Antibody or antigen binding fragment of targeting CTGF and application of antibody or antigen binding fragment

Abstract

A CTGF binding molecule, in particular an antibody and fragment thereof that specifically recognizes CTGF. Also provided are nucleic acids or host cells comprising such antibodies or fragments thereof, medicaments comprising such antibodies or fragments thereof, and therapeutic and diagnostic methods or uses of using these antibodies and fragments.

Description

An antibody targeting CTGF or its antigen-binding fragment and its application Technical Field

[0001] The present invention relates to the field of antibodies and, more specifically, to CTGF-binding molecules, particularly antibodies and fragments thereof that specifically recognize CTGF. Furthermore, the present invention relates to nucleic acids or host cells containing such antibodies or fragments thereof, medicaments containing such antibodies or fragments thereof, and therapeutic and diagnostic methods or uses utilizing such antibodies and fragments. Background Art

[0002] Idiopathic pulmonary fibrosis (IPF) is an irreversible, progressive, and fatal chronic pulmonary fibrosis characterized by diffuse interstitial fibrosis accompanied by low-grade inflammation, the development of fibroblastic foci, extensive proliferation and deposition of extracellular matrix, and the formation of cellulite. IPF patients often experience progressive exertional dyspnea and decreased lung function. As the disease progresses, these symptoms become irreversible, ultimately leading to death from respiratory failure. The average survival after diagnosis of IPF is only 2.8 years, with a 5-year survival rate of less than 30%. It has been dubbed "a cancer that isn't cancer." It predominantly affects individuals over 50 years old and is more common in men than in women. There are approximately 150,000 cases in the United States, with at least 30,000 new cases annually. China is estimated to have over 500,000 patients. Given my country's high smoking rates, which are likely higher than those in other countries, the number of patients is steadily increasing. Given its relatively low prevalence, IPF is considered a rare disease in most countries, and the global market is projected to exceed US$2 billion in 2018.

[0003] Human connective tissue growth factor (CTGF), also known as CCN2 (cellular communication network factor 2), is a cysteine-rich, secreted growth factor composed of 349 amino acids and belongs to the CCN (cyr61, CTGF, nov) family. Its protein structure comprises: domain 1, the insulin-like growth factor binding region (IGFBP); domain 2, the von Willebrand factor C-type repeat region (VWC); domain 3, the thrombospondin type 1 repeat region (TSP); and domain 4, the C-terminal cysteine-rich binding region. These four domains interact with multiple cytokines and cell surface proteins, including IGF, TGF-β, BMPs, and LRP, to stimulate fibroblast proliferation, promote the phenotypic transformation of various cell types into myofibroblasts, mediate cell adhesion and migration, and participate in and promote fibrosis.

[0004] There is evidence that TGF-β-driven accumulation of myofibroblasts is a major feature of idiopathic pulmonary fibrosis. Based on the enhanced extracellular stress caused by cell remodeling behavior and changes in extracellular matrix structure, TGF-β activates interstitial intrinsic fibroblasts and catalyzes the transdifferentiation of epithelial cells to mesenchymal cells.

[0005] The plasma concentration of connective tissue growth factor (CTGF) is significantly higher in patients with idiopathic pulmonary fibrosis than in healthy controls. In particular, CTGF is strongly correlated with decreased forced vital capacity in patients with idiopathic pulmonary fibrosis, indicating a positive correlation between CTGF concentration and the severity of idiopathic pulmonary fibrosis, making it a biomarker for IPF. CTGF is the primary TGF-β-driven factor in idiopathic pulmonary fibrosis. In lung epithelial cells, CTGF is regulated by TGF-β, with CTGF concentration increasing significantly with TGF-β. Under TGF-β regulation, the fibrosis-associated molecule SMA is significantly elevated, while the mesenchymal cell transition marker E-cad is significantly decreased. Inhibition of the CTGF gene reverses SMA and E-cad expression. Given its downstream TGF-β pathway, CTGF possesses greater selectivity, making it an effective therapeutic target for idiopathic pulmonary fibrosis.

[0006] Although some CTGF antibodies exist, the field still needs new antibodies specific for CTGF, especially therapeutic antibodies that can be effectively used for the clinical purpose of alleviating CTGF-related diseases, and can be applied clinically to achieve the purpose of treating or alleviating diseases.

[0007] Summary of the Invention

[0008] The present invention discloses an antibody or an antigen-binding fragment thereof targeting CTGF and applications thereof.

[0009] The present invention therefore provides a novel antibody that binds to CTGF, and an antigen-binding fragment thereof.

[0010] In some embodiments, the anti-CTGF antibodies of the invention have one or more or all of the following properties:

[0011] (1) Ability to bind to human or rhesus monkey CTGF with high affinity;

[0012] (2) capable of binding to human or rhesus monkey CTGF expressed on the cell membrane surface with high affinity, such as CTGF domain 2;

[0013] (3) can block the binding of TGF-β1 and CTGF, such as CTGF domain 2;

[0014] (4) It can bind to FcRn in an acidic environment (approximately pH 6.0) but not in a neutral environment (approximately pH 7.4);

[0015] (5) can effectively treat pulmonary fibrosis; or

[0016] (6) It has a good serum half-life.

[0017] In some embodiments, the present invention provides nucleic acids encoding the antibodies or fragments thereof of the present invention, vectors comprising the nucleic acids, and host cells comprising the vectors.

[0018] In some embodiments, the present invention provides methods of making an antibody or fragment thereof of the present invention.

[0019] In some embodiments, the present invention provides immunoconjugates, pharmaceutical compositions, and combination products comprising the antibodies of the invention.

[0020] The present invention also provides methods for blocking CTGF-mediated signaling pathways in an individual using the antibodies of the present invention, as well as methods for preventing or treating CTGF-related diseases, such as immune system diseases (eg, autoimmune diseases or inflammation).

[0021] The present invention also relates to a method for detecting CTGF in a sample.

[0022] The present invention is further illustrated in the following drawings and specific embodiments. However, these drawings and specific embodiments should not be considered to limit the scope of the present invention, and changes that are readily apparent to those skilled in the art will be included within the spirit of the present invention and the protection scope of the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows antibody binding to CHOK1 cells expressing CTGF domain 1;

[0024] Figure 2 shows antibody binding to CHOK1 cells expressing CTGF domain 2;

[0025] Figure 3 shows antibody binding to CHOK1 cells expressing CTGF domain 3;

[0026] Figure 4 shows antibody binding to CHOK1 cells expressing CTGF domain 4;

[0027] Figure 5 shows that antibodies block the binding of TGF-β1 to CTGF domain 2 on the surface of CHOK1 cells;

[0028] Figure 6 shows the effect of CTGF antibody on total cell number and differential cell count in BALF of mice with BLM-induced pulmonary fibrosis;

[0029] FIG7 shows the effect of CTGF antibody on soluble collagen in BALF of mice with BLM-induced pulmonary fibrosis;

[0030] FIG8 shows the results of the lung tissue pathological scoring of mice with BLM-induced pulmonary fibrosis treated with CTGF antibodies;

[0031] FIG9 shows the effect of CTGF antibody on lung function in mice with BLM-induced pulmonary fibrosis;

[0032] Figure 10 shows the serum drug concentration of 2101Z25m21 after tail vein administration to SD rats (10 mg / kg, n=3);

[0033] FIG11 shows the serum drug concentration of 2101Z25m23 after tail vein administration to SD rats (10 mg / kg, n=3).

[0034] Detailed Description of the Invention

[0035] The present invention therefore relates to a novel anti-CTGF antibody or antigen-binding fragment thereof.

[0036] In some embodiments, the anti-CTGF antibodies or fragments thereof of the invention bind to mammalian (e.g., mouse), e.g., primate CTGF (e.g., human CTGF or rhesus monkey CTGF) with high affinity, e.g., with an equilibrium dissociation constant (K D ) binds to CTGF (eg, human CTGF), the K D Less than about 10 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, 0.05 nM, 0.04 nM or 0.03 nM, or within a range of about 0.01 nM, 0.02 nM or 0.05 nM to any of the above values, or within a range of any of the above values.

[0037] In some embodiments, the antibodies or fragments thereof of the present invention are capable of binding to domain 2 of CTGF, such as binding to cells expressing domain 2 of CTGF.

[0038] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can block the binding of TGF-β1 to CTGF (eg, CTGF domain 2), such as blocking the binding of TGF-β1 to CTGF (eg, CTGF domain 2) on the cell surface.

[0039] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention are capable of binding to FcRn in an acidic environment (approximately pH 6.0), but do not bind to FcRn in a neutral environment (approximately pH 7.4).

[0040] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention can effectively treat pulmonary fibrosis, such as reducing the total white blood cell count in the lungs, reducing the number of neutrophils, lymphocytes and / or macrophages; reducing inflammation, and / or improving lung function (e.g., vital capacity), etc.

[0041] In some embodiments, the antibodies or antigen-binding fragments thereof of the invention have a good serum half-life, such as about 1 day or 2 days, such as greater than or equal to 1 day.

[0042] In some embodiments, an anti-CTGF antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3.

[0043] In some embodiments, an anti-CTGF antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0044] In some embodiments, an anti-CTGF antibody or antigen-binding fragment thereof of the invention comprises three complementarity determining regions (HCDRs) from a heavy chain variable region and three complementarity determining regions (LCDRs) from a light chain variable region.

[0045] In some aspects, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH). In some aspects, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention comprise a light chain variable region (VL). In some aspects, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region comprises three complementary determining regions (HCDRs) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3, such as HCDR1, HCDR2, and HCDR3 determined by the Chothia scheme. In some embodiments, the light chain variable region comprises three complementary determining regions (LCDRs) from the light chain variable region, LCDR1, LCDR2, and LCDR3, such as LCDR1, LCDR2, and LCDR3 determined by the Chothia scheme.

[0046] In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody heavy chain constant region. In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention further comprise an antibody light chain constant region. In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention further comprise a heavy chain constant region and a light chain constant region.

[0047] In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention comprise an antibody heavy chain (HC). In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention comprise an antibody light chain (LC). In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention comprise a heavy chain and a light chain.

[0048] In some embodiments, the antibody heavy chain of the present invention comprises an antibody heavy chain variable region and an antibody heavy chain constant region. In some embodiments, the antibody light chain of the present invention comprises an antibody light chain variable region and an antibody light chain constant region.

[0049] In some embodiments, the heavy chain variable region of the present invention

[0050] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 1, 10, 23 or 27; or

[0051] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 1, 10, 23 or 27; or

[0052] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 1, 10, 23 or 27, preferably, the amino acid changes do not occur in the CDR regions.

[0053] In some embodiments, the light chain variable region of the present invention

[0054] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 2, 9, 21, 25, 29 or 31; or

[0055] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 2, 9, 21, 25, 29 or 31; or

[0056] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 2, 9, 21, 25, 29 or 31, preferably, the amino acid changes do not occur in the CDR regions.

[0057] In some embodiments, the three complementarity determining regions (HCDRs) of the present invention, HCDR1, HCDR2, and HCDR3, from the heavy chain variable region are selected from

[0058] (i) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1, 10, 23 or 27, or

[0059] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three HCDR regions relative to the sequence of any one of (i), preferably, the HCDRs are determined according to Chothia.

[0060] In some embodiments, the three complementarity determining regions (LCDRs) of the present invention, LCDR1, LCDR2, and LCDR3, from the light chain variable region are selected from

[0061] (i) three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2, 9, 21, 25, 29 or 31, or

[0062] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three LCDR regions relative to the sequence of any one of (i), preferably, the LCDR is determined according to Chothia.

[0063] In some embodiments, HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 14, or HCDR1 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 14.

[0064] In some embodiments, HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 15, 24 or 28, or HCDR2 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 15, 24 or 28.

[0065] In some embodiments, HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 16, or HCDR3 comprises an amino acid sequence having one, two or three alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 16.

[0066] In some embodiments, LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 11 or 22, or LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 11 or 22.

[0067] In some embodiments, LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 12, 26, 30 or 32, or LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 12, 26, 30 or 32.

[0068] In some embodiments, LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 13, or LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 13.

[0069] In some embodiments, the heavy chain constant region of the antibody of the present invention is a heavy chain constant region of (human) IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of (human) IgG1. In some embodiments, the light chain constant region of the antibody of the present invention is a (human) lambda or kappa light chain constant region, preferably a (human) kappa light chain constant region.

[0070] In some preferred embodiments, the heavy chain constant region of the antibody of the present invention is

[0071] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3;

[0072] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3; or

[0073] (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 3.

[0074] In some embodiments, the amino acid changes occur in the Fc region.

[0075] In some embodiments, the antibody light chain constant region of the invention is

[0076] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4;

[0077] (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or

[0078] (iii) comprises an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO: 4, or consists of said amino acid sequence.

[0079] In some embodiments, the heavy chain (HC) of the present invention

[0080] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 18, 20, 37 or 39; or

[0081] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 18, 20, 37 or 39; or

[0082] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 18, 20, 37 or 39, or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region, preferably, said amino acid changes do not occur in the heavy chain variable region.

[0083] In some embodiments, the light chain (LC) of the present invention

[0084] (i) comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 17, 19, 38, 40, 41 or 42; or

[0085] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 17, 19, 38, 40, 41 or 42; or

[0086] (iii) comprises an amino acid sequence having one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO: 17, 19, 38, 40, 41 or 42, or consists of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR region, preferably, said amino acid changes do not occur in the light chain variable region.

[0087] In some specific embodiments of the present invention, the anti-CTGF antibody or antigen-binding fragment thereof of the present invention comprises: complementary determining regions HCDR1, HCDR2 and HCDR3, and LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2 and HCDR3 and LCDR1, LCDR2 and LCDR3 respectively comprise or consist of the amino acid sequence shown in the following SEQ ID NO:

[0088] In some embodiments of the present invention, the anti-CTGF antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0089] (1) VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 1, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 2, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2;

[0090] (2) VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 10, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 9, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9;

[0091] (3) VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 23, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 23; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21;

[0092] (4) VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 25, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 25;

[0093] (5) VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 29, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 29;

[0094] (6) VH comprises, or consists of, the amino acid sequence of SEQ ID NO: 27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 27; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO: 21, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 21; or

[0095] (7) VH comprises, or consists of, the amino acid sequence of SEQ ID NO:27, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:27; and VL comprises, or consists of, the amino acid sequence of SEQ ID NO:31, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:31.

[0096] In some embodiments of the present invention, the anti-CTGF antibody or antigen-binding fragment thereof comprises VH and VL, wherein

[0097] 1) VH comprises or consists of the amino acid sequence of SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence of SEQ ID NO: 21;

[0098] 2) VH comprises or consists of the amino acid sequence of SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence of SEQ ID NO: 31;

[0099] 3) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 2;

[0100] 4) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9;

[0101] 5) VH comprises or consists of the amino acid sequence of SEQ ID NO: 23, and VL comprises or consists of the amino acid sequence of SEQ ID NO: 21;

[0102] 6) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; or

[0103] 7) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 29.

[0104] In some embodiments of the present invention, the anti-CTGF antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

[0105] (1) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 18, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 18; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 17, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 17;

[0106] (2) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:20, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:20; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:19, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:19;

[0107] (3) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 37, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 37; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO: 38, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 38;

[0108] (4) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:39; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:40, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO:40;

[0109] (5) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:39; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:41, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:41;

[0110] (6) the heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:39; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:38, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:38; or

[0111] (7) The heavy chain comprises, or consists of, the amino acid sequence of SEQ ID NO:39, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:39; and the light chain comprises, or consists of, the amino acid sequence of SEQ ID NO:42, or an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:42.

[0112] In some embodiments of the present invention, the anti-CTGF antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein

[0113] 1) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 38;

[0114] 2) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 42;

[0115] 3) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17;

[0116] 4) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 20, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 19;

[0117] 5) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 38;

[0118] 6) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 40; or

[0119] 7) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 41.

[0120] In one embodiment of the invention, the amino acid changes described herein are conservative changes. In one embodiment of the invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0121] As used herein, "conservative changes" include substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, conservative changes are conservative substitutions. A conservative substitution refers to the replacement of one amino acid with another within the same class, such as the replacement of an acidic amino acid with another acidic amino acid, the replacement of a basic amino acid with another basic amino acid, or the replacement of a neutral amino acid with another neutral amino acid. For example, conservative substitutions often result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following lists 8 groups of amino acids containing conservative substitutions for each other: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine ​​(C), Methionine (M). In some embodiments, the term "conservative change" when applied to an antibody molecule amino acid sequence refers to an amino acid modification that does not significantly affect or change the target antigen binding characteristics of the antibody molecule of the present invention containing the amino acid sequence. For example, a conservatively changed variant maintains at least 80%, 85%, 90%, 95%, 98%, 99% or higher, such as 100-110% or higher, binding affinity for the target antigen relative to the parent antibody.

[0122] In some embodiments, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). For example, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region.

[0123] In certain embodiments, the change, for example, substitution occurs in the CDR regions of the antibody. Typically, the variant obtained has modifications (for example, improvements) relative to the parent antibody in certain biological properties (for example, increased affinity) and / or will have certain biological properties that are substantially retained of the parent antibody.

[0124] In some embodiments, the present invention provides affinity variants of antibodies of the present invention. Preferably, the affinity variants exhibit one or more amino acid changes in the amino acid sequence relative to the parent antibody from which it originates, wherein the affinity variants have a changed binding affinity for the target antigen compared to the parent antibody. Typically, affinity variants exhibit improved antigen-binding affinity that is superior to the parent. The improvement may be, but is not limited to, a lower KD value, a faster dissociation rate, or an increase (or decrease) in cross-reactivity with non-human species homologous proteins of the target antigen. Affinity variants often have one or more amino acid residue substitutions in the CDRs compared to the parent. The substitutions may be conservative substitutions or non-conservative substitutions. In one embodiment, the affinity variants of the present invention have no more than 10, no more than 6, or 1-5, such as 1, 2, 3, 4 or 5 amino acid substitutions in CDR1-3 relative to the parent. In some embodiments, affinity variants introduce mutations (changes) in CDR2 relative to the parent. In another embodiment, affinity variants introduce mutations (changes) in CDR1 relative to the parent. Affinity variants can be obtained by various affinity maturation methods known in the art, including mutating CDRs, chain shuffling, using E. coli mutator strains, DNA shuffling, phage display, yeast display, etc.

[0125] In some embodiments, the affinity mature variant comprises the introduction of G30E, T31N and / or A34V (Chothia numbering) in the CDR1 of the light chain variable region, preferably the introduction of G30E, T31N and A34V (Chothia numbering) in the CDR1 of the light chain variable region. In some embodiments, the affinity mature variant comprises the introduction of any one or more of the substitutions A51S, N53Y or Y55F in the CDR2 of the light chain variable region, for example, the introduction of A51S, or the introduction of N53Y and Y55F, or the introduction of A51S, N53Y and Y55F. In some embodiments, the affinity mature variant comprises the introduction of any one or more of the substitutions K52bN or K52bR, A52cR, N53K or Y55S in the CDR2 of the heavy chain variable region, for example, the introduction of K52bN, A52cR and N53K, or the introduction of K52bR, N53K and Y55S.

[0126] In some embodiments, the affinity matured variant comprises the following substitutions:

[0127] (1) G30E, T31N, and A34V were introduced into CDR1 of the light chain variable region, and K52b N, A52c R, and N53K were introduced into CDR2 of the heavy chain variable region;

[0128] (2) N53Y and Y55F were introduced into CDR2 of the light chain variable region, and K52b R, N53K, and Y55S were introduced into CDR2 of the heavy chain variable region;

[0129] (3) A51S, N53Y, and Y55F were introduced into the CDR2 of the light chain variable region, and K52b R, N53K, and Y55S were introduced into the CDR2 of the heavy chain variable region;

[0130] (4) introducing G30E, T31N, and A34V into CDR1 of the light chain variable region, and introducing K52bR, N53K, and Y55S into CDR2 of the heavy chain variable region; or

[0131] (5) G30E, T31N, A34V were introduced into CDR1 of the light chain variable region, A51S was introduced into CDR2 of the light chain variable region, and K52bR, N53K, Y55S were introduced into CDR2 of the heavy chain variable region.

[0132] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0133] In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0134] In some embodiments, the antibodies of the present invention are humanized. Humanization can be achieved by replacing one or more amino acid residues, particularly framework region sequences, in the heavy and light chain variable regions of non-human natural antibodies with residues at corresponding positions in the variable regions of conventional antibodies from humans. Methods for humanizing antibodies are well known in the art. Typically, humanizing substitutions are performed in a manner that maintains the favorable binding properties of the antibody. Tests for determining the biological properties of humanized antibodies, such as binding affinity, are well known in the art to determine and select suitable humanized residue mutations or combinations of mutations.

[0135] In some embodiments, the humanized antibodies of the present invention can be obtained by a method comprising the following steps:

[0136] 1) determining the CDR loop structure of a parent antibody (e.g., an antibody produced from a hybridoma);

[0137] 2) Find the closest homologous sequence for each V / J region in a human germline sequence database (e.g., IMGT database);

[0138] 3) Screening for the human germline framework that best matches the heavy chain and the lowest amount of back mutations, in one embodiment of the invention,

[0139] The human germline gene is IGHV3-72*01 or IGHJ4*01; the CDR regions of the chimeric antibody are constructed onto the human framework regions;

[0140] 4) Screening for the human germline framework that best matches the light chain and the lowest amount of back mutations. In one embodiment of the invention,

[0141] The human germline gene is IGKV3-20*02 or IGKJ4*01; the CDR regions of the chimeric antibody are constructed onto the human framework regions;

[0142] 5) Using sequence and structural features, determine the amino acid positions in the framework regions that maintain CDR function;

[0143] 6) Perform back mutations (return to the input amino acid type) at sequence positions identified as important;

[0144] 7) Optionally, optimizing amino acids at risk sites, such as removing post-translational modification (PTM) sites; and

[0145] 8) Obtaining humanized antibodies and optionally sequencing the antibody sequence.

[0146] In some embodiments, the anti-CTGF antibodies or antigen-binding fragments thereof of the present invention further include antibodies or antigen-binding fragments having one or more of the following properties:

[0147] (i) exhibiting the same or similar binding affinity and / or specificity for CTGF as the antibodies of the present invention;

[0148] (ii) inhibiting (e.g., competitively inhibiting) the binding of an antibody of the invention to CTGF;

[0149] (iii) binds to the same or overlapping epitope as an antibody of the invention;

[0150] (iv) competing with the antibodies of the present invention for binding to CTGF;

[0151] (v) possess one or more biological properties of an antibody of the invention.

[0152] As defined herein, an "antibody that binds to the same or overlapping epitope as a reference antibody" is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

[0153] As defined herein, an antibody that "competes for binding to its antigen" with a reference antibody is an antibody that blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), sandwich competition assays, biointerferometry (e.g., Fortebio), or surface plasmon resonance (Biacore).

[0154] As defined herein, an "antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen" refers to an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90%, or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0155] An "antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody" refers to an antibody that has at least 50%, 60%, 70%, 80%, 90% or more of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0156] In some embodiments, the anti-CTGF antibody of the present invention is an antibody in the form of IgG1, an antibody in the form of IgG2, an antibody in the form of IgG3, or an antibody in the form of IgG4.

[0157] In some embodiments, the anti-CTGF antibody is a monoclonal antibody.

[0158] In some embodiments, the anti-CTGF antibody is a chimeric antibody or a humanized antibody.

[0159] In one embodiment, the anti-CTGF antibodies of the present invention also encompass antibody fragments thereof, preferably selected from the following antibody fragments: antigen-binding fragments including but not limited to Fab, Fab', Fab'-SH, (Fab')2, Fv, single-chain antibodies such as scFv, diabodies, single-domain antibodies (sdAb), nanobodies, sc(Fv)2.

[0160] In certain embodiments, the anti-CTGF antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody can bind to two different epitopes of the CTGF protein. In one embodiment, the bispecific antibody can bind to the CTGF binding site and another protein.

[0161] Immunoconjugates

[0162] In some embodiments, the invention provides immunoconjugates comprising any of the anti-CTGF antibodies provided herein and other substances.

[0163] In some embodiments, the immunoconjugate is used to prevent or treat a CTGF-related disease.

[0164] Pharmaceutical compositions and drug combinations

[0165] In some embodiments, the present invention provides a composition comprising any anti-CTGF antibody or fragment thereof (preferably an antigen-binding fragment thereof) or an immunoconjugate thereof as described herein, preferably a pharmaceutical composition or pharmaceutical preparation. In one embodiment, the composition further comprises a pharmaceutical excipient. In one embodiment, the composition, e.g., a pharmaceutical composition, comprises an anti-CTGF antibody or fragment thereof or an immunoconjugate thereof of the present invention, and a combination of one or more other therapeutic agents. The present invention also includes a composition (including a pharmaceutical composition or pharmaceutical preparation) comprising a polynucleotide encoding an anti-CTGF antibody.

[0166] The term "pharmaceutical composition" refers to a composition that is in form permitting the biological activity of the active ingredient contained therein to be effective, and that contains no additional ingredients that are unacceptably toxic to a subject to which the composition would be administered.

[0167] In certain embodiments, the compositions of the present invention comprise one or more antibodies or fragments thereof that bind to CTGF, or one or more polynucleotides encoding one or more anti-CTGF antibodies or fragments thereof. The compositions of the present invention may also comprise suitable pharmaceutical excipients, such as pharmaceutical carriers and pharmaceutical excipients known in the art, including buffers. Pharmaceutical formulations comprising the antibodies described herein can be prepared by mixing an antibody of the present invention having the desired purity with one or more optional pharmaceutical excipients, preferably in the form of a lyophilized formulation or aqueous solution.

[0168] As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible.

[0169] As used herein, the term "pharmaceutical excipient" refers to a diluent, adjuvant (e.g., Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, etc., which is administered together with the active substance. For the use of pharmaceutical excipients and their applications, see also "Handbook of Pharmaceutical Excipients", 8th edition, RC Rowe, PJ Eskey and S C Owen, Pharmaceutical Press, London, Chicago.

[0170] The compositions of the present invention can be in a variety of forms. These forms include, for example, liquid, semisolid and solid dosage forms, such as liquid solutions (e.g., injectable solutions and infusible solutions), powders or suspensions, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0171] The compositions of the present invention, such as pharmaceutical compositions or formulations, may also contain more than one active ingredient, the active ingredients being required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other therapeutic agents. The active ingredients are suitably combined in amounts effective for the intended use.

[0172] In some embodiments, the present invention also provides a pharmaceutical combination or pharmaceutical combination product comprising an anti-CTGF antibody or fragment thereof (preferably an antigen-binding fragment), or an immunoconjugate thereof, of the present invention, and one or more other therapeutic agents.

[0173] As used herein, the term "pharmaceutical combination or combination product" refers to a non-fixed combination product or a fixed combination product, including but not limited to a kit / test kit, a pharmaceutical composition. The term "non-fixed combination" means that the active ingredients (e.g., (i) an antibody of the present invention, and (ii) other therapeutic agent) are administered to a patient simultaneously, without specific time restrictions, or at the same or different time intervals, in separate entities, wherein such administration provides two or more active agents with prophylactic or therapeutically effective levels in the patient's body. The term "fixed combination" means that two or more active agents are administered to a patient simultaneously in the form of a single entity. The dosage and / or time interval of the two or more active agents are preferably selected so that the combined use of the parts can produce an effect greater than that achieved by using any one component alone when treating a disease or condition. Each component can be in the form of a separate formulation, which can be the same or different.

[0174] Another object of the present invention is to provide a kit comprising the pharmaceutical combination of the present invention, preferably in the form of a pharmaceutical dosage unit, whereby dosage units can be provided according to a dosing regimen or a drug administration interval.

[0175] In one embodiment, the kit of parts of the present invention comprises in the same package:

[0176] - a first container containing a pharmaceutical composition comprising an anti-CTGF antibody or fragment thereof;

[0177] - A second container containing a pharmaceutical composition comprising an additional therapeutic agent.

[0178] In some embodiments, the composition or pharmaceutical combination or combination product or kit is used to prevent or treat a CTGF-related disease.

[0179] Treatment, diagnosis and testing

[0180] In one aspect, the present invention provides a method for preventing or treating a CTGF-related disease in an individual, comprising administering to the individual an effective amount of an anti-CTGF antibody or antigen-binding fragment thereof, immunoconjugate, pharmaceutical composition or combination product of the present invention.

[0181] As used herein, "treatment" refers to slowing down, interrupting, blocking, alleviating, stopping, reducing or reversing the onset of symptoms, complications, or biochemical signs of a disease, alleviating symptoms or preventing or inhibiting the further development of a disease, condition or illness. As used herein, the term "prevention" includes the inhibition of the occurrence or development of a disease or illness or the symptoms of a specific disease or illness. As used herein, the term "individual" or "subject" includes mammals. Mammals include, but are not limited to, domestic animals (e.g., cattle, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human. As used herein, the term "effective amount" refers to an antibody or fragment or composition of the present invention or such amount or dosage of a combination that, after being administered to a patient in a single or multiple doses, produces a desired effect in a patient in need of treatment or prevention.

[0182] In some embodiments, a "therapeutically effective amount" refers to an amount that is effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. Relative to an untreated subject, a "therapeutically effective amount" preferably inhibits a measurable parameter or improves a measurable parameter (e.g., total white blood cell count, e.g., neutrophil count, lymphocyte count, and / or macrophage count) by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0183] In some embodiments, a "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result at the required dosage and for the required period of time. Typically, since a prophylactic dose is used in a subject before or at an earlier stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0184] In other aspects, the present invention provides the use of an anti-CTGF antibody or fragment thereof in the production or preparation of a medicament for treating the relevant diseases or disorders mentioned herein, such as a CTGF-related disease.

[0185] In some embodiments, the antibodies or antibody fragments or immunoconjugates or compositions or products of the invention delay the onset of a disorder and / or symptoms associated with the disorder.

[0186] In some embodiments, the antibodies, antibody fragments, immunoconjugates, compositions, or products of the present invention reduce lung infection, reduce total white blood cell counts in the lungs, reduce neutrophil counts, lymphocyte counts, and / or macrophage counts; reduce lung inflammation, and / or improve lung function, such as increasing vital capacity.

[0187] In some embodiments, the prophylactic or therapeutic methods described herein further comprise combination therapy, e.g., comprising administering to the individual or individuals in combination an antibody molecule or pharmaceutical composition or immunoconjugate disclosed herein, and one or more other therapies, e.g., therapeutic modalities and / or other therapeutic agents.

[0188] In some embodiments, the antibodies described herein can be combined with other antibodies for separate administration, eg, each as a separate antibody, or when linked (eg, as a bispecific or multispecific antibody molecule).

[0189] Combination therapy encompasses combined administration (e.g., two or more therapeutic agents contained in the same formulation or separate formulations), and separate administration, in which case administration of the antibodies of the invention can occur before, concurrently with, and / or after administration of the other therapeutic agents and / or agents.

[0190] The term "combination therapy" as used herein refers to the administration of two or more therapeutic agents or treatment modalities to treat diseases described herein. This administration includes co-administering these therapeutic agents in a substantially simultaneous manner, such as in a single capsule with a fixed ratio of active ingredients. Alternatively, this administration includes co-administration of each active ingredient in a variety of or separate containers (such as tablets, capsules, powders, and liquids). Powders and / or liquids can be reconstituted or diluted to the desired dose before administration. In addition, this administration also includes using each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In either case, the therapeutic regimen will provide a beneficial effect of the drug combination in treating illnesses or conditions described herein.

[0191] The anti-CTGF antibodies of the present invention (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) can be administered by any suitable method, including parenteral administration and, if desired for local treatment, intralesional administration. Parenteral injection or infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous injection or infusion. Depending to some extent on whether the administration is short-term or long-term, the administration can be by any suitable route, for example, by injection, such as intravenous or subcutaneous injection. Various administration schedules are contemplated herein, including, but not limited to, single administration or multiple administrations at multiple time points, bolus administration, and pulse infusion.

[0192] For the prevention or treatment of disease, the appropriate dosage of the anti-CTGF antibodies or fragments thereof (and immunoconjugates, compositions, pharmaceutical compositions, formulations, combination products, etc. comprising the same) of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether it is administered for preventive or therapeutic purposes, previous treatment, the patient's clinical history and response to the antibody, the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; the use of any concomitant therapy and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments.

[0193] In other aspects, the present invention provides the use of the anti-CTGF antibody or fragment thereof or immunoconjugate or composition comprising the same of the present invention in the production or preparation of a medicament for the uses described herein, such as for preventing or treating the related diseases or disorders mentioned herein, such as CTGF-related diseases.

[0194] In some embodiments, anti-CTGF antibodies or fragments thereof (as well as immunoconjugates, compositions, pharmaceutical compositions, formulations, etc. comprising the same) can also be administered in combination with one or more other therapies, such as treatment modalities and / or other therapeutic agents, for the uses described herein, e.g., for preventing and / or treating the relevant diseases or conditions mentioned herein, such as CTGF-related diseases.

[0195] In one aspect, the present invention also relates to methods for diagnosis and detection of the antibodies or antigen-binding fragments thereof of the present invention and compositions for diagnosis and detection comprising the same.

[0196] In certain embodiments, any of the anti-CTGF antibodies or antigen-binding fragments thereof provided herein can be used to detect the presence of CTGF in a biological sample.

[0197] The term "detection" as used herein includes quantitative or qualitative detection, and exemplary detection methods can involve immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads of antibody molecule complexes, ELISA assays, PCR-techniques (e.g., RT-PCR). In certain embodiments, the biological sample is other liquid samples of blood, serum, plasma, or biological origin. In certain embodiments, the biological sample comprises cells or tissues. In some embodiments, the biological sample is from lung tissue. In some embodiments, the biological sample is from blood, such as plasma.

[0198] In one embodiment, an anti-CTGF antibody or antigen-binding fragment thereof is provided for use in a method of diagnosis or detection.

[0199] In another aspect, a method for detecting the presence of CTGF in a biological sample is provided. In certain embodiments, the method comprises detecting the presence of CTGF protein in a biological sample. In certain embodiments, CTGF is human CTGF or rhesus monkey CTGF or mouse CTGF. In certain embodiments, the method comprises contacting the biological sample with an anti-CTGF antibody as described herein under conditions that allow the anti-CTGF antibody to bind to CTGF, and detecting whether a complex is formed between the anti-CTGF antibody and CTGF. The formation of a complex indicates the presence of CTGF. The method can be an in vitro or in vivo method. In one embodiment, the anti-CTGF antibody is used to select a subject suitable for treatment with the anti-CTGF antibody, for example, where CTGF is a biomarker for selecting the subject.

[0200] In certain embodiments, there is provided a labeled antibody or a fragment thereof. As used herein, the term "label" refers to a compound or composition that is directly or indirectly conjugated or fused to a reagent (such as a polynucleotide probe or antibody) and promotes the detection of the reagent it is conjugated or fused to. The label itself can be detectable (e.g., radioisotope labeling or fluorescent labeling) or can catalyze the chemical alteration of a detectable substrate compound or composition in the case of enzymatic labeling. The term is intended to encompass direct labeling of a probe or antibody by coupling a detectable substance (i.e., physical connection) to a probe or antibody and indirect labeling of a probe or antibody by reacting with another reagent of a direct label.

[0201] In some embodiments, labels include, but are not limited to, labels or moieties that are directly detected (such as fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), as well as moieties that are indirectly detected, such as enzymes or ligands, e.g., by an enzymatic reaction or molecular interaction.

[0202] In some embodiments provided herein, the sample is obtained prior to treatment with an antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained prior to treatment with an alternative therapy. In some embodiments, the sample is obtained during treatment with an alternative therapy, or after treatment with an alternative therapy.

[0203] In some embodiments, CTGF is detected prior to treatment, eg, prior to initiation of treatment or prior to a treatment after a treatment interval.

[0204] In some embodiments, a method for treating a disease of the present invention is provided, comprising: testing an individual (e.g., a sample) (e.g., an individual sample, such as an individual's blood, such as blood) for the presence of CTGF, thereby determining a CTGF value (e.g., the protein concentration or protein content of CTGF), comparing the CTGF value with a control value (e.g., a value in a healthy individual or normal tissue), and if the CTGF value is greater than the control value, administering to the individual a therapeutically effective amount of an antibody or fragment thereof, or an immunoconjugate, composition, pharmaceutical composition, formulation, combination product, etc., of the present invention, optionally in combination with one or more other therapies, thereby treating the disease.

[0205] As used herein, "subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. The source of a tissue or cell sample can be solid tissue, such as an organ or tissue sample or a biopsy sample or a puncture sample from a fresh, frozen and / or preserved organ; blood or any blood component; body fluids, such as tears, vitreous humor, cerebrospinal fluid, amniotic fluid (amniotic fluid), peritoneal fluid (ascites), or interstitial fluid; cells from any time during the subject's pregnancy or development. Tissue samples may contain compounds that are not naturally contaminated with tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

[0206] Nucleic acids, expression vectors, and host cells

[0207] In one aspect, the invention provides nucleic acids encoding any of the above anti-CTGF antibodies or fragments thereof or either chain thereof.

[0208] For example, nucleic acids of the invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 9, 10, 17, 18, 19, 20, 21, 23, 25, 27, 29, 31, 37, 38, 39, 40, 41, or 42, or nucleic acids encoding an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from any one of SEQ ID NOs: 1, 2, 9, 10, 17, 18, 19, 20, 21, 23, 25, 27, 29, 31, 37, 38, 39, 40, 41, or 42. As will be apparent to those skilled in the art, due to codon degeneracy, each antibody or polypeptide amino acid sequence can be encoded by multiple nucleic acid sequences. The nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, such as de novo solid-phase DNA synthesis or PCR amplification. To facilitate production and purification, a secretory signal peptide and / or a tag peptide that facilitates purification can be fused to the N-terminus of the heavy and / or light chains of the antibody.

[0209] In one aspect, the present invention provides nucleic acids encoding any of the above antibodies or fragments thereof or any of their chains. When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human (and / or rhesus monkey and / or mouse) CTGF antigen binding ability. For example, in some embodiments, the nucleic acid encoding the variable region of the heavy chain and / or light chain is operably linked in frame with the nucleic acid encoding the constant region of the heavy chain and / or light chain, thereby producing a nucleic acid encoding the antibody heavy chain and / or light chain when expressed from a suitable expression vector.

[0210] In one embodiment, one or more vectors comprising the nucleic acid are provided. In one embodiment, the vector is an expression vector, such as a eukaryotic expression vector. As described herein, the term "vector" refers to a nucleic acid molecule capable of proliferating another nucleic acid connected thereto. The term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the genome of the host cell into which it has been introduced. The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence that is effectively connected to the nucleotide sequence to be expressed. The expression vector comprises enough cis-acting elements for expression; other elements for expression can be provided by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporated therein.

[0211] In some embodiments, vectors, such as expression vectors, include but are not limited to viruses, plasmids, cosmids, lambda phages or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pCDNA vector, such as pCDNA3.4.

[0212] In one embodiment, a host cell comprising the nucleic acid or the vector is provided. The term "host cell" refers to a cell into which an exogenous polynucleotide has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells", which include primary transformed cells and progeny derived therefrom, without considering the number of passages. In some embodiments, the host cell is any type of cell system that can be used to produce the antibody molecule of the present invention, including eukaryotic cells, for example, mammalian cells, insect cells, yeast cells; and prokaryotic cells, for example, Escherichia coli cells. Host cells include cultured cells, as well as cells within transgenic animals, transgenic plants, or cultured plant tissues or animal tissues.

[0213] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. For example, fungal and yeast strains whose glycosylation pathways have been "humanized" result in the production of antibodies with partially or fully human glycosylation patterns. Host cells suitable for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Vertebrate cells can also be used as hosts. For example, mammalian cell lines modified to be suitable for suspension growth can be used. Other examples of useful mammalian host cell lines are monkey kidney CV1 lines (COS-7) transformed with SV40; human embryonic kidney lines (HEK293, 293F or 293T cells), etc. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including CHO-S cells or CHO-K cells, etc.; and myeloma cell lines such as Y0, NS0 and Sp2 / 0. Suitable mammalian host cell lines for producing antibodies are known in the art.

[0214] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells (e.g., CHO cells or 293 cells) or other cells suitable for preparing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, e.g., bacteria, e.g., E. coli.

[0215] Antibody production, purification, and assays

[0216] In one embodiment, the present invention provides a method for producing an anti-CTGF antibody or fragment thereof (preferably an antigen-binding fragment), wherein the method comprises culturing the host cell under conditions suitable for expressing a nucleic acid encoding the antibody or fragment thereof (preferably an antigen-binding fragment), or either or both chains thereof, and optionally isolating the antibody or fragment thereof (preferably an antigen-binding fragment). In a certain embodiment, the method further comprises recovering the anti-CTGF antibody or fragment thereof (preferably an antigen-binding fragment) from the host cell.

[0217] The polynucleotide encoding the polypeptide chains of the antibodies of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Methods well known to those skilled in the art can be used to construct expression vectors. Once an expression vector comprising one or more nucleic acid molecules of the present invention has been prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. A variety of techniques can be used to achieve this purpose, for example, protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, liposome-based transfection or other conventional techniques.

[0218] Antibodies prepared as described herein can be purified by known techniques such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography, e.g., Protein A, size exclusion chromatography, etc. The actual conditions used to purify a particular protein will also depend on factors such as net charge, hydrophobicity, hydrophilicity, etc., and these will be apparent to those skilled in the art.

[0219] The purity of the antibody molecules of the present invention can be determined by any of a number of well-known analytical methods, including size exclusion chromatography, gel electrophoresis, high performance liquid chromatography, and the like.

[0220] With respect to the CTGF antibodies of the present invention, the anti-CTGF antibodies provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activities by various assays known in the art.

[0221] On the one hand, the antibody of the present invention is tested for its antigen-binding activity, methods known in the art can be used to measure. In some embodiments, radioimmunoassay (RIA) or biofilm thin layer interferometry (BLI) or electrochemiluminescence (ECL) or surface plasmon resonance (SPR) or ELISA or flow cytometry (FACS) are used to measure.

[0222] The present invention also provides assays for identifying anti-CTGF antibodies with biological activity. The biological activity is selected from the properties of the antibodies of the invention and can include, for example, binding to CTGF (e.g., binding to human and / or rhesus monkey and / or mouse CTGF), blocking the binding of TGFβ1 to CTGF, etc.

[0223] For example, the binding activity of the antibody molecules of the present invention to CTGF or cells expressing CTGF can be determined by methods known in the art, such as Fortebio, flow cytometry, Octet or plasma resonance (Biacore), or the exemplary methods disclosed in Examples 3, 7 or 8 herein.

[0224] For example, the blocking effect of the antibody molecules of the present invention on the binding of TGFβ to CTGF (e.g., CTGF domain 2) or cells expressing the same can be determined by methods known in the art, such as flow cytometry, or the exemplary method disclosed in Example 8 herein.

[0225] For example, the therapeutic effect of a composition comprising an antibody molecule of the present invention on pulmonary fibrosis (e.g., a reduction in the number of total leukocytes in the lungs, a reduction in the number of neutrophils, lymphocytes and / or macrophages, a reduction in lung inflammation, and / or an improvement in lung function, such as vital capacity) can be determined by methods known in the art, such as the exemplary methods disclosed in Example 9 herein.

[0226] For example, the pharmacokinetic properties of the antibody molecules of the present invention, such as blood concentration or half-life, can be determined by methods known in the art, such as the exemplary method disclosed in Example 10 herein.

[0227] Cells for use in any of the above in vitro assays include cell lines that naturally express CTGF or are engineered to express CTGF. Such cells also include cell lines that express CTGF and cell lines that are not normally expressing CTGF and are transfected with a nucleic acid encoding CTGF.

[0228] It will be appreciated that any of the above assays can be performed using the immunoconjugates of the invention in place of or in addition to anti-CTGF antibodies.

[0229] It will be appreciated that any of the above assays can be performed using an anti-CTGF antibody in combination with an additional active agent.

[0230] Other definitions

[0231] It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0232] Herein, terms used in the singular may also include the plural, and vice versa, whenever appropriate. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0233] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0234] As used herein, the term "and / or" means any one of the alternatives or two or more of the alternatives.

[0235] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, combinations of the recited elements, integers, or steps are also encompassed unless otherwise indicated. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0236] An "isolated" antibody or molecule is one that has been separated from a component of its natural environment. In some embodiments, the antibody or molecule is purified to greater than 95% or 99% purity, as determined by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC).

[0237] As used herein, "CTGF" refers to any native CTGF polypeptide (e.g., human CTGF polypeptide) or variant thereof. The term "CTGF" encompasses "full-length" unprocessed CTGF polypeptide as well as any form of CTGF polypeptide produced by processing within the cell. The term also encompasses naturally occurring variants of CTGF, such as those encoded by splice variants and allelic variants. The CTGF polypeptides described herein can be isolated from a variety of sources, such as from human tissue or from another source, such as rhesus monkeys, or prepared by recombinant or synthetic methods. In one embodiment of the invention, the human CTGF protein is as shown in Uniport Primary accession P29279,349. In one embodiment, the human CTGF gene is as shown in NCBI Gene ID: 1490. In one embodiment of the invention, the rhesus monkey CTGF protein is as shown in Unoport Primary accession H9FQD5. In one embodiment, the human CTGF gene is as shown in NCBI Gene ID: 714520.

[0238] As used herein, the terms "anti-CTGF antibody," "anti-CTGF," "CTGF antibody," or "CTGF-binding antibody" refer to an antibody that binds (primate, e.g., human or rhesus monkey) CTGF or a fragment thereof with sufficient affinity.

[0239] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules with the structure of natural immunoglobulin molecules. In the case of conventional four-chain IgG antibodies, the full-length antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of heavy chain antibodies having only heavy chains and lacking light chains, the full-length antibody comprises two heavy chains (H) interconnected by disulfide bonds. For conventional four-chain IgG antibodies, the full-length antibody heavy chain is generally composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, wherein the heavy chain constant region comprises at least three domains CH1, CH2 and CH3. The full-length antibody light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region, wherein the light chain constant region consists of one domain CL. Each heavy chain variable region VH and each light chain variable region are composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In one embodiment, the antibodies of the invention are full-length antibodies.

[0240] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0241] The term "antigen-binding fragment" of an antibody is a molecule that is different from a full-length antibody and comprises a portion of a full-length antibody, but is capable of binding to the antigen of the full-length antibody or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment is derived) for antigen binding. Antigen-binding fragments can be prepared by recombinant DNA technology, or by enzymatic or chemical cleavage of intact antibodies. For example, Fab fragments can be obtained by digesting a full-length antibody with papain. In addition, digesting a full-length antibody with pepsin below the disulfide bonds in the hinge region produces F(ab')2, which is a dimer of Fab' and is a divalent antibody fragment. F(ab')2 can be reduced under neutral conditions by destroying the disulfide bonds in the hinge region, thereby converting the F(ab')2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab fragment with a hinge region. The Fv fragment consists of the VL and VH domains of a single arm of an antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but can also be produced as a single protein chain using recombinant methods, using a synthetic linker peptide to connect the two domains. In the single protein chain, the VL region and the VH region are paired to form a single-chain Fv (scFv). sc(Fv)2 is a small antibody in which two VH and two VL are connected by a linker to form a single chain.

[0242] The term "single-chain antibody (scAb)" is used in the broadest sense herein and specifically covers antibodies with monospecificity or multispecificity (e.g., bispecific) that were initially produced as a single continuous polypeptide chain. Such single-chain antibodies include, but are not limited to, antibodies having two linked VL and VH regions. In one embodiment, a single-chain antibody is an scFv. A "diabody" is a small bivalent antibody constructed by gene fusion, for example, a dimer consisting of two polypeptide chains. The VL and VH domains of each polypeptide chain of the diabody are bound by a linker, thereby forming a dimer with different single-chain variable region fragments encoded in the same polypeptide chain. Diabodies generally have two antigen binding sites.

[0243] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment schemes, including, for example, Chothia based on the three-dimensional structure of antibodies and the topology of the CDR loops (Chothia et al. (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0244] CDRs using Kabat, AbM, Chothia, Contact, and IMGT schemes can be defined based on AbYsis (http: / / www.abysis.org / abysis / ).

[0245] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above ways. CDRs can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., any of the exemplary CDRs of the present invention). Unless otherwise indicated, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to the numbering position according to the Chothia numbering system (http: / / www.abysis.org / abysis / ).

[0246] In one embodiment, the CDRs of the heavy chain variable region of an antibody of the present invention are determined according to Chothia. In some embodiments, the CDRs of the light chain variable region of an antibody of the present invention are determined according to Chothia. In some embodiments, the Chothia definition under Chothia numbering is as follows:

[0247] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment schemes may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment schemes may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment scheme rules or combinations).

[0248] The term "chimeric antibody" is an antibody molecule in which (a) the constant region or a portion thereof is changed, replaced or exchanged so that the antigen binding site is connected to a constant region of a different or altered class, effector function and / or species or a completely different molecule (e.g., enzyme, toxin, hormone, growth factor, drug) that imparts new properties to the chimeric antibody; or (b) the variable region or a portion thereof is changed, replaced or exchanged with a variable region having a different or altered antigenic specificity. For example, a mouse antibody can be modified by replacing its constant region with a constant region from a human immunoglobulin. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing the antigen while having reduced immunogenicity in humans as compared to the original mouse antibody.

[0249] A "humanized antibody" is an antibody that retains the antigen-specific reactivity of a non-human antibody (e.g., a mouse monoclonal antibody) while being less immunogenic when administered to humans as a therapeutic agent. This can be achieved, for example, by retaining the non-human antigen-binding site and replacing the remaining portions of the antibody with their human counterparts (i.e., replacing the constant region and portions of the variable region not involved in binding with the corresponding portions of a human antibody).

[0250] As used herein, the terms "anti," "binding," or "specific binding" mean that the binding is selective for the target or antigen and can be distinguished from unwanted or non-specific interactions. The ability of a binding site to bind to a specific target or antigen can be determined by flow cytometry or enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0251] An "immunoconjugate" is an antibody conjugated to one or more other substances (eg, macromolecular substances, radioactive elements, cytotoxic agents, etc.).

[0252] "Percent identity (%)" of an amino acid sequence refers to the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the specific amino acid sequence set forth in this specification, after aligning the candidate sequence with the specific amino acid sequence set forth in this specification and introducing gaps, if necessary, to achieve the maximum percentage identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, the present invention contemplates variants of the antibody molecules of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98% or 99% or more, relative to the antibody molecules and sequences thereof specifically disclosed herein. Such variants may comprise conservative changes.

[0253] The term "therapeutic agent" as used herein encompasses any substance that is effective in preventing or treating the diseases described herein, e.g., CTGF-related diseases such as pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis). In some embodiments, the other therapeutic agent described herein is selected from, for example, a chemotherapeutic agent or an immunomodulator, such as an anti-inflammatory agent or an immunosuppressant.

[0254] The term "CTGF-related disease" as described herein refers to any disease associated with abnormal CTGF. In some embodiments, the CTGF-related disease refers to a disease in which CTGF protein expression or protein concentration is elevated in an individual (e.g., in tissue or plasma, e.g., compared to a healthy individual, e.g., compared to the plasma of a healthy individual). In some embodiments, the CTGF-related disease refers to a disease in which CTGF nucleic acid is elevated in an individual. In some embodiments, the CTGF-related diseases described herein include, for example, fibrotic diseases. In some embodiments, the fibrotic disease is selected from pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis).

[0255] All publications, patent applications, patents and other references mentioned herein are fully incorporated by reference. Any or all features discussed above and throughout this application can be combined in various embodiments of the present invention. In addition, the materials, methods and examples described herein are only illustrative and are not intended to be restrictive. Other features, purposes and advantages of the present invention will become apparent from this specification and accompanying drawings and from the appended claims. Example

[0256] Example 1 Preparation of mouse anti-CTGF antibody

[0257] 1.1 Immunization of animals

[0258] For the initial immunization, CTGF protein (Acrobiosystem CTF-H52H5) was emulsified in complete Freund's adjuvant at a 1:1 ratio and injected intraperitoneally and subcutaneously at multiple sites into 6- to 8-week-old female Balb / c mice or SD rats at a rate of 50 μg per mouse. Booster immunizations were then performed two to three weeks apart, with 25 μg of antigen plus incomplete Freund's adjuvant per mouse. Three immunizations were performed in total. Two weeks after the final immunization, tail blood was collected to determine serum anti-human CTGF antibody titers. Those with the highest titers were then given a boost immunization with 25 μg of antigen plus incomplete Freund's adjuvant. Splenocytes were harvested three days later.

[0259] 1.2 Construction and selection of mouse immune phage antibody library

[0260] Spleens of immunized rats and mice with serum titers meeting the requirements were obtained and splenocytes were isolated. Total RNA was extracted using TRIzol (Invitrogen, Cat. No. 15596026) and analyzed using PrimeScript. TMcDNA preparation was completed using the 1st Strand cDNA Synthesis Kit (TAKARA, Catalog No. 6110A). High-quality antibody light and heavy chain variable region genes were amplified using mouse-specific primers. The spliced ​​scFv gene was integrated into the pComb3xss phage plasmid (synthesized in-house) using molecular cloning methods and electroporated into SS320 competent cells (Lucigen, Catalog No. 60512-1) to construct a library with a capacity of 1.2*10 8 Collect the cells, add glycerol to a final concentration of 10%, and store in a refrigerator at -80 degrees Celsius.

[0261] Use glycerol bacteria of antibody library to prepare titer 1*10 13 2 mL of phage from a 100 cfu / ml phage library was pre-bound to 150 μL Streptavidin Magnetic Beads (Thermo Fisher, Catalog No. 88817) and incubated at room temperature for 90 minutes to remove nonspecific binding. The background-removed phage library was then added with 10 μg biotinylated human CTGF (Bipsys, Catalog No. CTF-H82E6, hereinafter referred to as H CTGF-Bio) and 150 μL Streptavidin Magnetic Beads and incubated at room temperature for 15 minutes. The cells were then washed 14 times with PBST (PBS containing 0.05% Tween-20) to remove unbound phage.

[0262] Antigen-specific phage were eluted with 450 μL of 100 mM hydrochloric acid, neutralized with 50 μL of 1 M Tris-HCl (pH 11), and infected with E. coli SS320 cells in logarithmic growth phase to generate and purify phage for the second round of screening. The screening method was the same as the first round, except that the amount of antigen was reduced to 4 μg. After two rounds of screening, positive enrichment was achieved.

[0263] 1.3 Yeast display library construction and screening

[0264] Using the phage plasmid enriched after two rounds of panning against human CTGF as a template, primers were designed to amplify the gene fragments of the single-chain antibody scFv library; the amplified gene fragments were recovered and co-transformed with the yeast display plasmid into the cerevisiae strain EBY100 (purchased from ATCC). Through homologous recombination in cerevisiae, the scFv gene was inserted into the yeast display plasmid, thereby realizing the display of the single-chain antibody scFv on the yeast cell wall surface. The displayed single-chain antibody library was named JYYDL231. After electroporation, the library JYYDL231 was cultured in 100 mL of SD-Trp medium (Clontech, Cat. No. 630308) at 30°C and 225 rpm overnight; 5.0×10 8The cells were resuspended in 100 mL YPGP liquid medium (2% galactose, 2% peptone, 1% yeast extract, 0.54% Na2HPO4, 0.86% NaH2PO4·H2O), cultured at 20°C and 225 rpm for 24 h, and placed in a 4°C refrigerator until use.

[0265] After induction of JYYDL231 library, measure the OD of the bacterial solution 600 , 1OD is 1.0×10 7 The cell number was calculated as 3.0×10 7 The cells were sorted by flow cytometry and then spread on SD-Trp solid medium (Clontech, product number: 630309) plates and cultured at 30°C for 3 days to grow yeast monoclonal colonies.

[0266] 1.4 Monoclonal yeast colony sequencing and flow cytometry identification

[0267] 92 clones were selected for sequencing analysis, ultimately yielding 25 unique single-chain antibody (scFv) sequences. Pamrevlumab was displayed on the yeast surface as a positive control. Yeast clones with unique sequences were flow cytometrically analyzed according to the staining schemes in Table 1. In Scheme 1, a higher mean fluorescence intensity (MFI) for PE reflects stronger binding to human CTGF. Similarly, Schemes 2, 3, and 4 can assess the binding of each clone to monkey CTGF, mouse CTGF, and unrelated antigens. In Scheme 5, the MFI for APC reflects the strength of competition with the positive control antibody. Lower APC signals indicate stronger competition with the positive control antibody, and vice versa.

[0268] Table 1 Monoclonal flow cytometry identification scheme

[0269] The yeast monoclonal staining results are shown in Table 2. Based on the staining results of each clone, clones that did not bind to human, monkey, or mouse CTGF, clones that nonspecifically bound to irrelevant antigens, and antibodies that did not compete with the control antibody were excluded. Finally, the sequence of clone Y129C1 was selected for plasmid construction for subsequent antibody expression and identification.

[0270] Table 2 Summary of yeast monoclonal flow cytometry staining results

[0271] Example 2 Expression of chimeric anti-CTGF antibodies

[0272] According to the sequencing results, the amino acid sequence of the heavy chain variable region of monoclonal Y129C2 is:

[0273] >Y129C2 VH (SEQ ID NO: 1)

[0274] The amino acid sequence of the light chain variable region of monoclonal Y129C2 is:

[0275] >Y129C2 VL (SEQ ID NO: 2)

[0276] The heavy chain variable region (VH) sequence of the Y129C2 clone was fused to the human IgG1 heavy chain constant region (SEQ ID NO: 3), and the light chain variable region (VL) sequence was fused to the human kappa constant region (SEQ ID NO: 4). The gene fragments expressing the heavy and light chains were cloned and synthesized into the expression vector pcDNA3.4 (Life Technologies). The plasmids were transiently transfected into CHO cells to express the chimeric antibody Ab2101.67, which was then purified using protein A (Shanghai Bio-Technology Co., Ltd.). The expression levels of the obtained antibodies are shown in Table 3.

[0277] The expression information is shown in Table 3.

[0278] Table 3 Candidate antibody expression and purification data

[0279] Example 3 Anti-CTGF chimeric antibody affinity determination

[0280] Octet RED96e (Fortebio) was used to determine the affinity of Ab2101.67 and the control antibody pamrevlumab (heavy chain: SEQ ID NO: 43; light chain SEQ ID NO: 44, prepared as Ab2101.67) to human CTGF (Acro, Catalog No.: CTF-H52H5), mouse CTGF (Acro, Catalog No.: CTF-M52Hc), and rhesus monkey (Acro, Catalog No.: GTF-R52H4). Both antigens and antibodies were diluted in 1×PBST (1×PBS: Gibco, 10010-023; 0.02% Tween 20: Sigma, P1379). The antigen concentration was 11.6 nM, and the antibody concentration was 5 μg / mL. The mouse antibody sample was added to a 96-well plate (Greiner bio-one, catalog number: 655209) at a rate of 200 μL / well. The software parameters were set to 30°C and a frequency of 5.0 Hz for collecting standard kinetic signals. The AHC sensor (Fortebio, catalog number: 18-5060) was pre-wetted with 1×PBST for 10 minutes and then tested on the machine. Each cycle included the following steps: 1) immersion in buffer for 60 seconds; 2) detection of non-specific binding of the antigen to the sensor; 3) regeneration with 10 mM glycine solution at pH 1.7; 4) immersion in buffer for 60 seconds; 5) antibody immobilization on the sensor for 17 seconds; 6) sensor immersion in buffer for 180 seconds; 7) antigen binding to antibody for 180 seconds; 8) antigen-antibody dissociation for 5 minutes; 9) sensor regeneration. Finally, Fortebio's Data Analysis 12.0 software was used to determine the binding rate (K) of the antigen-antibody in a 1:1 binding mode. on ) and dissociation rate (K off ), and the equilibrium dissociation constant (K D ).

[0281] The affinity results of Ab2101.67 for human, mouse and rhesus monkey CTGF are shown in Tables 4, 5 and 6, respectively, indicating that murine anti-CTGF antibodies all bind to human, mouse and rhesus monkey CTGF, among which the affinity of Ab2101.67 for human, mouse and rhesus monkey CTGF is comparable to that of Pamrevlumab.

[0282] Table 4 Affinity determination of candidate antibodies to human CTGF

[0283] Table 5 Affinity determination of candidate antibodies to mouse CTGF

[0284] Table 6 Affinity determination of candidate antibodies to rhesus monkey CTGF

[0285] Example 4 Humanization of anti-CTGF chimeric antibody

[0286] Through sequence alignment, the human antibody germline gene with the highest homology (data source: IMGT) was selected as the humanization design framework. The light chain framework was IGKV3-20*02 or IGKJ4*01, and the heavy chain framework was IGHV3-72*01 or IGHJ4*01.

[0287] Humanized amino acid design of the antibody light and heavy chain variable regions was performed based on sequence alignment and variable region structure information;

[0288] 1. Design expression vectors, gene synthesis, mammalian cell expression and purification of recombinant antibodies, compare the activity and physicochemical properties of humanized antibodies and chimeric antibodies, and perform 1-2 rounds of humanization optimization;

[0289] 2. Human germline antibody gene amino acid sequence information:

[0290] IGKV3-20*02 (SEQ ID NO: 5):

[0291] IGHV3-72*01 (SEQ ID NO: 6):

[0292] IGKJ4*01 (SEQ ID NO: 7):

[0293] IGHJ4*01 (SEQ ID NO: 8):

[0294] The following optimized design of the light chain variable region is based on the human antibody germline gene sequence IGKV3-20*02 and IGKJ4*01 as the framework, and 2101ZVL 0 (Ab2101.67 VL) is the light chain variable region sequence of the chimeric antibody for CDR transplantation and restoration.

[0295] Mutated humanized sequence:

[0296] 2101ZVL 0 light chain variable region sequence (SEQ ID NO: 2)

[0297] 2101ZVL 5 light chain variable region sequence (SEQ ID NO: 9)

[0298] The following optimized design of the heavy chain variable region is based on the human antibody germline gene sequences IGHV3-72*01 and IGHJ4*01 as the framework, and 2101ZVH0 (Ab2101.67 VH) as the heavy chain variable region sequence of the chimeric antibody for CDR transplantation and back mutation of the humanized sequence.

[0299] 2101ZVH0 heavy chain variable region sequence (SEQ ID NO: 1)

[0300] 2101ZVH2 heavy chain variable region sequence (SEQ ID NO: 10)

[0301] The antibody light and heavy chain variable regions were numbered using Chothia to define the antibody CDR regions. The CDRs defined in this way are shown in Table 7:

[0302] Table 7 CDR sequences and numbers of each antibody

[0303] 3. Heavy and light chain combinations and antibody expression of humanized antibodies

[0304] The humanized light chain variable region sequence was combined with the human kappa chain constant region (SEQ ID NO: 4) to form the humanized antibody light chain, and the humanized heavy chain variable region sequence was combined with the human IgG1 constant region (SEQ ID NO: 3) to form the humanized antibody heavy chain. The constant region sequences are shown in Table 8.

[0305] Table 8 Human light chain and heavy chain constant regions

[0306] The 2101ZVH0 heavy chain variable region and the 2101ZVL0 light chain variable region were combined with the human heavy chain constant region and the human light chain constant region, respectively, and then paired to form the chimeric antibody 2101Z00 (Ab2101.67); the 2101ZVH2 heavy chain variable region and the 2101ZVL5 light chain variable region were combined with the human heavy chain constant region and the human light chain constant region, respectively, and then paired to form the humanized antibody 2101Z25.

[0307] The full-length sequence information of the light and heavy chains of the above chimeric and humanized antibodies is shown in Table 9 below:

[0308] Table 9 Antibody light and heavy chains

[0309] After sequence optimization, the light and heavy chain protein sequences of the full-length recombinant monoclonal antibody were codon-optimized, and the codon-optimized DNA fragments were synthesized. The synthesized gene fragments were cloned into the expression vector pcDNA3.4 (Life Technologies), transiently transfected into CHO cells, expressed, and purified by Protein A affinity chromatography. The humanized antibody expression results are shown in Table 10, indicating that anti-CTGF antibodies can be expressed using the above method.

[0310] Table 10 Expression data

[0311] Example 5 Affinity determination of humanized anti-CTGF antibodies

[0312] Referring to the method described in Example 3, the affinity of the humanized anti-CTGF antibody for human CTGF (Acro, Catalog No. CTF-H52H5), mouse CTGF (Acro, Catalog No. CTF-M52Hc), and rhesus monkey CTGF (Acro, Catalog No. GTF-R52H4) was determined. The results, shown in Tables 11-13, demonstrate that the affinity of 2101Z25 for human CTGF is approximately 4.1-fold higher than that of pamrevlumab. The affinity of 2101Z25 for mouse and rhesus monkey CTGF is comparable to that of pamrevlumab.

[0313] Table 11 Affinity determination of humanized antibodies to human CTGF

[0314] Table 12 Affinity determination of humanized antibodies and mouse CTGF

[0315] Table 13 Affinity determination of humanized antibodies to rhesus monkey CTGF

[0316] Example 6 Affinity maturation of humanized antibody 2101Z25

[0317] 6.1 Design and Construction of 2101Z25 Single CDR Mutation Library

[0318] Random amino acid mutations were performed at the antigen-binding determinant (CDR) sites of 2101Z25 to construct mutation libraries for each CDR region. High-throughput screening for sequences with strong antigen-specific binding was performed using yeast display technology. The amino acid sequence of the variable region of 2101Z25 was encoded according to the Chothia coding rules, and the CDR regions were defined according to Chothia. Polymerase chain reaction (PCR) amplification of gene fragments from each CDR mutation library was performed using NNK mutagenesis primers designed for the light chain variable region CDR1, CDR2, and CDR3, and the heavy chain variable region CDR2. After the amplified gene fragments were recovered, they were co-transformed with the yeast display plasmid into the Saccharomyces cerevisiae strain EBY100. The mutant library genes were inserted into the yeast display plasmid through homologous recombination of Saccharomyces cerevisiae, thereby realizing the display of mutant antibodies on the yeast cell wall surface. The library number is JYYDL247-250. After electroporation, the library JYYDL247-250 was cultured in 250 mL of SD-Trp Leu medium (Clontech, product number: 630316) at 30°C and 225 rpm overnight. 1.0×10 9 The bacteria were resuspended in 200 mL YPGP induction medium, cultured at 20°C and 225 rpm for 24 hours, and placed in a 4°C refrigerator for use. At the same time, the parent sequence of 2101Z25 was displayed on the yeast surface as a parent control.

[0319] 6.2 Screening of 2101Z25 Single CDR Mutation Library

[0320] After induction of JYYDL247-250 library, 1.0×10 9 Cells were enriched using a magnetic bead separation system in the first round: 1. Wash once with 50 mL of 1× PBSA, centrifuge at 3000 rpm for 3 minutes, and discard the supernatant; 2. Incubate with 20 mL of 1× PBSA containing 50 nM H-CTGF-Bio at room temperature for 30 minutes; 3. After washing, add anti-biotin magnetic beads (miltenyi, catalog number: 130-090-485), mix well, incubate for 10 minutes, and collect positive cells through a magnetic column (Quadro MACS Starting Kit).

[0321] After magnetic bead screening, positive cells were cultured and induced again, and 1.0×10 7The cells were subjected to a second round of flow cytometry sorting: 1. Centrifuge with 1 mL of 1× PBSA and discard the supernatant; 2. Incubate with 1 mL of 1× PBSA containing 10 nM R-CTGF-Bio and mouse anti-V5 antibody at room temperature for 30 minutes; 3. Centrifuge and discard the supernatant, then wash once with 1 mL of 1× PBSA; 4. Add 200 μL of 1× PBSA containing fluorescent antibodies (SA-PE, diluted 1:200; goat anti-mouse-647, diluted 1:400) and incubate on ice in the dark for 20 minutes; 5. Repeat step 3, add 1 mL of 1× PBSA to resuspend the cells, and collect the cell population with strong 647 and PE fluorescent signals using a flow cytometer.

[0322] For the second round of screening products of JYYDL247-250, 92 single clones were picked for sequencing, and the obtained single sequences were analyzed to remove the mutant sequences containing post-translational modification (PTM) sites.

[0323] 6.3 Construction, Screening, and Single-Clone Identification of 2101Z25 Light and Heavy Chain CDR Mutation Combinatorial Libraries

[0324] The plasmids containing mutations in each CDR of the light and heavy chains obtained above were mixed and physically and chemically transformed into yeast strains to construct a combinatorial library of light chain CDR1, 2, 3 and heavy chain CDR2, with the library number being JYYDL252.

[0325] After the JYYDL252 library was cultured and induced, 3.0×10 7 The cells were screened using the second round of screening described in Example 6.2. The library was further flow cytometry sorted using 10 nM R CTGF-Bio to increase its affinity for monkey CTGF. After sorting, an appropriate amount of cells was plated onto SD-Trp-Leu solid medium (Clontech, Catalog No. 630317) and incubated at 30°C for 3 days.

[0326] In the first round of screening of JYYDL252, 92 monoclonal clones were selected for sequencing, and finally monoclonal clones with a single sequence were obtained for flow cytometry analysis. 5 Individual cells were stained and evaluated according to the scheme in Table 1: Scheme 1 evaluated the binding level of each clone to human CTGF, and the stronger the PE mean fluorescence signal intensity (MFI), the stronger the binding ability; similarly, schemes 2 and 3 evaluated the binding level of each clone to monkey and mouse CTGF, and the stronger the PE MFI value, the stronger the binding ability.

[0327] Table 14 Monoclonal flow cytometry identification scheme

[0328] Based on the staining results of each clone, the sequences of clones YC341H2, YC341A6, and YC340A4 were finally selected for antibody expression. The corresponding mutation sites and staining results of each clone are summarized in Table 15.

[0329] Table 15 Yeast monoclonal colony flow staining results

[0330] 6.4 Affinity Maturation and Expression of Candidate Antibodies

[0331] The heavy chain variable region VH sequence of the YC341H2, YC341A6, and YC340A4 clones were fused with the human IgG1 heavy chain constant region, and the light chain variable region VL sequence was fused with the human kappa constant region to prepare antibodies in IgG format, numbered 2101Z25m08, 2101Z25m13, and 2101Z25m17. In addition, the light chain of 2101Z25m08 and the heavy chain of 2101Z25m17 were expressed in combination and named 2101Z25m21. Based on 2101Z25m21, the light chain CDR2A51S mutation was introduced and the antibody was named 2101Z25m23. Plasmid construction, transient transfection into CHO cells, expression, and purification were performed (see Example 2), and the antibodies finally obtained are shown in Table 16.

[0332] Table 16 Affinity matured candidate antibody expression data

[0333] Example 7 Affinity determination of affinity matured antibodies

[0334] The affinity of affinity-matured anti-CTGF antibodies to human CTGF (Acro, Catalog No.: CTF-H52H5), mouse CTGF (Acro, Catalog No.: CTF-M52Hc) and rhesus monkey (Acro, Catalog No.: GTF-R52H4) was determined using Biacore T200 (cytiva). A multi-cycle kinetic method was used with software parameters set at 25°C. Both antigen and antibody were treated with 1×HBS-EP + Buffer (10×HBS-EP + : Cytiva, Catalog No.: BR100669) dilution. The starting concentration of human, mouse and rhesus monkey CTGF was 12.5nM and diluted 2-fold. First, place the S series sensor chip Protein A (cytiva, Catalog No.: 18-5060) and 1×HBS-EP +Buffer, prime and enter standby mode, and then be tested on the machine. Each cycle contains the following steps: 1) Capture: Antibody flows into channels 2, 3, and 4, antibody is added, flow rate 10μL / min, time is 30s. 2) Analyte: Inject analyte antigen, flow into channels 1, 2, 3, and 4, flow rate 60μL / min, contact time 180s, dissociation 300s. 3) Regeneration: Inject 10mM glycine-HCl, pH 1.5, sample flows into channels 1, 2, 3, and 4, flow rate 30μL / min, time 30s, The data obtained from the experiment were fitted with a 1:1 binding model using Biacore T200 Evaluation 3.2.1 software. The results are shown in Tables 17-19, which show that the affinity of affinity matured antibodies 2102Z25m21 and 2102Z25m23 to human CTGF was increased by approximately 5.3 times and 2.4 times, respectively, compared with Pamrevlumab, the affinity to mouse CTGF was increased by approximately 5.5 times and 9.5 times, respectively, compared with Pamrevlumab, and the affinity to rhesus monkey CTGF was increased by approximately 1.7 times and 2.0 times, respectively, compared with Pamrevlumab.

[0335] Table 17 Affinity determination of candidate antibodies to human CTGF

[0336] Table 18 Affinity determination of candidate antibodies to mouse CTGF

[0337] Table 19 Affinity determination of candidate antibodies to rhesus monkey CTGF

[0338] Example 8 Affinity Maturation Anti-CTGF Antibody Epitope Analysis

[0339] 8.1 Epitope Grouping of Affinity Matured Anti-CTGF Antibodies

[0340] Epitope grouping of affinity-matured antibodies was performed using the Octet RED96e (Fortebio). Human biotinylated CTGF (Acro, Catalog No. CTF-H82E6) and the antibody were diluted in 1×PBST (1×PBS: Gibco, 10010-023; 0.02% Tween 20: Sigma, P1379). The antigen concentration was 5 μg / mL, and the antibody concentration was 100 nM. Candidate antibody samples were added to a 96-well plate (Greiner bio-one, 655209) at a rate of 200 μL / well. Software parameters were set to 30°C and a standard kinetic signal acquisition frequency of 5.0 Hz. A SA sensor (Fortebio, Catalog No. 18-5020) was pre-wetted with 1×PBST for 10 minutes before detection. Each cycle consisted of the following steps: 1) immersion in buffer for 30 seconds; 2) immobilization of biotinylated antigen on the sensor for 18 seconds; 6) immersion of the sensor in buffer for 180 seconds; 7) binding of the antigen to the first antibody for 380 seconds; 8) binding of the antigen to the second antibody for 380 seconds. Data were further corrected and inhibition rates calculated using Fortebio's Data Analysis 12.0 software. The results are shown in Table 20. These results demonstrate that 2101Z25m21, 2101Z25m23, and pamrevlumab bind to similar or overlapping epitopes on the antigen.

[0341] Table 20 Humanized anti-CTGF antibody epitope analysis

[0342] 8.2 Binding of the 2101Z25 affinity-matured antibody to CHOK1 cells expressing different CTGF domains

[0343] A CHO-K1 cell line expressing human CTGF domains 1-4 (domain 1: insulin-like growth factor binding region, IGFBP; domain 2: von Willebrand factor C-type repeat region, VWC; domain 3: thrombospondin type 1 repeat region, TSP; domain 4: C-terminal binding region) was constructed (Jiman Biotechnology (Shanghai) Co., Ltd.), and the binding of antibodies to various domains of CTGF was further determined.

[0344] Domain 1:

[0345] Domain 2:

[0346] Domain 3:

[0347] Domain 4:

[0348] FACS was used to detect the binding of CTGF-targeted antibodies to CHOK1 cells expressing different CTGF domains. The specific steps are as follows:

[0349] The cells were collected, washed with PBS, and then resuspended in Assay buffer (PBS + 2% FBS) to 1*10 6 cell / mL, add 50 μL of 1*10 6 cell / mL cells (i.e., the number of cells per well is 5*10 4 The antibody was diluted to a final concentration of 100 μg / mL (prepared at 200 μg / mL), and then diluted 5-fold in a 5-fold gradient (7 gradients, 1 at 0 concentration). Then, 50 μL of the corresponding concentration of antibody (hIgG1, i.e., RSV-IgG1: heavy chain SEQ ID NO: 45; light chain: SEQ ID NO: 46) was added to each well. After incubation at 4°C for 1 hour, the cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and 200 μL of PBS was added to each well for washing 3 times. Alexa Fluor TM 488 goat anti-human IgG (H+L) (Thermo Fisher, Catalog No.: A11013) was diluted 1:200, and 100 μL was added to each well. After incubation at 4°C for 1 hour, the cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed three times with 200 μL of PBS added to each well. The cells were then resuspended in 200 μL of PBS per well and analyzed. The results are shown in Figures 1, 2, 3, and 4. The 2101Z25 affinity matured antibodies 2101Z25m21 and 2101Z25m23 specifically bound to CHOK1 cells expressing CTGF domain 2, with binding abilities comparable to pamrevlumab.

[0350] 8.3 In vitro blocking activity of affinity-matured antibodies

[0351] FACS was used to detect the binding of antibodies blocking biotin-labeled TGF-β1 (biotin-TGF-β1, KACTUS, Catalog No.: TG1-HM10MB) to CTGF domain 2 on the surface of CHOK1 cells expressing human CTGF domains 1-4 in Example 8.2. The specific steps are as follows:

[0352] The cells were collected, washed with PBS, and then resuspended in Assay buffer (PBS + 2% FBS) to 1*10 6The starting concentration of the prepared antibody was 160 μg / ml, and 50 μL was added to each well. The starting concentration of the prepared antibody was 160 μg / ml. The mixture was diluted 2-fold (10 concentration points + 1 zero concentration point). 25 μL of the corresponding concentration of antibody was added to the cells and incubated at 4°C for 1 hour. 25 μL of 160 μg / ml biotin-TGF-β1 was added to the mixture of cells and antibody, and the mixture was incubated at 4°C for 1 hour. The mixture was centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and 200 μL of PBS was added to each well for washing three times. Streptavidin PE Conjugate (eBioscience, Catalog No.: 12-4317-87) was diluted 1:200 and 100 μL was added to each well. The mixture was incubated at 4°C for 1 hour, centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and 200 μL of PBS was added to each well for washing three times. The mixture was then resuspended in 200 μL PBS per well and analyzed on an analyzer. The results are shown in Figure 5.

[0353] The results showed that antibodies 2101Z25m21, 2101Z25m23 and Pamrevlumab could block the binding of TGF-β1 and CTGF domain 2.

[0354] 8.4 Determination of the affinity of the 2101Z25 affinity-matured antibody for human and cynomolgus monkey FcRn

[0355] Octet RED96e (Fortebio) was used to determine the affinity of candidate antibodies to human FcRn (ACRO, Catalog No.: FCN-H52W7) and cynomolgus monkey FcRn (ACRO, Catalog No.: FCM-C52W9). TM His Tag Antibody [Biotin] (GenScript, Catalog No.: A00613) were diluted with 1xPBS, the antibody was diluted with 1xPBST (PBS with 0.02% Tween 20: Sigma, P1379), the human FcRn concentration was 5 μg / mL, The TM The His Tag Antibody [Biotin] was used at a concentration of 2 μg / mL, starting at 1000 nM and diluted 2-fold.

[0356] First, add the sample to a 96-well plate (Greiner bio-one, 655209) at a volume of 200 μL / well. Then, set the software parameters: plate temperature to 30°C, and collect standard kinetic signals at a frequency of 5.0 Hz. Next, pre-wet a Streptavidin (SA) sensor (Fortebio, Cat. No. 18-5020) with 1x PBS for 10 minutes before testing on the instrument.

[0357] First immerse in buffer solution for 60s; solidify TM His Tag Antibody [Biotin], 290 seconds. Each subsequent cycle consisted of the following steps: 1) regeneration with 10 mM glycine solution, pH 1.7; 2) immersion in buffer for 60 seconds; 3) FcRn immobilization on the sensor, 60 seconds; 4) sensor immersion in buffer for 60 seconds; 5) FcRn binding to the antibody, 60 seconds; 6) antigen-antibody dissociation, 60 seconds; 7) sensor regeneration. Fortebio's Data Analysis 12.0 software was used to measure the association rate (Ka) and dissociation rate (Kd) for a 1:1 antigen-antibody binding reaction, and the equilibrium dissociation constant (KD) was calculated. The results are shown in Tables 21 and 22 below. As can be seen, the sensor binds to both human and cynomolgus monkey FcRn at pH 6.0, but not to both human and cynomolgus monkey FcRn at pH 7.4.

[0358] Table 21 Affinity of Antibodies to Human FcRn

[0359] Table 22 Affinity of Antibodies to Cynomolgus Monkey FcRn

[0360] Example 9 Pharmacodynamic Study of Affinity Matured Anti-CTGF Antibodies on Bleomycin-Induced Pulmonary Fibrosis in C57 Mice

[0361] C57BL / 6J mice were purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., 5-7 weeks old, male, a total of 37. After the acclimation period, the animals were randomly divided into the following four groups (N = 7 or 10) according to the BioBook random allocation function: G1 was a normal control group given PBS (IP, Q2D), G2 was a model group given an isotype control RSV-IgG1 (RSV-IgG1-H: SEQ ID NO: 45; RSV-IG1-L: SEQ ID NO: 46; 10 mg / kg, IP, Q2D) (Isotype Ctrl group), G3 and G4 were given the antibodies of the present invention 2101Z25m21 (10 mg / kg, IP, Q2D) and 2101Z25m23 (10 mg / kg, IP, Q2D), respectively. The specific groupings are shown in Table 23.

[0362] On the first day of the experiment, the animals were anesthetized by inhalation of 1-4% isoflurane. Based on body weight, animals in the G2-G4 groups were given 2 mg / kg of bleomycin (Nippon Kayaku Co., Ltd., batch number: X01900) by intratracheal aerosolization to establish the model, and the G1 blank control animals were treated with an equal volume of normal saline. Animals in the G2-G4 groups were injected intraperitoneally with the corresponding isotype control or antibody treatment immediately after bleomycin modeling on the first day (animals in the G1 group were given PBS at the same time), and then administered every other day until the end of the experiment. The experimental period was 21 days, with a total of 11 intraperitoneal administrations. On the 22nd day, the animals were anesthetized with Zotai (IP, 25-50 mg / kg, containing 1 mg / mL xylazine) and intubated. The lung function of all animals was tested using a small animal pulmonary function tester (DSI, model: PFT). After the pulmonary function test, the lungs were lavaged for the first time with 0.4 mL of PBS (containing 1% FBS), and the lungs were lavaged for the second time with another 0.4 mL of PBS (containing 1% FBS). After collecting the lavage fluid, the animals were euthanized by cervical dislocation, and the left lung tissue of the mice was collected and fixed with 4% neutral formalin for H&E staining (hematoxylin-eosin staining) and Masson's staining.

[0363] 9.1 Total Cell and Differential Cell Counts and Soluble Collagen Detection in BALF (Bronchoalveolar Lavage Fluid)

[0364] 100 μL of BALF suspension was collected and stained with trypan blue before microscopic white blood cell count. The remaining BALF was centrifuged (4°C, 300g, 5 min), and the supernatant was collected and assayed for soluble collagen using a commercial kit (Sigma, Cat. No. CS0006, Lot No. 0000174002). The cell pellet obtained after centrifugation was resuspended for smear preparation and stained with Wright-Giemsa staining solution to distinguish eosinophils, neutrophils, macrophages, and lymphocytes. The number of each cell type was randomly counted in 200 cells under a light microscope.

[0365] Figures 6B–E show the total and differential leukocyte counts in the BALF of mice. Compared with the normal mice in group G1, the total leukocyte count in the BALF of the model group (Isotype Ctrl group) was significantly increased (P<0.001). Compared with the model group (Isotype Ctrl group), treatment with the antibodies of the present invention, 2101Z25m21 (P<0.001 vs. Isotype Ctrl group) and 2101Z25m23 (P<0.001 vs. Isotype Ctrl group), significantly reduced the total leukocyte count in the BALF of mice, effectively alleviating inflammation (Figure 6A).

[0366] The results of soluble collagen detection in BALF are shown in Figure 7. The soluble collagen in BALF of mice in the model group (Isotype Ctrl group) was significantly increased (P<0.01 vs. normal control group G1). After treatment with antibodies 2101Z25m21 (P<0.001 vs. Isotype Ctrl group) and 2101Z25m23 (P<0.05 vs. Isotype Ctrl group), the soluble collagen in BALF of mice was significantly decreased compared with the model group.

[0367] 9.2 Lung Histopathology

[0368] The left lungs of mice were collected and fixed in 4% neutral formalin. Each animal's left lung was transversely sectioned into upper, middle, and lower sections. The sections were then embedded in paraffin and prepared for ultrathin sections at 5 μm thickness. Two sections were prepared from each block for histopathological evaluation after H&E and Masson's staining.

[0369] The results of mouse lung tissue pathology are shown in Figures 8A and 8B. After bleomycin modeling, severe fibrosis and inflammatory infiltration occurred in the lungs of mice in the model group. After treatment with antibodies 2101Z25m21 and 2101Z25m23, the lung fibrosis and inflammatory symptoms of mice were significantly improved compared with those in the model group.

[0370] Table 23 Animal grouping and dosing

[0371] 9.3 Improvement of Lung Function Indices in Mice

[0372] To test the lung function of mice, a certain pressure was applied to the mouse lungs, and the changes in the lung volume under the corresponding pressure were measured, thereby reflecting the degree of lung damage and fibrosis in the mice. Compared with normal mice without modeling, the lung volume of the model group mice treated with isotype control after pressure application was significantly reduced after bleomycin modeling. Compared with the model group mice, the lung volume of mice treated with the antibodies of the present invention 2101Z25m21 and 2101Z25m23 was significantly improved (Figure 9A); similarly, VC (vital capacity) results showed that treatment with 2101Z25m21 (P < 0.05 vs. Isotype Ctrl group) and 2101Z25m23 (P < 0.05 vs. Isotype Ctrl group) significantly increased the lung capacity of mice (Figure 9B).

[0373] Taken together, the above results indicate that antibodies 2101Z25m21 and 2101Z25m23 exhibited a strong inhibitory effect in the bleomycin-induced mouse pulmonary fibrosis model. Treatment with 2101Z25m21 and 2101Z25m23 significantly reduced the total number of white blood cells, neutrophils, lymphocytes, and macrophages in the alveolar lavage fluid of mice, effectively inhibited the formation of pulmonary fibrosis in mice, reduced inflammation, and significantly improved lung function.

[0374] Example 10 Study on the blood concentration of humanized anti-CTGF antibody in SD rats

[0375] Six male Sprague-Dawley rats, aged 6-7 weeks, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. After the acclimation period, the animals were randomly divided into the 2101Z25m21 group (10 mg / kg, IV, single dose) and the 2101Z25m23 group (10 mg / kg, IV, single dose), with three rats in each group, according to the EXCEL complete randomization method. The specific groupings are shown in Table 24. The day of the experiment was designated D0. Whole blood was collected from the animals before drug administration (Pre) by jugular vein blood sampling. Subsequently, a single IV dose of the corresponding drug was administered. Whole blood was also collected from the animals 5 minutes, 2 hours, 6 hours, 24 hours, and on days 2, 3, 4, 7, 10, 14, 21, 28, 35, and 42 after drug administration. Serum was collected by centrifugation (4°C, 1000 g, 15 minutes) and stored at -80°C for determination of drug concentrations in the serum.

[0376] Table 24 Animal grouping and dosing

[0377] The indirect ELISA method was used to detect the concentration of antibodies in serum. The plate was coated with 1 μg / ml CTGF protein (Arco, CTF-H52H5) and incubated overnight at 2-8°C. The plate was then washed with a plate washer (1×PBS+0.05% Tween20) and blocked with 1×PBST (1×PBS+0.05% Tween20+1% BSA) containing 1% BSA at 37°C for 1 hour. The plate was washed and the final concentration of the standard protein was set to 100 ng / ml to 0.78125 ng / ml. The standard protein and serum sample were first diluted with rat serum (Sbjbio, SBJ-SE-RAT003-100 mL) to the appropriate concentration, then diluted 50-fold with 1×PBST containing 1% BSA and added to the blocked ELISA plate. The plate was incubated at 37°C for 1 hour. The plate was washed and HRP-labeled anti-human IgG Fc secondary antibody (Abcam, Ab99759) was added. The plate was incubated at 37°C for 1 hour. The plate was washed and TMB (sera) was added. The reaction was terminated with a stop solution (Solarbio, C1058) (care, 5120-0077) and color developed at 37°C for 2-5 minutes. OD (450 nm) was read on an M3 microplate reader (Molecular Devices). The results were analyzed using softmax software, and serum drug concentrations were calculated by curve back-calculation. Pharmacokinetic parameter analysis of drug concentrations was performed using WinNonlin software. The plasma drug concentration results are shown in Figures 10-11 and Table 25, indicating that the T1 / 2 half-lives of 2101Z25m21 and 2101Z25m23 were 1.882 days and 0.962 days, respectively.

[0378] Table 25 Serum drug concentrations of 2101Z25m21 and 2101Z25m23 after tail vein administration to SD rats (10 mg / kg, n=3)

[0379] Sequence and Summary

[0380] Exemplary antibody sequences:

[0381] Sequence Listing:

Claims

1. An antibody or antigen-binding fragment thereof that binds to CTGF, the antibody comprising 1) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 27, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 21; 2) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO:27, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO:31; 3) three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in VH as shown in SEQ ID NO: 1 or 10, and three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in VL as shown in SEQ ID NO: 2 or 9; 4) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 23, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 21; or 5) the three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the VH as shown in SEQ ID NO: 27, and the three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the VL as shown in SEQ ID NO: 25 or 29.

2. An antibody or antigen-binding fragment thereof that binds to CTGF, the antibody comprising three complementary determining regions HCDR1, HCDR2 and HCDR3 of the heavy chain variable region, and three complementary determining regions LCDR1, LCDR2 and LCDR3 of the light chain variable region, wherein the HCDR1, HCDR2 and HCDR3 and the LCDR1, LCDR2 and LCDR3 respectively comprise the amino acid sequence shown in the following SEQ ID NO, or respectively consist of the amino acid sequence shown in the following SEQ ID NO composition: 1) SEQ ID NO: 14, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 12, SEQ ID NO: 13; 2) SEQ ID NO: 14, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 32, SEQ ID NO: 13; 3) SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13; 4) SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 16, SEQ ID NO: 22, SEQ ID NO: 12, SEQ ID NO: 13; 5) SEQ ID NO: 14, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 11, SEQ ID NO: 26, SEQ ID NO: 13; or 6) SEQ ID NO: 14, SEQ ID NO: 28, SEQ ID NO: 16, SEQ ID NO: 11, SEQ ID NO: 30, SEQ ID NO:

13.

3. An antibody or antigen-binding fragment thereof that binds to CTGF as described in claim 1 or 2, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 10, 23 or 27, or consists of the sequence; or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 10, 23 or 27, or consists of the sequence.

4. An antibody or antigen-binding fragment thereof that binds to CTGF as described in claim 1 or 2, wherein the antibody comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 9, 21, 25, 29 or 31, or consists of the sequence; or comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 9, 21, 25, 29 or 31, or consists of the sequence.

5. The antibody or antigen-binding fragment thereof that binds to CTGF according to claim 1 or 2, wherein the antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein 1) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; 2) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 31; 3) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 1, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 2; 4) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 10, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 9; 5) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 23, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 21; 6) VH comprises or consists of the amino acid sequence shown in SEQ ID NO: 27, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO: 25; or 7) VH comprises or consists of the amino acid sequence shown in SEQ ID NO:27, and VL comprises or consists of the amino acid sequence shown in SEQ ID NO:

29.

6. The antibody or antigen-binding fragment thereof that binds to CTGF according to any one of claims 1 to 5, wherein the antibody comprises a heavy chain constant region, such as a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, such as a heavy chain constant region of IgG1, (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 3; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:

3.

7. An antibody or antigen-binding fragment thereof that binds to CTGF as claimed in any one of claims 1 to 6, wherein the antibody comprises a light chain constant region, such as a lambda or kappa light chain constant region, such as the light chain constant region (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:4; (ii) comprises or consists of the amino acid sequence of SEQ ID NO: 4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2, or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence of SEQ ID NO:

4.

8. The antibody or antigen-binding fragment thereof that binds to CTGF according to any one of claims 1 to 7, wherein the antibody comprises a heavy chain constant region and a light chain constant region, wherein The heavy chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:3; and the light chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

4.

9. The antibody or antigen-binding fragment thereof that binds to CTGF of any one of claims 1-8, which comprises a heavy chain comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 18, 20, 37 or 39.

10. An antibody or antigen-binding fragment thereof that binds to CTGF as described in any one of claims 1-9, which comprises a light chain, which comprises or consists of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 17, 19, 38, 40, 41 or 42.

11. The antibody or antigen-binding fragment thereof that binds to CTGF according to any one of claims 1 to 10, comprising a heavy chain and a light chain, wherein 1) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 39, and The light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:38; 2) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:42; 3) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 18, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; 4) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 20, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 19; 5) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 37, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 38; 6) the heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:40; or 7) The heavy chain comprises or consists of the amino acid sequence shown in SEQ ID NO:39, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:

41.

12. The antibody or antigen-binding fragment thereof that binds to CTGF according to claim 1 or 2, which has one or more of the following characteristics: 1) Ability to bind human or rhesus monkey CTGF with high affinity; 2) capable of binding to domain 2 of human or rhesus monkey CTGF, for example, binding to cells expressing domain 2 of CTGF; 3) being able to block the binding of TGF-β1 to CTGF (e.g., CTGF domain 2), such as blocking the binding of TGF-β1 to CTGF (e.g., CTGF domain 2) on the cell surface; 4) Effectively treat pulmonary fibrosis, such as reducing lung white blood cells, reducing inflammation, and / or improving lung function (such as vital capacity); 5) having a good serum half-life, such as about 1 day or 2 days, such as greater than or equal to 1 day; 6) showing the same or similar binding affinity and / or specificity for CTGF as any of the antibodies listed in claim 11; 7) Inhibiting the binding of any antibody listed in claim 11 to CTGF; 8) binding to the same or overlapping epitope as any of the antibodies of claim 11; 9) competing with any antibody of claim 11 for binding to CTGF; or 10) having one or more biological properties of any antibody molecule listed in claim 11.

13. The antibody or antigen-binding fragment thereof of any one of claims 1-12, wherein the antibody is a monoclonal antibody.

14. The antibody or antigen-binding fragment thereof of any one of claims 1 to 13, wherein the antibody is a humanized antibody or a chimeric antibody.

15. The antibody or antigen-binding fragment thereof of any one of claims 1 to 14, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, single-chain antibody, sc(Fv) 2 , (Fab') 2 , single domain antibodies, diabodies, nanobodies or linear antibodies.

16. The antibody or antigen-binding fragment thereof of claim 15, wherein the single-chain antibody is a scFv.

17. The antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the antibody is a bispecific antibody or a multispecific antibody.

18. An isolated nucleic acid encoding the anti-CTGF antibody or antigen-binding fragment thereof of any one of claims 1 to 17.

19. An expression vector comprising the nucleic acid of claim 18.

20. The expression vector of claim 19, wherein the expression vector is a pcDNA3.4 vector.

21. A host cell comprising the nucleic acid of claim 18 or the expression vector of claim 19 or 20.

22. The host cell of claim 21, wherein the host cell is prokaryotic or eukaryotic.

23. The host cell of claim 22, wherein the host cell is selected from Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for producing antibodies or antigen-binding fragments thereof.

24. The host cell of claim 23, wherein the host cell is a 293 cell or a CHO cell.

25. A method for preparing an anti-CTGF antibody or an antigen-binding fragment thereof, the method comprising culturing the host cell of any one of claims 21-24 under conditions suitable for expressing a nucleic acid encoding the anti-CTGF antibody or antigen-binding fragment thereof of any one of claims 1 to 17, optionally isolating the antibody or antigen-binding fragment thereof, and optionally the method further comprises recovering the anti-CTGF antibody or antigen-binding fragment thereof from the host cell.

26. An immunoconjugate comprising the antibody or antigen-binding fragment thereof of any one of claims 1 to 17.

27. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or the immunoconjugate according to claim 26, and optionally a pharmaceutically acceptable excipient.

28. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 17 or the immunoconjugate according to claim 26, and other therapeutic agents, and optionally pharmaceutically acceptable excipients.

29. Use of an effective amount of an anti-CTGF antibody or antigen-binding fragment thereof according to any one of claims 1 to 17, or an immunoconjugate according to claim 26, or a pharmaceutical composition according to claim 27 or 28 in the preparation of a medicament for preventing or treating a CTGF-related disease in a subject or individual, wherein the CTGF-related disease is a fibrotic disease.

30. The use according to any one of claims 29, wherein the fibrotic disease is pulmonary fibrosis (eg idiopathic pulmonary fibrosis).

31. The use of claim 29 or 30, further comprising co-administering one or more other therapeutic agents to the subject.

32. The use of claim 31, wherein the other therapeutic agent is selected from a chemotherapeutic agent or an immunomodulatory agent, such as an anti-inflammatory agent or an immunosuppressant.

33. A method for detecting CTGF in a sample, the method comprising (a) contacting a sample with any anti-CTGF antibody or antigen-binding fragment thereof according to any one of claims 1 to 17; and (b) detecting formation of a complex between the anti-CTGF antibody or antigen-binding fragment thereof and CTGF; optionally, the anti-CTGF antibody is detectably labeled.

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