and Rh001-22 antibody groups with different concentrations. The treatment groups were incubated at room temperature for 30 minutes.

3.2.51mL PBS stops the antigen-antibody combination reaction, centrifuges for 5 minutes at 4 ℃ and 2000rpm, abandons and washes the excess antibody; the washing was repeated once to wash out the remaining antibody.

3.2.6100. Mu.l PBS re-suspended THP-1 cells, fluorescent antibody (CD 45 APC. Cy7 and CD62P BV 605) was added and mixed gently and incubated at room temperature for 30 minutes in the dark.

3.2.7 Add 1mL PBS to stop the antigen-antibody binding reaction, wash away excess antibody, 4 degrees C, 2000rpm centrifugal 5 minutes, repeated washing.

3.2.8 add 200 u l PBS heavy suspension, flow cytometry detection, use FCS Express 6 Flow Research Edition data statistics, statistics each group CD62P + CD45 +/total CD45+ cell percentage.

As shown in fig. 4B and 4C, flow cytometry results reflecting the binding of each set of THP-1 cells to the recombinant human P-selectin/CD62P Fc chimera are shown. The results show an approximately 2-fold decrease in CD62P + CD45 +/total CD45+ cells at a concentration of 10. Mu.g/mL of Rh001-6 antibody; there was also an approximately 1.5-fold reduction in CD62P + CD45 +/total CD45+ cells at a concentration of 10. Mu.g/mL of the Rh001-22 antibody. Thus, it was shown that both antibodies, rh001-6 and Rh001-22, were able to block the binding capacity of THP-1 cells to the recombinant human P-selectin/CD62P Fc chimera and had concentration-dependent effects.

Example four: antibody Rh001-6 treatment significantly improved Dextran Sodium Sulfate (DSS) -induced chronic colitis (IBD)

4.1 establishment of dextran sodium sulfate induced mouse chronic colitis model

In this example, female Balb/c mice (6-8 weeks old) were randomly divided into five groups (5 per group), the different groups of mice were weighed and labeled, placed in a 12 hour light/dark cycle environment, and allowed to access standard food and water ad libitum. Mice were given 2% DSS in water for 7 days, followed by 1% DSS in water for 10 days, and then 2% DSS in water for 7 days, and control mice were given normal water.

Disease Activity Index (DAI) was recorded every three days starting on the first day of DSS administration: including animal body weight, fecal consistency/diarrhea, and colonic bleeding using a fecal occult blood test kit. According to the clinical parameters DAI scoring the disease: the percentage of weight loss is 10-20%, loose stool/diarrhea and occurance of recessive/major hemorrhage, and whether the IBD model is successfully constructed is judged.

4.2Rh001-6 antibody can significantly relieve colitis symptoms of mice

To verify whether Rh001-6 antibodies could alleviate colitis-related inflammatory symptoms, following the success of the mouse-induced IBD model, mice were divided into a control group, a DSS +6-TG group administered with 2mg/kg of IBD-positive therapeutic drug 6-thioguanine (6-TG, as an immunosuppressive drug, has been shown to be useful as a positive therapeutic for chronic colitis), an anti-hSGL antibody (Rh 001-6) 1mg/kg dose group (i.e., DSS +1mg/kg Rh001-6 group), and an anti-hSGL antibody (Rh 001-6) 10mg/kg dose group (i.e., DSS +10mg/kg Rh001-6 group). The drug was administered by tail vein injection once every three days for a total of three times. Animals were weighed daily before starting treatment and before the end of treatment. Animals were checked daily for consistency of their stool and for blood in the stool.

As shown in fig. 5A to 5D, the results of disease activity index analysis of different groups of mice in the DSS-induced mouse chronic colitis model are shown. The results show that the DSS-induced mice have obvious weight loss, diarrhea and colonic hemorrhage, the DAI score reaches 4 points, and after the DSS-induced mice are treated by 6-TG and 10mg/kg Rh001-6, the DAI score is obviously reduced, and the clinical symptoms of the colitis of the mice are obviously relieved.

4.3Rh001-6 antibody can effectively reduce the degree of colon involvement of mice

At the end of treatment, animals were sacrificed using tribromoethane deep anesthesia. The colon was dissected and the entire colon was removed from the cecum to the anus and measured as an indirect marker of inflammation.

As shown in fig. 5E and 5F, colonic shortening and engorgement was shown for different groups of mice in the DSS-induced mouse chronic colitis model. The results show that after DSS induction, the colon of a mouse is obviously shortened and congestion and swelling appear, the colon of a 6-TG treatment group mouse is not obviously shortened and the congestion and swelling are obviously weakened, the colon of a 1mg/kg Rh001-6 treatment group mouse is obviously shortened and the congestion and swelling of the colon are weakened, the colon of a 10mg/kg Rh001-6 treatment group mouse is not obviously shortened and the congestion and swelling are obviously weakened, and the 10mg/kg Rh001-6 treatment group mouse can effectively reduce the colonic involvement degree.

4.4Rh001-6 antibody can obviously reduce the percentage of neutrophils and monocytes accounting for white blood cells after treatment

Peripheral blood from mice was collected, blood samples were lysed with 1 XBD FACS lysate and stained for Ly6C, ly6G, CD11b, CD45 and isotype matched controls with FITC-, APC-, perCP cy 5.5-and APC. Cy7-binding Abs on ice in the dark and fluorescently labeled antibody for 30 minutes to identify the percentage of neutrophils and monocytes in leukocytes in different groups of whole blood cells. The labeled and fixed samples were analyzed on the FACSCanto III system either immediately or within 24 hours by flow cytometry. Before each run, the BD cytometer set up and track the beads for internal calibration. Appropriate quality controls were prepared for each sample to compensate and detect non-specific binding. Cell fluorescence was quantified as the mean fluorescence intensity or percentage of double positive cells at each time point.

As shown in fig. 6A, flow cytometry analysis results of whole blood cells, neutrophil and monocyte percentage of leukocytes, of different groups of mice in the DSS-induced mouse chronic colitis model are shown. The results show that the percentage of the neutrophils and the monocytes accounting for the white blood cells in the whole blood cells of the mice after the DSS induction is obviously increased, and the percentage of the neutrophils and the monocytes accounting for the white blood cells is obviously reduced after the treatment of 6-TG and 10mg/kg Rh 001-6.

4.5Rh001-6 antibody treatment reduces inflammatory cell infiltration of colon tissue in mice

Two 0.3-0.5 cm sections of the distal-most colon were transversely and longitudinally cut and fixed in 10% neutral buffered formalin. The remainder of the colon is cut longitudinally and the contents of the distal section are removed and frozen on dry ice. Mucus from the distal colon was scraped with a spatula and placed in 100. Mu.l Tris buffer (50mM Tris,5mM EDTA, 50mM NaCl, pH 8) and frozen on dry ice. The frozen colon samples were stored at-80 ℃ until used for further experiments.

Fixed tissue samples were dehydrated in ethanol, isopropanol, and xylene and embedded in paraffin. The tissue was cut to a thickness of approximately 5 μm using a rotary microtome and stained with hematoxylin and eosin (H & E). The image is taken from a selected sample and digitized using an image scanner. The extent of inflammation, edema and surface necrosis, as well as infiltration of granulocytes, lymphocytes and macrophages into the tissue were assessed from the stained sections.

As figure 6B, the results of staining colon tissue of different groups of mice in the DSS-induced mouse chronic colitis model are shown. The results show that after the DSS induction, the colon tissues of the mice have obvious inflammatory cell infiltration, crypt deformation and most of epithelium are damaged, after the mice are treated by 6-TG and 10mg/kg Rh001-6, the inflammatory cell infiltration of the colon tissues of the mice is obviously reduced, and the epithelium and the crypt are complete and orderly.

Neutrophil Myeloperoxidase (MPO) and monocyte/macrophage (F4/80) expression level detection: leukocyte infiltration, especially of neutrophils and macrophages, is an important process leading to the development of IBD. This example therefore examined the infiltration of leukocytes in colon tissue of different groups of mice using immunohistochemical methods. Paraffin embedded tissues were deparaffinized in xylene (twice for 5 minutes each), rehydrated in 100%, 95% and 70% ethanol (5 minutes each), and blocked with 2% normal goat serum in TPBS bovine serum albumin for 30 minutes at room temperature. They were then diluted with the antibody MPO, F4/80 at 1. Sections were washed with PBS and then detected using a general two-step detection kit. And (3) performing hematoxylin dyeing after DAB color development is finished, and performing dehydration and mounting after all dyeing is finished. Finally the digital image is captured and processed using a fluorescence microscope.

As shown in FIG. 6B, the results showed that the mice in the DSS-induced group had significantly more MPO-positive and F4/80-positive leukocyte infiltrates than the control group, and that the leukocyte infiltrates were significantly reduced after the administration of 6-TG and 10mg/kg Rh 001-6.

4.6Rh001-6 antibody for treating significant decrease in MPO activity

MPO activity assay in plasma and colon tissues: whole blood was centrifuged at 4000rpm for 30 minutes to obtain plasma, and tissue samples from control, DSS-treated or treated groups of animals, including tissue specimens from the middle to distal colon (adjacent to the tissue used for histology), were stored at-80 ℃ until use. MPO activity assay was performed using a mouse myeloperoxidase enzyme linked immunosorbent assay kit (MPO).

As shown in FIG. 6C, the results of the measurement of MPO activity in plasma and colon tissues of different groups of mice in the DSS-induced mouse chronic colitis model are shown. The results show that the MPO activity in the plasma and colon tissues of the DSS induction group is obviously improved, and the MPO activity of the 6-TG and 10mg/kg Rh001-6 treatment group is reduced by more than 2 times compared with that of the DSS group.

4.7Rh001-6 antibody reduces production of Th1 and Th17 cytokines

Levels of the inflammatory cytokines interferon-gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in plasma were measured using an enzyme-linked immunosorbent assay. The detection is carried out by using a mouse IFN-gamma enzyme-linked immunosorbent assay kit, a mouse TNF-alpha enzyme-linked immunosorbent assay kit and a mouse interleukin-6 beta enzyme-linked immunosorbent assay kit respectively. Briefly, serum was added to wells of 96-well plates coated with IFN-. Gamma.TNF-. Alpha.or IL-6 monoclonal antibodies, incubated for 2 hours at 37 ℃, washed 4 times with wash solution for 3 minutes each, and then incubated for 30 minutes at 37 ℃ in a dark room with horseradish peroxidase-linked streptavidin solution. All samples were tested in triplicate and absorbance measured at 450nm using a microplate reader.

As figure 6D, results of the analysis of inflammatory cytokine levels in plasma of different groups of mice in the DSS-induced mouse chronic colitis model are shown. The results show that the production of Th1 (IFN-gamma and TNF-alpha) and Th17 (IL-6) cytokines is obviously increased in the DSS induction group, and the treatment groups of 6-TG and 10mg/kg Rh001-6 can relieve DSS colitis and reduce the production of Th1 and Th17 cytokines.

Example five: antibody Rh001-6 treatment significantly improves Acute Pancreatitis (AP) induced by rana japonica peptide

4.1 establishment of Rana Nigromaculata peptide-induced mouse acute pancreatitis model

In the embodiment, 20 healthy male Balb/c mice (6-8 weeks old) are selected, the body weight is 20-25g,5 mice are distributed in an SPF (specific pathogen free) barrier system in a box mode, the temperature is 22 +/-2 ℃, the humidity is 50% -60%, the illumination is carried out in a 12-hour circulating mode, and the mice can take food and water freely.

50 mu g of Rana temporaria peptide (AP) model is adopted for inducing acute pancreatitis by intraperitoneal injection ^-1 .h ^-1 The induction dose of (4) was 1 injection every 1 hour and 7 injections were given in total. Mice were sacrificed 24 hours from the first injection. After the experiment is finished, the mass of the mouse body is weighed, the mouse body is anesthetized by bromoform intraperitoneal injection, the mouse body is placed on an animal experiment table in a supine position, the four limbs are fixed, the chest and the abdomen are exposed after depilation, and the inferior vena cava blood sampling method is adopted. After standing at room temperature for 30 minutes, the mixture was centrifuged at 3,000rpm at 4 ℃ for 15 minutes, and the serum was separated to determine the amylase level. Dissect the mouse abdominal cavity, take the pancreas tissue, weigh the mass after the filter paper is dried, and one of them is performed HE staining. The rest pancreas tissues were sub-packaged in 1.5mL centrifuge tubes and stored at-80 ℃.

After mice successfully induced the AP model, the mice were randomized into a control group, an AP group, an anti-hPSGL antibody (Rh 001-6) 1mg/kg dose group (i.e., AP +1mg/kg Rh001-6 group), and an anti-hPSGL antibody (Rh 001-6) 10mg/kg dose group (i.e., AP +10mg/kg Rh001-6 group). Administration was by tail vein injection, starting 1 hour after the first needle injection of ranolamin, once every 3 hours for a total of three administrations.

4.2Rh001-6 antibody can remarkably relieve inflammatory reaction and acinar injury of pancreatitis of mice

At the end of treatment, animals were sacrificed using tribromoethane deep anesthesia. The mouse abdominal cavity was dissected, pancreatic tissue was taken, mass-weighed after blotting with filter paper, and fixed in 10% neutral buffered formalin, and the fixed tissue sample was embedded in paraffin and stained with hematoxylin and eosin (H & E).

As figure 7A, staining results of different groups of mouse pancreatic tissues in a rana peptide-induced mouse acute pancreatitis model are shown. The results show that the pancreatic tissue structure of the mice is destroyed after induction of the rana peptides, obvious edema and leukocyte infiltration appear in the tissues, and acinar cells are widely necrotic, while the pancreatic tissue structure of the mice is intact after the treatment of 10mg/kg of Rh001-6 antibody, and interstitial edema, acinar cell necrosis and inflammatory cell infiltration of the pancreas are obviously weakened.

In fig. 7B, analysis results of plasma amylase levels of different groups of mice in the rana japonica peptide-induced mouse acute pancreatitis model are shown. The level of the plasma amylase of the mice after induction of the rana japonica peptide is obviously higher than that of a control group, and the plasma amylase is obviously reduced after treatment by 10mg/kg of Rh001-6 antibody.

In fig. 7C, the results of analysis of inflammatory cytokines in plasma of different groups of mice in the rana grahami peptide-induced mouse acute pancreatitis model are shown. Compared with AP mice, the level of proinflammatory cytokine IL-6 in the blood plasma of the mice treated by the 10mg/kg Rh001-6 antibody is obviously reduced.

4.3 percentage of neutrophils and monocytes leukocytes significantly decreased after treatment with Rh001-6 antibody

Peripheral blood of mice is taken, after cell lysis solution is used for lysis, ly6C, ly6G, CD11b, CD45 and isotype matching control are dyed for 30 minutes on ice in a dark place, and the percentage of neutrophils and monocytes in the whole blood cells of different groups to leukocytes is analyzed.

As shown in fig. 7D, the results of analysis of the percentage of neutrophils and monocytes in whole blood cells of different groups of mice in the rana grahami peptide-induced mouse acute pancreatitis model are shown. The results show that the percentage of the neutrophils and the monocytes in the whole blood cells of the mice is obviously increased after the induction of the rana japonica peptide, and the percentage of the neutrophils and the monocytes in the white blood cells is obviously reduced after the treatment of 10mg/kg of Rh 001-6.

Example six: analysis of biocompatibility of antibody Rh001-6

Hearts, livers, kidneys, lungs and spleens of different groups of mice were individually fixed in 10% neutral buffered formalin and embedded in paraffin, and the tissues were cut to a thickness of about 5 μm and stained with hematoxylin and eosin (H & E).

As in fig. 8A, stained sections of organs of different groups of mice are shown. Microscopic observations showed that no significant pathological lesions were observed in sections of heart, kidney, liver, lung and spleen. Any organ-related toxicity caused by rat tail intravenous injection of different Rh001-6 was also assessed by plasma biochemical parameters.

In this example, organ toxicity was evaluated in vivo in different Rh001-6 treated groups. As fig. 8B, the analysis results of the cardiotoxicity test, liver function test, and kidney function test for different groups of mice are shown. The results showed that cardiotoxicity tests (CK), liver function tests (ALT, AST and TBIL levels) and kidney function tests (BUN and CRE levels) were similar for all groups. This indicates that Rh001-6 is biocompatible in vivo, has no toxicity to organs, and is highly safe.

The novel monkey monoclonal antibody targeting PSGL-1 provided by the embodiment of the specification has the advantages of high specificity, stronger inflammatory response blocking, high affinity, low immunogenicity, strong stability and the like. In addition, in vivo experiments prove that the novel PSGL-1 targeting monkey monoclonal antibody provided by the embodiment of the specification can be used for treating inflammation, has no toxic or side effect on organs, and has a very great application value in clinical treatment.

Claims (10)

1. An antibody targeting human PSGL-1 comprising a light chain and a heavy chain, wherein:

the light chain comprises the amino acid sequence of SEQ ID NO. 1 and the heavy chain comprises the amino acid sequence of SEQ ID NO. 5;

alternatively, the light chain comprises the amino acid sequence of SEQ ID NO 9 and the heavy chain comprises the amino acid sequence of SEQ ID NO 13.

2. An antibody targeting human PSGL-1 comprising a light chain variable region and a heavy chain comprising a heavy chain variable region, wherein:

the light chain variable region comprises CDR1 shown in SEQ ID NO. 2, CDR2 shown in SEQ ID NO. 3 and CDR3 shown in SEQ ID NO. 4; and the heavy chain variable region comprises CDR1 shown in SEQ ID NO. 6, CDR2 shown in SEQ ID NO. 7 and CDR3 shown in SEQ ID NO. 8;

alternatively, the light chain variable region comprises CDR1 of SEQ ID NO. 10, CDR2 of SEQ ID NO. 11 and CDR3 of SEQ ID NO. 12; and the heavy chain variable region comprises CDR1 of SEQ ID NO. 14, CDR2 of SEQ ID NO. 15 and CDR3 of SEQ ID NO. 16.

3. The antibody of claim 1 or 2, further comprising a heavy chain constant region of an IgG1 subtype and a light chain constant region of a kappa subtype.

4. The antibody of claim 1 or 2, which inhibits adhesion of endothelial cells to leukocytes; and/or blocking binding of THP-1 cells to recombinant human P-selectin/CD62P Fc chimeras.

5. A pharmaceutical composition comprising the antibody of claim 1 or 2 and a pharmaceutically acceptable excipient, diluent or carrier.

6. A polynucleotide encoding the antibody of claim 1 or 2.

7. A vector comprising the polynucleotide of claim 6.

8. Use of an antibody according to claim 1 or 2 or a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the treatment or prophylaxis of inflammatory diseases, such as hypersensitivity pneumonitis, atopic dermatitis, asthma, crohn's disease, psoriatic arthritis, or multiple sclerosis.

9. Use of an antibody according to claim 1 or 2 or a pharmaceutical composition according to claim 5 for the manufacture of a medicament for the treatment or prophylaxis of an inflammatory disease which is chronic colitis induced by sodium dextran sulphate; the treatment or prevention comprises: relieving colitis clinical symptoms, relieving colonic involvement, inhibiting inflammatory infiltration, relieving edema, and reducing surface necrosis.

10. Use of the antibody of claim 1 or 2 or the pharmaceutical composition of claim 5 for the preparation of a medicament for the treatment or prevention of an inflammatory disease, said inflammatory disease being acute pancreatitis induced by rana peptides; the treatment or prevention comprises: inhibiting inflammatory infiltration, improving pancreatic interstitial edema, and reducing acinar cell necrosis.

Images