Anti-pspb antibodies and uses thereof

Abstract

The present disclosure provides an antibody or antigen-binding fragment thereof that binds to PSPB (pulmonary surfactant-associated protein B) or a fragment thereof. The present disclosure also relates to a detection kit for detecting PBPS and applications thereof.

Description

TECHNICAL FIELD

[0001] The present disclosure belongs to the field of biotechnology, and particularly relates to an antibody or antigen-binding fragment thereof of pulmonary surfactant-associated protein B (PSPB), a detection kit and application thereof. BACKGROUND

[0002] Interstitial lung disease (ILD) is a group of chronic lung diseases mainly involving the lung interstitial tissue. The main feature of this group of diseases is diffuse fibrosis of the alveolar septum in the lung tissue, accompanied by inflammatory cell infiltration and lung structure destruction, eventually leading to progressive damage of lung tissue and decline of lung function.

[0003] Pulmonary surfactant-associated protein B (PSPB) is a kind of essential amphiphilic surfactant protein closely related to the function of pulmonary surfactant. The main functions of PSPB include helping to maintain normal alveolar tension, reducing surface tension, and promoting respiratory gas exchange. By reducing the surface tension at the gas-liquid interface in the alveoli, PSPB can prevent lung collapse and thus maintain the stability of the alveoli. It is mainly expressed in lung type II alveolar epithelial cells and exists in the alveoli, but it can penetrate from the alveoli into the blood along the concentration gradient when the alveolar capillary barrier is destroyed.

[0004] In the pathogenesis of ILD, inflammation, fibrosis and functional damage in the lung often lead to the destruction of the alveolar capillary barrier, resulting in abnormal levels of PSPB in the blood. Therefore, by detecting the content of PSPB in the blood, it can not only help to determine whether the patient has the possibility of ILD, but also can evaluate the progression of ILD patients, providing a basis for timely adjustment of treatment plan. SUMMARY

[0005] In order to solve one of the above technical problems in the prior art, the present disclosure provides an antibody or antigen-binding fragment thereof against PSPB protein, which can specifically bind to PSPB, can be used for preventing or treating PSPB-related diseases, and can also be used for effectively detecting PSPB in the blood, thereby providing an efficient and accurate diagnostic basis for the clinical diagnosis of ILD.

[0006] The present disclosure first provides an antibody or antigen-binding fragment thereof binding to PSPB or a fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises:

[0007] (1) 3 heavy chain variable region complementarity determining regions (HCDRs):

[0008] HCDR1 having an amino acid sequence of HCDR1 contained in a heavy chain variable region as shown in any one of SEQ ID NOs: 1 to 4, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of HCDR1 contained in the heavy chain variable region, or having substitution, deletion, or addition of one or several amino acids;

[0009] HCDR2 having an amino acid sequence of HCDR2 contained in a heavy chain variable region as shown in any one of SEQ ID NOs: 1 to 4, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of HCDR2 contained in the heavy chain variable region, or having substitution, deletion, or addition of one or several amino acids;

[0010] HCDR3 having an amino acid sequence of HCDR3 contained in a heavy chain variable region as shown in any one of SEQ ID NOs: 1 to 4, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of HCDR3 contained in the heavy chain variable region, or having substitution, deletion, or addition of one or several amino acids;

[0011] and / or

[0012] (2) 3 light chain variable region complementarity determining regions (LCDRs):

[0013] LCDR1 having an amino acid sequence of LCDR1 contained in a light chain variable region as shown in any one of SEQ ID NOs: 17 to 20, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of LCDR1 contained in the light chain variable region, or having substitution, deletion, or addition of one or several amino acids;

[0014] LCDR2 having an amino acid sequence of LCDR2 contained in a light chain variable region as shown in any one of SEQ ID NOs: 17 to 20, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of LCDR2 contained in the light chain variable region, or having substitution, deletion, or addition of one or several amino acids;

[0015] LCDR3 having an amino acid sequence of LCDR3 contained in a light chain variable region as shown in any one of SEQ ID NOs: 17 to 20, or an amino acid sequence having at least 80% sequence identity to the amino acid sequence of LCDR3 contained in the light chain variable region, or having substitution, deletion, or addition of one or several amino acids.

[0016] In some embodiments, the heavy chain variable region comprises, in order from N-terminus to C-terminus, HCDR1, HCDR2, and HCDR3.

[0017] In some embodiments, the light chain variable region comprises, in order from N-terminus to C-terminus, LCDR1, LCDR2, and LCDR3.

[0018] In some embodiments, the 3 HCDRs contained in the heavy chain variable region and the 3 LCDRs contained in the light chain variable region are defined by the rules of Kabat, Chothia, IMGT, or a combination thereof.

[0019] In some embodiments, the antibody or antigen-binding fragment thereof comprises the 3 HCDRs contained in the heavy chain variable region as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises the 3 HCDRs contained in the heavy chain variable region as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region as set forth in SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0021] In some embodiments, the antibody or antigen-binding fragment thereof comprises the 3 HCDRs contained in the heavy chain variable region as set forth in SEQ ID NO: 3, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region as set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof comprises the 3 HCDRs contained in a heavy chain variable region as set forth in SEQ ID NO: 4, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in a light chain variable region as set forth in SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0023] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a HCDR1 as set forth in any one of SEQ ID NOs: 5-8, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions; and / or a HCDR2 as set forth in any one of SEQ ID NOs: 9-12, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions; and / or a HCDR3 as set forth in any one of SEQ ID NOs: 13-16, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0024] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises a LCDR1 as set forth in any one of SEQ ID NOs: 21-24, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions; and / or a LCDR2 as set forth in any one of SEQ ID NOs: 25-28, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions; and / or a LCDR3 as set forth in any one of SEQ ID NOs: 29-32, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0025] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0026] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0027] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0028] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0029] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 29, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0030] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 30, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0031] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment thereof comprises a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 23, SEQ ID NO: 27, and SEQ ID NO: 31, respectively, or an amino acid sequence having at least 80% sequence identity thereto or having one or several amino acid substitutions, deletions, or additions.

[0032] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment thereof comprises a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions; and, a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 29, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions; and, a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 30, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions.

[0035] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions; and, a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 23, SEQ ID NO: 27, and SEQ ID NO: 31, respectively, or an amino acid sequence that has at least 80% sequence identity thereto or that has one or several amino acid substitutions, deletions, or additions.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a HCDR1, a HCDR2, and a HCDR3 as set forth in SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16, respectively, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions; and, a LCDR1, a LCDR2, and a LCDR3 as set forth in SEQ ID NO: 24, SEQ ID NO: 28, and SEQ ID NO: 32, respectively, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 1-4, or an amino acid sequence having at least 80% sequence identity thereto; and / or, a light chain variable region having an amino acid sequence as set forth in any one of SEQ ID NOs: 17-20, or an amino acid sequence having at least 80% sequence identity thereto.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto; and, a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto; and, a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereto.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having an amino acid sequence as set forth in SEQ ID NO: 3, or an amino acid sequence having at least 80% sequence identity thereto; and, a light chain variable region having an amino acid sequence as set forth in SEQ ID NO: 19, or an amino acid sequence having at least 80% sequence identity thereto.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region having an amino acid sequence of SEQ ID NO: 4 or an amino acid sequence having at least 80% sequence identity thereto; and, a light chain variable region having an amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having at least 80% sequence identity thereto.

[0042] It will be understood by one of ordinary skill in the art that when referring to an amino acid sequence having at least 80% sequence identity to a heavy chain variable region or a light chain variable region, there is typically one or several amino acid substitutions, deletions, or additions to the amino acid sequence of the framework regions (FRs) or the non-complementarity determining regions (CDRs) of the heavy chain variable region or the light chain variable region.

[0043] In some embodiments, the antibody is of the IgA, IgD, IgE, IgG, or IgM type.

[0044] In some embodiments, the antibody or fragment thereof is an scFv, Fab, Fab', (Fab')2, Fv fragment, Fd, dsFv, diabody, bispecific antibody, and multispecific antibody.

[0045] In some embodiments, the antibody is a chimeric antibody or a humanized antibody.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and / or a light chain constant region.

[0047] In some embodiments, the heavy chain constant region is selected from an IgG1, IgG2, IgG3, or IgG4 heavy chain constant region.

[0048] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50of less than 1000 nM, less than 800 nM, less than 600 nM, less than 400 nM, less than 200 nM, or lower. 50 binds to human PSPB.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof has an EC50of less than 100 nM, less than 100 nM, less than 80 nM, less than 60 nM, less than 40 nM, less than 20 nM, less than 10 nM, or lower. 50 binds to human PSPB.

[0050] In another aspect of the present disclosure, there is provided an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof described above.

[0051] In yet another aspect of the present disclosure, there is provided a vector comprising the isolated nucleic acid molecule described above.

[0052] In yet another aspect of the present disclosure, a host cell comprising the isolated nucleic acid molecule or the vector is provided.

[0053] In yet another aspect of the present disclosure, a chimeric antigen receptor comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain is provided, wherein the extracellular antigen binding domain comprises the antibody or antigen binding fragment thereof.

[0054] In yet another aspect of the present disclosure, a modified immune cell comprising the chimeric antigen receptor is provided.

[0055] In yet another aspect of the present disclosure, an antibody drug conjugate comprising the antibody or antigen binding fragment thereof; and a drug covalently linked to the antibody or antigen binding fragment thereof is provided.

[0056] In yet another aspect of the present disclosure, a pharmaceutical composition comprising the antibody or antigen binding fragment thereof, or the antibody drug conjugate; and a pharmaceutically acceptable carrier is provided.

[0057] In yet another aspect of the present disclosure, a kit comprising the antibody or antigen binding fragment thereof is provided.

[0058] In some embodiments, the kit comprises: a first antibody or antigen binding fragment thereof; and a second antibody or antigen binding fragment thereof.

[0059] In some embodiments, the first antibody or antigen binding fragment thereof is selected from the 3 HCDRs contained in the heavy chain variable region as set forth in SEQ ID NO: 1, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region as set forth in SEQ ID NO: 17, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13, respectively, or an amino acid sequence that has at least 80% sequence identity thereto, or that has one or several amino acid substitutions, deletions, or additions; and, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 29, respectively, or an amino acid sequence that has at least 80% sequence identity thereto, or that has one or several amino acid substitutions, deletions, or additions.

[0061] In some embodiments, the first antibody or antigen-binding fragment thereof is selected from the 3 HCDRs contained in the heavy chain variable region as set forth in SEQ ID NO: 3, or an amino acid sequence that has at least 80% sequence identity thereto, or that has one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region as set forth in SEQ ID NO: 19, or an amino acid sequence that has at least 80% sequence identity thereto, or that has one or several amino acid substitutions, deletions, or additions. In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, and HCDR3 as set forth in SEQ ID NO: 7, SEQ ID NO: 11, and SEQ ID NO: 15, respectively, or an amino acid sequence that has at least 80% sequence identity thereto, or that has one or several amino acid substitutions, deletions, or additions; and, LCDR1, LCDR2, and LCDR3 as set forth in SEQ ID NO: 23, SEQ ID NO: 27, and SEQ ID NO: 31, respectively, or an amino acid sequence that has at least 80% sequence identity thereto, or that has one or several amino acid substitutions, deletions, or additions.

[0062] In some embodiments, the second antibody or antigen-binding fragment thereof is selected from the 3 HCDRs contained in the heavy chain variable region set forth as SEQ ID NO: 2, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region set forth as SEQ ID NO: 18, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions. In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NO: 6, SEQ ID NO: 10, and SEQ ID NO: 14, respectively, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions; and, LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NO: 22, SEQ ID NO: 26, and SEQ ID NO: 30, respectively, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions.

[0063] In some embodiments, the second antibody or antigen-binding fragment thereof is selected from the 3 HCDRs contained in the heavy chain variable region set forth as SEQ ID NO: 4, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions, and the 3 LCDRs contained in the light chain variable region set forth as SEQ ID NO: 20, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions. In some embodiments, the antibody or antigen-binding fragment thereof comprises: HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NO: 8, SEQ ID NO: 12, and SEQ ID NO: 16, respectively, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions; and, LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NO: 24, SEQ ID NO: 28, and SEQ ID NO: 32, respectively, or an amino acid sequence having at least 80% sequence identity thereto, or having one or several amino acid substitutions, deletions, or additions.

[0064] In some embodiments, the first antibody or antigen fragment thereof and / or the second antibody or antigen fragment thereof is coupled to a detectable label or magnetic beads.

[0065] In some embodiments, the detectable label comprises fluorescein isothiocyanate, 7-amino-4-methylcoumarin, 5-carboxyfluorescein, rhodamine B, luminol, isoluminol, acridinium ester, trispyridyl ruthenium, horseradish peroxidase, alkaline phosphatase, biotin.

[0066] In some embodiments, the kit comprises a first antibody or antigen-binding fragment thereof coated on magnetic beads and a second antibody or antigen-binding fragment thereof labeled with acridinium ester. In some embodiments, the kit comprises a second antibody or antigen-binding fragment thereof coated on magnetic beads and a first antibody or antigen-binding fragment thereof labeled with acridinium ester.

[0067] In another aspect of the present disclosure, a method for detecting PSPB in a sample is provided, which comprises the step of detection with the antibody or antigen-binding fragment thereof or the kit.

[0068] In some embodiments, the sample is whole blood, red blood cell concentrate, platelet concentrate, white blood cell concentrate, tissue, bone marrow aspirate, plasma, serum, cerebrospinal fluid, stool, urine, cultured cells, saliva, oral secretion, and / or nasal secretion.

[0069] In another aspect of the present disclosure, the use of the antibody or antigen-binding fragment thereof, the nucleic acid molecule, the chimeric antigen receptor, the immune cell, the antibody-drug conjugate, or the pharmaceutical composition in the diagnosis, prevention, and / or treatment of a PSPB-related disease is provided.

[0070] In some embodiments, the PSPB-related disease comprises interstitial lung disease, pulmonary carcinoid, lung adenocarcinoma, squamous carcinoma, neonatal respiratory distress syndrome, congenital pulmonary alveolar proteinosis, chronic obstructive pulmonary disease, acute lung injury, or pulmonary fibrosis. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 ELISA results showing the binding of four exemplary anti-human PSPB antibodies to human PSPB protein are shown.

[0072] Figure 2 Epitope binning results of exemplary anti-human PSPB antibodies are shown.

[0073] Figure 3 Chemiluminescence detection results of paired antibodies (113 & 114 and 115 & 116) on free huPSPB protein are shown.

[0074] Figure 4 Detection results of paired antibodies (113 & 114 and 115 & 116) on blood samples of interstitial lung disease patients, other lung disease patients, and healthy people are shown. DETAILED DESCRIPTION

[0075] In order to make the objects, technical solutions and advantages of the present application clearer, the following further describes the present application with reference to the embodiments. The specific embodiments described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of the known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0076] PSPB can penetrate from the alveoli into the blood along the concentration gradient when the alveolar capillary barrier is destroyed. Plasma usually contains little detectable PSPB, but in diseases that damage the alveolar capillary barrier, the PSPB content in the blood can serve as a biomarker. In pathology, the application of PSPB is quite extensive. For example, in the differential diagnosis of pulmonary sclerosing pneumocytoma and lung adenocarcinoma, and in the differential diagnosis of lung adenocarcinoma and squamous carcinoma, PSPB plays an important role. The plasma biomarker PSPB also has important significance in the diagnosis of ILD.

[0077] Definitions

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein and, unless otherwise specified, terms used in the singular will include the plural and vice versa, as appropriate for the context in which it is used. For purposes of interpreting this specification, the words "comprises," "comprising," "includes," "including," "has," "having" or the like are included in the description like a "consisting of" or "consisting essentially of."

[0079] The expressions "a" and "an" as used herein include plural references unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so on.

[0080] As used herein, the term "about" means a range of ±20% of the numerical value stated thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value stated thereafter. In some embodiments, the term "about" means a range of ±5% of the numerical value stated thereafter.

[0081] The term "antibody" or "full-length antibody" as used herein refers to an immunoglobulin molecule that typically consists of two pairs of polypeptide chains (each pair comprising one light (LC) and one heavy (HC) chain). Antibody light chains can be assigned to a kind, kappa (kappa) and lambda (lambda) light chains. The heavy chains can be assigned to alpha, delta, epsilon, gamma, or mu. Antibodies include five types, IgM, IgD, IgG, IgA, and IgE. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains do not directly participate in antibody binding to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the antigen binding site.

[0082] Natural IgG class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy chain domain or a heavy chain variable domain, and three constant domains (CH1, CH2 and CH3), also called a heavy chain constant region. From N- to C-terminus, each light chain has a variable region (VL), also called a variable light chain domain or a light chain variable domain, and a light chain constant domain (CL), also called a light chain constant region.

[0083] The term "complementarity determining region" or "CDR" as used herein refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The precise boundaries of these regions can be defined according to various numbering systems known in the art, such as defined according to the Kabat numbering system, the Chothia numbering system, the IMGT numbering system, or a combination thereof. For a given antibody, a skilled artisan will readily be able to identify the CDRs defined by each numbering system. And, the correspondence between different numbering systems is well known to the skilled artisan.

[0084] "Framework" or "FR" refers to variable domain residues other than hypervariable region (HVR) residues. The FR of a variable domain is generally comprised of four FR domains: FR1, FR2, FR3, and FR4. Thus, the HVR and FR sequences generally appear in the following sequence in VHand (or VL): FR1-HCDR1 (LCDR1)-FR2-HCDR2 (L2)-FR3-HCDR3 (LCDR3)-FR4.

[0085] The term "specific binding protein" refers broadly to molecules that specifically bind to an antigenic determinant. Antigen binding molecules or specific binding proteins include, for example, antibodies, antibody fragments, and scaffold antibodies or antigen binding fragments thereof.

[0086] The term "antigen binding fragment" of an antibody as used herein refers to a polypeptide fragment of an antibody, e.g., a polypeptide fragment of a full length antibody, that retains the ability to bind to the same antigen as the full length antibody, and / or has the ability to compete with the full length antibody for specific binding to the antigen, which is also referred to as an "antigen binding portion". Antigen binding fragments of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Non-limiting examples of antigen binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity determining region (CDR) fragments, single chain antibodies (e.g., scFv), chimeric antibodies, diabodies, linear antibodies, nanobodies, domain antibodies, and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding ability to the polypeptide.

[0087] Herein, unless the context clearly indicates otherwise, when referring to the term "antibody", it includes not only intact antibodies, but also antigen binding fragments of antibodies.

[0088] The term "Fd fragment" as used herein means an antibody fragment consisting of a VH and CH1 domain; the term "Fab fragment" means an antibody fragment consisting of a VL, VH, CL and CH1 domain; the term "F(ab')2 fragment" means an antibody fragment comprising two Fab fragments connected by a disulfide bridge over the hinge region; the term "Fv fragment" means an antibody fragment consisting of a VL and VH domain of a single arm of an antibody. Fv fragments are generally considered to be the minimum antibody fragments that can form a complete antigen binding site. It is generally considered that the six CDRs confer the antigen binding specificity of an antibody. However, even a single variable region (e.g., an Fd fragment, which contains only three CDRs specific for an antigen) is able to recognize and bind antigen, albeit perhaps at a lower affinity than the intact binding site.

[0089] The term "Fc fragment" means an antibody fragment formed by disulfide bonds between the second, third constant regions of the first heavy chain and the second, third constant regions of the second heavy chain of an antibody. The Fc fragment of an antibody has a variety of different functions, but is not involved in antigen binding.

[0090] The term "scFv" as used herein refers to a single polypeptide chain comprising a VL and a VH domain, wherein the VL and VH are connected by a linker. Such scFv molecules can have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. In some cases, a disulfide bond can also exist between the VH and VL of the scFv. The term "di-scFv" as used herein refers to an antibody fragment formed by the linkage of two scFv.

[0091] Each of the above-mentioned antibody fragments retains the ability to bind to the same antigen as the full-length antibody, and / or has the ability to compete with the full-length antibody for specific binding to the antigen. Antigen-binding fragments of antibodies (e.g., the above-mentioned antibody fragments) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those of skill in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage) and screened for specificity in the same manner as is done for whole antibodies.

[0092] The term "monoclonal antibody" of the present application refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind the same epitope except for possible variants that can exist in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different antigenic determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen.

[0093] The term "diabodies" as used herein refer to antibody fragments whose VHand VLdomains are expressed on a single polypeptide chain, but using a too-short linker that does not allow for pairing between the domains on the same chain, thereby forcing the domains to pair with the complement domains of another chain and creating two antigen binding sites.

[0094] A "humanized antibody" has an amino acid sequence corresponding to that of an antibody produced by a human or human cell or derived from an antibody of non-human origin that utilizes a human antibody repertoire or other human antibody-encoding sequences. This definition specifically excludes a humanized antibody comprising non-human antigen binding residues.

[0095] The present application also relates to amino acid sequence variants, which can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the molecule, or by peptide synthesis. Such modifications include, for example, deletions from, insertions into, and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution is made to arrive at the final construct, provided that the final constructs possess the desired characteristics, e.g., antigen binding activity. Sites of interest for substitutional mutagenesis include the HVRs and framework (FR) regions. As used herein, the terms "conservative" and "conservative substitution" can refer to the substitution of an amino acid residue by another amino acid residue having similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including families based on charge characteristics, such as basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a conservative substitution can be made by replacing an amino acid within a polypeptide with another amino acid from the same side chain family. In another aspect, a string of amino acids can be conservatively replaced with a string of structurally similar amino acids that differ in the order and / or composition of side chain family members.

[0096] "Percent sequence identity" or "percent identity" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions in a comparison window, taking into account any additions or deletions (i.e., gaps) that must be introduced in order to align the two sequences for optimal comparison. A matching position is any position in which the same nucleotide or amino acid is present in both the target and reference sequences. Gaps are not nucleotides or amino acids, and thus, the presence of a gap in the target sequence is not counted. Likewise, the presence of a gap in the reference sequence is not counted, as the nucleotides or amino acids from the target sequence are counted, but not those from the reference sequence.

[0097] The percentage of sequence identity can be calculated by determining the number of positions at which the same amino acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of the percentage of sequence identity between two sequences can be accomplished using software programs commonly available to those skilled in the art for online use and download. Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. One suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST suite of programs available from the National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm for comparison between two sequences. BLASTN is used for comparing nucleic acid sequences, while BLASTP is used for comparing amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk / Tools / psa.

[0098] As used herein, a sequence having "at least 80% sequence identity" to a sequence means having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the sequence.

[0099] The term "polynucleotide" or "nucleic acid" or "nucleotide sequence" of the present application refers to an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA). The polynucleotide can comprise conventional phosphodiester bonds or non-conventional bonds (e.g., amide, such as found in peptide nucleic acids (PNAs). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide.

[0100] The term "antigen binding domain" or "antigen binding site" of the present application refers to the part of an antigen binding molecule that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to the part of an antibody that comprises the region that specifically binds to and is complementary to a part or all of an antigen. In the case of an antigen molecule that is large, the antigen binding molecule can bind only to a specific part of the antigen, which is referred to as an epitope. The antigen binding domain can be provided by, for example, one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain is capable of binding to its antigen and blocking or partially blocking the function of said antigen.

[0101] The term "antigenic determinant" of the present application is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration composed of different regions of non-contiguous amino acids) to which an antigen binding moiety binds, thereby forming an antigen binding moiety-antigen complex. Antigenic determinants can be found, for example, on the surface of tumor cells, microbe-infected cells, other diseased cells, immune cells, free agents in serum, and / or extracellular matrix (ECM). Unless otherwise specified, proteins used as antigens in the present application can be any native form of protein of any vertebrate origin, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). The antigens can also be human proteins, or the antigens are "full-length," unprocessed proteins, as well as any form of the protein resulting from intracellular processing, or naturally occurring variants of the protein, e.g., splice variants or allelic variants.

[0102] "Specifically binds" refers to having binding selectivity for an antigen and can be distinguished from non-specific or unwanted binding. The ability of an antigen binding molecule to bind to a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one skilled in the art (e.g., surface plasmon resonance (SPR) techniques, biolayer interferometry (BLI), and traditional binding assays). In one embodiment, the extent of binding of an antigen binding molecule to an unrelated protein is less than about 10% of the extent of binding of the antigen binding molecule to the antigen, e.g., as measured by SPR. In certain embodiments, the dissociation constant (Kd) of a molecule that binds to an antigen is < 1 μM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g., 10 -7 M or lower, e.g., 10 -7 M to 10 -13 M, e.g., 10 -9 M to 10 -13 M).

[0103] Enzyme linked immunosorbent assay (ELISA) is an immunoassay method that combines antigens or antibodies on the surface of a solid carrier, uses the specific binding of antigens and antibodies, and the enzyme catalyzed by the labeled enzyme on the antibody or antigen to produce a color reaction of a specific substrate, to realize the detection of the target. Based on the ELISA method, the binding of antigens and antibodies (i.e. the affinity of antigens and antibodies) needs to have a clear four-parameter S-shaped curve, that is, there needs to be a signal curve that gradually changes with the concentration of the material to be detected (antibody, antigen or receptor, ligand). There are obvious upper and lower platform periods at low and high concentrations, and the antibody concentration corresponding to half of the signal value of the upper platform period (EC 50 ) value corresponds to the affinity KD value between antigens and antibodies. Therefore, under normal circumstances, the lower the EC 50 value, the higher the affinity of antigens and antibodies.

[0104] “Affinity” or “binding affinity” refers to the strength of the noncovalent interaction between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Binding affinity can generally be expressed as a dissociation constant (Kd), which is the ratio of the dissociation rate constant to the association rate constant (K off and K on , respectively). Thus, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by routine methods known in the art, such as surface plasmon resonance (SPR).

[0105] The term “isolated” nucleic acid molecule or polynucleotide of the present invention refers to a nucleic acid molecule, DNA or RNA, that has been separated from a naturally occurring environment. Recombinant polynucleotides encoding polypeptides contained in vectors are also isolated in the present invention. Other examples of isolated polynucleotides include a recombinant polynucleotide in a heterologous host cell or a purified polynucleotide in solution. An isolated polynucleotide includes a polynucleotide molecule that is contained in the cell in which it normally is found, but which polynucleotide molecule is present extrachromosomally or at a different chromosomal location than where it is normally found. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the present invention, in both positive and negative sense forms, and double-stranded forms. Isolated polynucleotides or nucleic acids of the present invention further include synthetically produced such molecules. In addition, the polynucleotide or nucleic acid can be or can include regulatory elements, such as promoters, ribosome binding sites, or transcription terminators.

[0106] The term "vector" or "expression vector" of the present invention is used interchangeably with "expression construct" and is a DNA molecule that directs the introduction of a particular gene with which it is operably linked into a target cell and directs expression. The vector includes vectors that are self-replicating nucleic acid structures as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. The expression vectors of the present invention comprise an expression cassette. The expression vector can be transcribed to produce a large number of stable mRNAs. Once the expression vector is within the target cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. The term "expression cassette" of the present invention refers to a recombinantly or synthetically generated polynucleotide having a series of nucleic acid elements that allow for the transcription of a particular nucleic acid in a target cell. The recombinant expression cassette can be introduced into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of the expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter.

[0107] The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, and also include the progeny of the original cell which has been transformed. Host cells include "transformants" and "transformed cells" and include primary transformed cells as well as progeny of the same. The nucleic acid of the progeny can not be identical to that of the parent cell from which it was derived, but can contain mutations. Host cells are any type of cell that can be used to produce the antibodies or antigen binding fragments thereof of the present invention. Host cells include cultured cells, e.g., cultured mammalian cells such as CHO cells, HEK293 cells, BHK cells, NSO cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells, yeast cells, insect cells, and plant cells, and also include cells contained within a transgenic animal, transgenic plant, or cultured plant or animal tissue.

[0108] The term "antibody drug conjugate" or "ADC" refers to a binding protein (such as an antibody or antigen binding fragment thereof) chemically linked to one or more chemical drugs. In preferred embodiments, the ADC comprises a binding protein, a drug, and a linker that links the binding protein to the drug.

[0109] The term "chimeric antigen receptor" or "CAR" refers to a receptor that has a desired antigen specificity and a signaling domain to propagate an intracellular signal upon antigen binding. For example, T lymphocytes recognize specific antigens via the interaction of the T cell receptor (TCR) with short peptides presented by class I or class II major histocompatibility complex (MHC) molecules. Naive T cells rely on antigen presenting cells (APCs) to provide additional costimulatory signals for initial activation and clonal expansion. In some embodiments, monocytes and macrophages can be engineered to, for example, express a chimeric antigen receptor (CAR). The modified cells can be recruited to the tumor microenvironment, where they act as potent immune effectors by infiltrating tumors and killing target cancer cells. CARs can include an antigen binding domain, a transmembrane domain, and an intracellular domain. The antigen binding domain binds to an antigen on a target cell. Examples of cell surface markers that can serve as antigens for antigen binding domains bound to CARs include those associated with viral, bacterial, parasitic infections, autoimmune diseases, and cancer cells (e.g., tumor antigens).

[0110] The term "modified immune cell" refers to an immune cell that has been genetically modified to express a CAR. In some embodiments, the immune cell is a T cell, or a cell derived therefrom. In some embodiments, the immune cell is a natural killer (NK) cell, or a cell derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cell is a B cell, or a cell derived therefrom. In some embodiments, the immune cell is a monocyte or macrophage, or a cell derived therefrom.

[0111] An "effective amount" of a drug refers to the amount necessary to effect a desired physiological change in a cell or tissue receiving administration thereof. An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disorder. An effective amount also means an amount sufficient to allow or facilitate diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health status of the patient, the method route and dose of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.

[0112] A "therapeutically effective amount" of a drug (e.g., a pharmaceutical composition) refers to the amount necessary to effect a desired therapeutic or prophylactic effect at dosages and intervals and time of administration. For example, a therapeutically effective amount of a drug eliminates, reduces, delays, minimizes, or prevents adverse effects of a disease.

[0113] The term "individual" or "subject" refers to a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Specifically, the individual or subject is a human.

[0114] The term "pharmaceutical composition" means a mixture of one or more antibodies of the disclosure or antigen-binding fragments thereof with other chemical components such as physiologically / pharmaceutically acceptable carriers or excipients. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to a subject and to facilitate absorption into the bloodstream thereby eliciting a biological activity.

[0115] The term "pharmaceutically acceptable carrier" refers to a component of a pharmaceutical composition other than the active ingredient that is not toxic to a subject. Pharmaceutically acceptable excipients include, but are not limited to, buffers, stabilizers, and / or preservatives.

[0116] The term "treatment" refers to the administration of an internal or external therapeutic agent, such as a composition comprising any of the antibodies of the disclosure or antigen-binding fragments thereof or nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, to a patient having one or more diseases or symptoms, to which the therapeutic agent has a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more diseases or symptoms in the treated patient or population to induce regression or inhibit progression of such symptoms to any clinically measurable extent.

[0117] Kabat antibody numbering system, an immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). Chothia antibody numbering system, an immunoglobulin numbering system proposed by Chothia et al., is a classic rule for identifying CDR region boundaries based on the location of structural loops (see, e.g., Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883). IMGT antibody numbering system, a numbering system based on the international ImMunoGeneTics information system (IMGT) initiated by Lefranc et al., can be found in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003.

[0118] Pulmonary surfactant-associated protein B (PSPB) is a homodimer synthesized by alveolar type II cells and secreted onto the surface of alveoli, containing 79 amino acids, with a molecular weight of about 18 kDa, rich in cysteine. Cysteine residues form a unique structure of PSPB: three intramolecular loops and one intermolecular disulfide bond, which limits the flexibility of PSPB while enhancing the thermal stability of the secondary structure, and has the effects of resisting acid and protease degradation.

[0119] The following examples and drawings are provided to aid in the understanding of the present application. It should be understood that these examples and drawings are illustrative only and do not limit the present application in any way. The true scope of the present application is set forth in the appended claims. It should be understood that any modifications and variations are possible without departing from the spirit of the present application.

[0120] Examples

[0121] Example 1: Preparation of anti-human PSPB antibody

[0122] Anti-human PSPB antibodies were obtained by immunizing Balb / c mice with huPSPB protein (NCBI RefSeq: NP_000533.4) as antigen, dissociating memory B cells that can recognize human PSPB from the spleen and lymph nodes of the mice, obtaining antibody variable region genes by PCR followed by sequencing, and reconstructing IgG type antibodies.

[0123] The obtained candidate antibodies comprise the amino acid sequences of the heavy chain variable region and the light chain variable region as shown in Table 1.

[0124] Table 1

[0125]

[0126]

[0127] The sequences of the heavy chain CDRs (HCDRs) and the light chain CDRs (LCDRs) of the above-mentioned antibodies are shown in Table 2 (defined according to the Kabat rule).

[0128] Table 2

[0129]

[0130] Example 2 Affinity determination of antibody binding to huPSPB recombinant protein

[0131] The day before the experiment, the antigen huPSPB protein solution was prepared using coating buffer (pH 9.6, 0.05M carbonate buffer) with a final concentration of 2 μg / mL. The prepared antigen huPSPB protein solution was added to the enzyme-labeled plate at a volume of 100 μL per well, and gently shaken until the liquid was evenly distributed on the bottom of each well. The enzyme-labeled plate containing the antigen was placed in a sealed bag and sealed in a 4°C refrigerator overnight to allow the antigen to be adsorbed on the enzyme-labeled plate.

[0132] The next day, the supernatant was discarded and dried on clean absorbent paper. 250 μL / well of washing solution (pH 7.4 PBST) was added, and each time was kept for 5 min. The supernatant was discarded and dried on clean absorbent paper, and the washing was repeated 3 times. 250 μL / well of blocking solution (PBST + 3% skim milk powder) was added, and the new sealed bag was placed in a 37°C incubator for 1 h. The supernatant was discarded and dried on clean absorbent paper, and 250 μL / well of washing solution was washed, each time for 5 min. The supernatant was discarded and dried on clean absorbent paper, and the washing was repeated 3 times.

[0133] The antibodies screened in Example 1 were gradient diluted (3-fold gradient dilution for 10 gradients starting from 10 pg / mL) using antibody diluent (pH 7.4 PBS). The diluted antibody samples were added to the washed microplate at 100 pL / well, placed into a new seal bag, and incubated at 37 °C for 1 h. The supernatant was discarded, patted dry on a clean absorbent paper, washed with 250 pL / well of washing solution for 5 min each time, and the supernatant was discarded and patted dry on a clean absorbent paper. The washing was repeated for 3 times.

[0134] The 1 :5000 diluted anti-human IgG-HRP secondary antibody (Sigma, #A0170) was added at 100 pL / well, placed into a new seal bag, and incubated at 37 °C for 1 h. The supernatant was discarded, patted dry on a clean absorbent paper, washed with 250 pL / well of washing solution for 5 min each time, and the supernatant was discarded and patted dry on a clean absorbent paper. The washing was repeated for 3 times. The TMB color developing solution was added at 100 pL / well, wrapped with tin foil to avoid light, and developed at room temperature for 15 min. The blue reaction was observed, and the stop solution (2 M H2SO4) was added at 50 pL / well. After mixing, the mixture was immediately placed into a microplate reader for reading at 450 nm wavelength.

[0135] The data were analyzed using GraphPad Prism 8 software, and the four-parameter nonlinear equation was used for curve fitting to calculate the EC 50 values (50% maximum effective concentration values) of the binding of huPSPB antigen and antibodies.

[0136] The results are shown in Table 3. Figure 1 and Table 3. The results show that the antibodies 91 & 92, 109 & 110, 113 & 114, and 115 & 116 all have high affinity to the huPSPB protein antigen.

[0137] Table 3

[0138] Antibodies EC 50 (μg / mL) 91&92 0.009225 109&110 0.008171 113&114 0.002921 115&116 0.005046

[0139] Example 3: Anti-human PSPB antibodies screened in Example 1 bind to different epitopes of the antigen

[0140] In this example, to investigate the antigen epitopes bound by the antibodies 91 & 92, 109 & 110, 113 & 114, and 115 & 116 screened in Example 1, an ELISA competition experiment was performed for evaluation, in which the biotin-labeled huPSPB protein antigen was used, and the unlabeled anti-human PSPB antibodies (antibodies 91 & 92, 109 & 110, 113 & 114, and 115 & 116) were used as test antibodies.

[0141] The day before the experiment, prepare coating antibody solutions (antibodies 91 & 92, 109 & 110, 113 & 114 or 115 & 116) using coating buffer (pH 9.6, 0.05M carbonate buffer) to a final concentration of 2 μg / mL. Add 100 μL of the prepared coating antibody solution to each well of the microplate and gently vortex until the liquid is evenly distributed at the bottom of each well. Place the microplate containing the coated antibody in a sealed bag and incubate at 4°C overnight to allow the antibody to adsorb onto the microplate.

[0142] The next day, discard the supernatant, pat dry on clean absorbent paper, add 250 μL / well washing buffer (pH 7.4 PBST), hold for 5 min each time, discard the supernatant, pat dry on clean absorbent paper, and repeat the washing 3 times. Add 250 μL / well blocking buffer (PBST + 3% skim milk powder), place in a new sealing bag, and incubate at 37°C for 1 h. Discard the supernatant, pat dry on clean absorbent paper, wash with 250 μL / well washing buffer, hold for 5 min each time, discard the supernatant, pat dry on clean absorbent paper, and repeat the washing 3 times.

[0143] Biotin-labeled huPSPB antigen was serially diluted using dilution buffer (pH 7.4 PBS). 100 μL of the diluted antigen was added to each well of the washed ELISA plate, which was then placed in a new sealing bag and incubated at 37°C for 1 h. The supernatant was discarded, and the plate was patted dry on clean absorbent paper. The plate was then washed with 250 μL of washing buffer for 5 min each time, and the supernatant was discarded again. The plate was then patted dry on clean absorbent paper. This process was repeated three times.

[0144] Add 100 μL / well of 1:2000 diluted hypersensitive streptavidin-HRP (Sigma, #S2438), place in a new sealed bag, and incubate at 37°C for 1 h. Discard the supernatant, blot dry on clean absorbent paper, and wash with 250 μL / well of washing buffer for 5 min each time. Discard the supernatant, blot dry on clean absorbent paper, and repeat the washing process 3 times. Add 100 μL / well of TMB chromogenic solution, wrap in aluminum foil, and develop at room temperature for 15 min in the dark. Observe the blue reaction. Add 50 μL / well of stop solution (2M H2SO4), mix well, and immediately place in a microplate reader for reading at 450 nm.

[0145] Data were analyzed using GraphPad Prism 8 software, and curve fitting was performed using a four-parameter nonlinear equation to calculate the 80% maximum effective concentration (EC50) of biotin-labeled huPSPB antigen binding to the antibody. 80 ) value. Obtain EC 80 After the value is obtained, a second round of ELISA is performed.

[0146] As in the first round of ELISA, the coating buffer (pH 9.6, 0.05 M carbonate buffer) was added with antibodies 91 & 92, 109 & 110, 113 & 114, 115 & 116 to a final concentration of 2 μg / mL, 5 plates for each antibody, 4 antibodies, 20 plates in total, and then placed in a refrigerator at 4°C for adsorption overnight, and washed and blocked the next day. In each of the 5 plates coated with the antibodies, 50 μg / mL of each of the four test antibodies 91 & 92, 109 & 110, 113 & 114, 115 & 116 and diluent (as a blank control) were added, followed by the addition of EC 80 to each well. The plates were placed in a new bag and incubated at 37°C for 1 h.

[0147] The plates were washed 3 times according to the washing procedure described above. 100 μL / well of 1:2000 diluted hypersensitive streptavidin-HRP was added, the plates were placed in a new bag and incubated at 37°C for 1 h. The plates were washed 3 times according to the washing procedure described above. 100 μL / well of TMB color developing solution was added, the plates were wrapped in tin foil and color developed at room temperature for 15 min. The blue reaction was observed, 50 μL / well of stop solution (2 M H2SO4) was added, mixed well and immediately placed in a microplate reader for reading at 450 nm wavelength to detect the biotin-labeled huPSPB antigen still bound to the plates.

[0148] In this example, competition between the test antibodies and the coating antibodies for the same or similar epitopes of the huPSPB antigen will reduce the binding of the biotin-labeled huPSPB antigen to the coating antibodies. The competition inhibition rate can be calculated by normalizing the values with the blank control wells without the addition of the test antibodies.

[0149] The results show that antibodies 91 & 92 and 113 & 114 partially compete, and 115 & 116 and 109 & 110 partially compete, and these competing antibodies all have a competition inhibition rate of > 20% ( Figure 2 ). This indicates that they bind to similar or similar epitopes on the huPSPB antigen. In contrast, neither of 91 & 92 and 113 & 114 compete with either of 115 & 116 and 109 & 110 ( Figure 2 ), proving that they bind to different epitopes of the huPSPB antigen.

[0150] Example 4 Chemiluminescence detection of free huPSPB protein by paired antibodies

[0151] In this embodiment, in order to verify the detection effect of the paired antibodies 113&114 and 115&116 on free huPSPB protein, the antibody 113&114 was used for magnetic bead coating, the 115&116 was labeled with acridinium ester, and then tested on a chemiluminescence platform.

[0152] (1) Antibody 113&114 coated magnetic beads

[0153] Place the magnetic beads (DynaBio, #MB1004) on the blood homogenizer until the magnetic beads are completely mixed. Prepare one centrifuge tube, add 1 mL of PBS to the centrifuge tube, use a pipette to remove 10 mg of magnetic beads, and mix by repeatedly blowing and beating 3-5 times. Place the centrifuge tube on the magnetic separator and let it stand for a period of time until the solution is completely clear. Use a pipette to remove the waste liquid and discard it. Remove the centrifuge tube from the magnetic separator and immediately add 1 mL of PBS. Mix by repeatedly blowing and beating 3-5 times with the pipette. Place the centrifuge tube on the magnetic separator and let it stand for a period of time until the solution is completely clear. Use a pipette to remove the waste liquid and discard it. Remove the centrifuge tube from the magnetic stand and immediately add 1 mL of PBS. Mix by repeatedly blowing and beating 3-5 times with the pipette and mix on the vortex mixer for 20 s. At this time, the concentration of magnetic beads is 10 mg / mL.

[0154] Add 25 μL of EDC solution with a concentration of 20 mg / mL and 25 μL of NHS solution with a concentration of 40 mg / mL to the above magnetic bead solution (EDC:NHS:magnetic beads = 0.05:0.1:1, m / m / m) in sequence, immediately mix by inverting, then place on a rotary homogenizer and react at room temperature for 15 min. Place the above solution on the magnetic separator and let it stand for a period of time until the solution is completely clear. Use a pipette to remove the waste liquid and discard it. Remove the centrifuge tube from the magnetic stand and immediately add 1 mL of PBS. Mix by repeatedly blowing and beating 3-5 times with the pipette and mix on the vortex mixer for 20 s. At this time, the concentration of magnetic bead solution is 10 mg / mL.

[0155] Add 100 μg of antibody 113&114 (final concentration 200 μg / mL) to the above 0.5 mL magnetic bead solution, immediately invert and mix, place on a rotary homogenizer and react at room temperature for 2 h. Directly add 100 μL of magnetic bead blocking agent ethanolamine to the above solution, immediately invert and mix, place on a rotary homogenizer and react at room temperature for 2 h. Place the above solution on the magnetic separator and let it stand for a period of time until the solution is completely clear. Use a pipette to remove the waste liquid and discard it. Remove the centrifuge tube from the magnetic stand and immediately add 1 mL of PBST. Mix by repeatedly blowing and beating 3-5 times with the pipette. Place the centrifuge tube on the magnetic separator and let it stand for a period of time until the solution is completely clear. Use a pipette to remove the waste liquid and discard it. Repeat this process 3 times.

[0156] Place the above magnetic bead solution on a touch-sensitive ultrasonic cell disruptor and sonicate at 8% power for 5 seconds, pause for 5 seconds, for a total of 2 minutes. Place the sonicated magnetic bead solution on a magnetic separator and let it stand for a period of time until the solution becomes completely clear. Use a pipette to remove and discard the waste liquid. Remove the centrifuge tube from the magnetic rack and add 1 mL of PBST to the centrifuge tube. Mix thoroughly by pipetting 3-5 times. Place the above magnetic bead solution back on the touch-sensitive ultrasonic cell disruptor and perform a second ultrasonic dispersion using the same procedure. Store the above magnetic bead solution at 2-8℃ for later use.

[0157] (2) Acridinium ester labeled antibodies 115 & 116

[0158] Take a centrifuge tube and add 2.7 μL of acridinium ester NSP-SA-NHS (Meilun Biotechnology, #MT0013-2) to the bottom. Next, add 72.3 μL of 0.2 M NaHCO3 (pH = 9.0) solution. Gently vortex the tube. Finally, add 100 μg of antibody 115 & 116 (final concentration 200 μg / mL). Pipette the tube repeatedly 3-5 times, gently vortex, seal the tube with sealing film, and centrifuge for 10 seconds. Then, incubate the mixture at 30°C for 30 minutes.

[0159] After the reaction is complete, add 2 μL of 0.2 M NaHCO3 (pH = 9.0) solution containing 10% (w / v) lysine to the above reaction solution. Pipette the solution repeatedly 3-5 times, gently vortex, seal the centrifuge tube with sealing film, centrifuge for 10 seconds in a handheld centrifuge, and then quench the reaction for 30 minutes in a constant temperature mixer. After quenching, use PBS as the dialysis buffer and dialyze the sample overnight using a 7 kDa dialysis bag.

[0160] Prepare a centrifuge tube, remove the dialysis bag, and gently shake the liquid from the dialysis bag into the waste container. Then, use absorbent paper to remove any remaining liquid from the dialysis bag and dialysis clamp. Loosen the dialysis clamp at one end of the dialysis bag and cut off this section of the dialysis bag along the clamp mark with scissors. Collect the labeled substance from the dialysis bag using a pipette and add it to the bottom of a brown centrifuge tube. Add 0.1M Na2HPO4-NaH2PO4 (pH=6.5) solution to the brown centrifuge tube until the concentration of acridinium ester labeled antibody is 0.25 mg / mL.

[0161] (3) Chemiluminescence detection of free huPSPB protein

[0162] huPSPB protein is diluted with PBS to 5 μg / mL, 1 μg / mL, 0.2 μg / mL three gradient concentrations as detection samples, PBS solution as blank control sample, magnetic bead coated antibody 113 & 114, acridinium ester labeled antibody 115 & 116 as detection kit, put into full-automatic chemiluminescence detector. Set 50 μL detection sample + 50 μL magnetic bead coated antibody + 50 μL acridinium ester labeled antibody detection program.

[0163] From the detection results, it can be seen that the detection ability of the kit for free PSPB protein has concentration dependence Figure 3 , which can realize accurate and effective detection of target protein.

[0164] Example 5 Diagnosis ability of paired antibodies to blood samples of patients with interstitial lung disease

[0165] Collect blood samples of 36 patients with interstitial lung disease (ILDs), 36 patients with other lung diseases (including asthma, COPD, lung cancer, pneumonia) (DC), 24 healthy people (HC), use double antibody sandwich detection kit of magnetic bead coated 113 & 114 antibody, acridinium ester labeled 115 & 116 antibody, and detect in full-automatic chemiluminescence detector.

[0166] The results show that the detection of PSPB content in blood not only significantly distinguishes interstitial lung disease patients from healthy people (p<0.0001) Figure 4 , but also effectively distinguishes interstitial lung disease patients from other lung disease patients (p<0.01) Figure 4 .

[0167] The technical scheme of the present application is not limited to the restriction of the above specific examples, and any technical modification made according to the technical scheme of the present application falls within the protection scope of the present application.

Claims

1. An antibody or antigen-binding fragment thereof that binds to PSPB, wherein, The antibody or its antigen-binding fragment comprises: (1) The three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 1, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 17; or (2) The three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 3, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO:

19. The heavy chain variable region, from the N-end to the C-end, includes HCDR1, HCDR2, and HCDR3 in sequence. The light chain variable region includes LCDR1, LCDR2 and LCDR3 sequentially from the N end to the C end; The three HCDRs contained in the heavy chain variable region and the three LCDRs contained in the light chain variable region are defined by the rules of Kabat, Chothia or IMGT.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody or its antigen-binding fragment includes: HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 5, SEQ ID NO: 9, and SEQ ID NO: 13 respectively; and LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 21, SEQ ID NO: 25, and SEQ ID NO: 29 respectively; or HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 7, SEQ ID NO: 11 and SEQ ID NO: 15 respectively; and LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 23, SEQ ID NO: 27 and SEQ ID NO: 31 respectively.

3. The antibody or its antigen-binding fragment according to claim 1 or 2, wherein, The antibody or its antigen-binding fragment comprises: The heavy chain variable region having an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region having an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 17; or The heavy chain variable region has an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO: 3; and the light chain variable region has an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:

19.

4. The antibody or its antigen-binding fragment according to claim 3, wherein, The antibody or its antigen-binding fragment comprises: The heavy chain variable region having the amino acid sequence shown in SEQ ID NO: 1; and the light chain variable region having the amino acid sequence shown in SEQ ID NO: 17; or The heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 3; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:

19.

5. The antibody or its antigen-binding fragment according to claim 1, wherein, The antibody is of type IgA, IgD, IgE, IgG, or IgM; The antibody or its antigen-binding fragment is scFv, Fab, Fab', (Fab')2, Fv fragment or dsFv.

6. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody is a chimeric antibody or a humanized antibody.

7. The antibody or antigen-binding fragment thereof according to claim 1, wherein, The antibody or its antigen-binding fragment further includes a heavy chain constant region and / or a light chain constant region.

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein, The heavy chain constant region is selected from the heavy chain constant regions of IgG1, IgG2, IgG3, or IgG4.

9. An isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1 to 8.

10. A vector comprising the isolated nucleic acid molecule of claim 9.

11. A host cell comprising the isolated nucleic acid molecule of claim 9, or the vector of claim 10.

12. The host cell according to claim 11, wherein, The cells are mammalian cells, yeast cells, or insect cells.

13. The host cell according to claim 12, wherein, The mammalian cells are CHO cells, HEK293 cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells, or hybridoma cells.

14. A kit comprising the antibody or antigen-binding fragment thereof as described in any one of claims 1 to 8.

15. The kit according to claim 14, wherein, The kit comprises: a first antibody or an antigen-binding fragment thereof; and a second antibody or an antigen-binding fragment thereof, wherein, The first antibody or its antigen-binding fragment includes: The three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 1; and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 17, and / or For example, the three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 3; and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 19; The second antibody or its antigen-binding fragment includes: The three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 2; and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 18; and / or For example, the three HCDRs contained in the heavy chain variable region shown in SEQ ID NO: 4, and the three LCDRs contained in the light chain variable region shown in SEQ ID NO: 20; The first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment bind to PSPB; The three HCDRs contained in the heavy chain variable region and the three LCDRs contained in the light chain variable region of the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment are defined by the rules of Kabat, Chothia or IMGT.

16. The kit according to claim 15, wherein, The first antibody or its antigen-binding fragment and / or the second antibody or its antigen-binding fragment are coupled with a detectable label or magnetic bead.

17. The kit according to claim 16, wherein, The detectable markers include fluorescein isothiocyanate, 7-amino-4-methylcoumarin, 5-carboxyfluorescein, rhodamine B, luminol, isoluminol, acridine ester, ruthenium tripyridine, horseradish peroxidase, alkaline phosphatase, and biotin.

18. The kit according to claim 17, wherein, The kit comprises a first antibody or its antigen-binding fragment coated with magnetic beads and a second antibody or its antigen-binding fragment labeled with a detectable marker, or the kit comprises a second antibody or its antigen-binding fragment coated with magnetic beads and a first antibody or its antigen-binding fragment labeled with a detectable marker.

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