CN113398270B - Method for treating bone giant cell tumor - Google Patents

Abstract

The invention relates to a method for treating giant cell tumor of bone. Specifically, the present invention provides the use of a CD44 inhibitor, and the CD44 inhibitor is used to prepare a medicine for treating giant cell tumor of bone. The experimental results of the present invention show that SRGN can be used as a new type of biomarker for predicting giant cell tumor of bone for diagnosis, classification and/or treatment of giant cell tumor of bone. In addition, in giant cell tumor of bone, spindle-shaped stromal cells secrete sericin (SRGN) glycoprotein, and interact with the CD44 receptor on monocytes, thereby promoting the differentiation of monocytes into multinucleated cells. giant cells. Therefore, CD44 can be used as a target for clinical treatment of giant cell tumor of bone.

Description

A method for treating giant cell tumor of bone

technical field

The invention relates to the fields of oncology and diagnosis, and in particular, the invention relates to a method for treating giant cell tumor of bone.

Background technique

Giant cell tumor ^of bone is a common primary bone tumor1 that typically occurs in the metaphysis of the long bones of the extremities, including the distal ^femur , proximal femur, and proximal tibia2. Although giant cell tumor ^of bone is generally considered a benign tumor and rarely metastasizes, it is locally aggressive and often results in severe bone destruction3,4.

Currently, the diagnosis and treatment methods for giant cell tumor of bone are very limited. The diagnosis of giant cell tumor of bone is mainly through histopathological and radiological evaluation, as well as the detection of H3.3G34W mutation.

Currently, treatment options for giant cell tumor of bone are limited. In terms of clinical treatment, surgery is the most commonly used method of treatment, but 27%-65% of patients will experience recurrence or metastasis after surgery8 ^. In addition to surgery, bisphosphonates ⁹ and the RANKL monoclonal antibody denosumab ¹⁰ are also used in the treatment of giant cell tumor of bone. However, both drugs have a range of side effects ^11-13 , and patients are at risk of relapse after discontinuation of the drug. Therefore, finding more therapeutic targets is helpful for the clinical treatment of giant cell tumor of bone.

To sum up, it is imperative to find new markers and therapeutic targets for giant cell tumor of bone, and to develop targeted drugs. Therefore, there is an urgent need in this field to develop targeted drugs that can be used for the treatment of giant cell tumor of bone.

Contents of the invention

The object of the present invention is to provide a targeted drug for treating giant cell tumor of bone and its application.

In the first aspect of the present invention, a use of a CD44 inhibitor is provided, and the CD44 inhibitor is used for preparing a medicine for treating giant cell tumor of bone.

In another preferred embodiment, the CD44 inhibitor is selected from the group consisting of small molecule drugs, specific antibodies, nucleic acid drugs, gene editing drugs or combinations thereof.

In another preferred example, the nucleic acid drug includes antisense RNA and microRNA.

In another preferred example, the CD44 inhibitor is a blocking inhibitor that blocks the binding or interaction between SRGN and CD44.

In another preferred example, the CD44 inhibitor is a CD44-specific antibody.

In another preferred example, the CD44-specific antibody is selected from the group consisting of IM7, bivatuzumab, RG-7356, H90, PF-3475952, and RO5429083.

In another preferred example, the CD44 inhibitor is selected from the group consisting of hyaluronan-cisplatinconjugate, Angstrom6, Verbascoside, and AMC303.

In another preferred example, the drug further contains an SRGN inhibitor, or the drug is used in combination with an SRGN inhibitor.

In another preferred embodiment, the SRGN inhibitor is administered before, after and/or simultaneously with the administration of the CD44 inhibitor.

The aforementioned SRGN inhibitors include: small molecule drugs that inhibit SRGN, specific antibodies against SRGN, nucleic acid drugs, gene editing drugs or combinations thereof. Preferably, the nucleic acid drug includes antisense RNA and microRNA.

In another preferred example, the SRGN inhibitor blocks the binding or interaction between SRGN and CD44.

In the second aspect of the present invention, there is provided a pharmaceutical composition for treating giant cell tumor of bone, said pharmaceutical composition contains (a) CD44 inhibitor, (b) SRGN inhibitor and (c) pharmaceutically acceptable carrier.

In a third aspect of the present invention, a kit is provided, which contains:

(i) a first pharmaceutical composition, which contains a CD44 inhibitor and a pharmaceutically acceptable carrier;

(ii) a second pharmaceutical composition, which contains an SRGN inhibitor and a pharmaceutically acceptable carrier.

In another preferred example, the first pharmaceutical composition and the second pharmaceutical composition are different or independent.

In another preferred example, the pharmaceutical composition is an oral preparation or a non-oral preparation (such as an injection).

In a fourth aspect of the present invention, a kit is provided, the kit comprising:

(Z1) A diagnostic reagent for detecting giant cell tumor of bone or a marker for typing giant cell tumor of bone, wherein the diagnostic reagent is selected from the group consisting of detection reagents for SRGN gene, mRNA, cDNA, or protein; and

(Z2) An active ingredient for treating giant cell tumor of bone, wherein the active ingredient is selected from the group consisting of CD44 inhibitors, SRGN inhibitors or combinations thereof.

In the fifth aspect of the present invention, there is provided an in vitro non-therapeutic method for inhibiting giant cell tumor cells of bone, comprising the steps of: cultivating the cells in a culture system containing an effective amount of a CD44 inhibitor and/or an SRGN inhibitor. The aforementioned giant cell tumor cells of bone, thereby inhibiting the giant cell tumor cells of bone.

In another preferred example, the mononuclear cells of the giant cell tumor of bone.

In another preferred example, said inhibiting giant cell tumor cells of bone includes: inhibiting the differentiation of monocytes into multinucleated giant cells.

In the sixth aspect of the present invention, a method for treating giant cell tumor of bone is provided, comprising the step of: administering an effective amount of a CD44 inhibitor and/or an SRGN inhibitor to a subject in need.

In another preferred example, the subject is a patient with giant cell tumor of bone.

In another preferred example, the subject is an SRGN positive patient.

In the seventh aspect of the present invention, a use of SRGN gene, mRNA, cDNA, or protein or its detection reagent is provided, which is used (a) as a marker for detecting giant cell tumor of bone or the risk of giant cell tumor of bone; (b) a marker for typing giant cell tumor of bone; and/or (c) for preparing a diagnostic reagent or kit for detecting giant cell tumor of bone or giant cell of bone tumor risk, or to classify giant cell tumor of bone.

In another preferred example, the diagnostic reagents include antibodies, primers, probes, sequencing libraries, nucleic acid chips (such as DNA chips) or protein chips.

In another preferred embodiment, the protein includes a full-length protein or a protein fragment.

In another preferred example, the SRGN gene, mRNA, cDNA, or protein is derived from mammals, preferably from humans and non-human mammals (such as primates), more preferably, from the diagnosed Patients with giant cell tumor of bone.

In another preferred example, the SRGN gene, mRNA, cDNA, or protein is derived from a patient with giant cell tumor of bone.

In another preferred example, the SRGN protein is human SRGN, preferably, its amino acid sequence is shown in SEQ ID No: 1.

In another preferred example, the detection includes serum detection, detection of tissue or cell samples.

In another preferred example, the tissue includes isolated giant cell tumor tissue of bone.

In another preferred example, the cells include spindle-shaped stromal cells of giant cell tumor of bone.

In another preferred example, the detection is blood sample detection and/or serum sample detection.

In another preferred example, the detection reagents include SRGN-specific antibodies, SRGN-specific binding molecules, specific amplification primers, probes or chips.

In another preferred example, the SRGN protein or its specific antibody or specific binding molecule is coupled or bears a detectable label.

In another preferred embodiment, the detectable label is selected from the group consisting of chromophores, chemiluminescent groups, fluorophores, isotopes or enzymes.

In another preferred example, the SRGN-specific antibody is a monoclonal antibody or a polyclonal antibody.

In the eighth aspect of the present invention, a diagnostic kit for detecting giant cell tumor of bone or typing giant cell tumor of bone is provided. The kit contains a container containing the SRGN gene for detecting , mRNA, cDNA, or protein detection reagents;

And a label or an instruction, which indicates that the kit is used for detecting giant cell tumor of bone or the risk of giant cell tumor of bone; or for typing giant cell tumor of bone.

In another preferred example, the detection includes prejudgment (prediction), typing detection, and prognosis detection.

In another preferred example, the classification includes dividing giant cell tumor of bone into SRGN type giant cell tumor of bone (i.e. SRGN positive giant cell tumor of bone) and non-SRGN type giant cell tumor of bone (i.e. SRGN negative giant cell tumor of bone). tumor).

In another preferred example, the detection reagents for detecting SRGN gene, mRNA, cDNA, or protein include:

(a). Specific antibodies against SRGN protein; and/or

(b). Specific primers for specifically amplifying mRNA or cDNA of SRGN.

In another preferred example, the detection is blood sample detection and/or serum sample detection.

In another preferred example, the label or instructions indicate the following:

(i) When the serum SRGN concentration of the subject (subject) is ≥0.8ng/ml (preferably, ≥0.9ng/ml), it indicates that the subject has a high risk of developing giant cell tumor of bone, and/or the subject The test subject is classified as SRGN-positive giant cell tumor of bone (or SRGN-type giant cell tumor of bone); and

(ii) When the serum SRGN concentration of the subject (subject) is <0.8ng/ml (preferably, <0.6ng/ml), it indicates that the subject has a low risk of giant cell tumor of bone, and/or the subject The type of test object is SRGN-negative giant cell tumor of bone (or non-SRGN giant cell tumor of bone).

In the ninth aspect of the present invention, a method of detecting giant cell tumor of bone or risk of giant cell tumor of bone is provided, the method comprising:

a) provide a test sample from the subject;

b) detecting the concentration of SRGN protein in the test sample; and

c) comparing the concentration of the SRGN protein determined in step b) with the control,

Wherein compared with the control, the concentration of SRGN protein in the sample is lower than the reference value, indicating that the subject may suffer from giant cell tumor of bone or the probability of developing giant cell tumor of bone is higher than that of normal population.

In another preferred example, the test samples include blood samples and/or serum samples of patients with giant cell tumor of bone.

In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

In another preferred example, the reference value is a cut-off value (cut-off value).

In a tenth aspect of the present invention, a method for determining a treatment plan for giant cell tumor of bone is provided, comprising:

a) provide a test sample from the subject;

b) detecting the concentration of SRGN protein or mRNA in the test sample; and

c) determining a treatment regimen based on the concentration of SRGN protein or mRNA in said sample.

In another preferred example, the sample is selected from the group consisting of blood, serum, and isolated giant cell tumor tissue or cells.

In another preferred example, the cells include spindle-shaped stromal cells of giant cell tumor of bone.

In another preferred example, when the concentration of serum SRGN in patients with giant cell tumor of bone is ≥ 0.8 ng/ml, the treatment plan of SRGN inhibitor, the treatment plan of CD44 inhibitor or the combination of SRGN inhibitor and CD44 inhibitor are selected Treatment prescription; when the concentration of serum SRGN in patients with giant cell tumor of bone is less than 0.8ng/ml, conventional giant cell tumor of bone treatment can be used.

In another preferred example, the SRGN inhibitors include: small molecule drugs that inhibit SRGN, specific antibodies against SRGN, and nucleic acid drugs.

In another preferred example, the nucleic acid drug includes antisense RNA and microRNA.

In another preferred example, the SRGN inhibitor blocks the binding or interaction between SRGN and CD44.

In another preferred example, the CD44 inhibitors include: small molecule drugs that inhibit CD44, specific antibodies against CD44, and nucleic acid drugs.

In another preferred example, the nucleic acid drug includes antisense RNA and microRNA.

In another preferred example, the CD44 inhibitor blocks the binding or interaction between SRGN and CD44.

In another preferred example, the conventional treatment regimen for giant cell tumor of bone is selected from the group consisting of bisphosphonates, RANKL monoclonal antibody (such as denosumab), surgical treatment, radiotherapy, or a combination thereof.

In another preferred example, the SRGN inhibitor is selected from the following group:

ShSRGN-2, its sequence is: CCAGGACTTGAATCGTATCTT (SEQ ID No: 2) or

ShSRGN-5, its sequence is: ACATGGATTAGAAGAGGATTT (SEQ ID No: 3).

In an eleventh aspect of the present invention, a diagnostic device is provided, and the diagnostic device includes:

an input module configured to input concentration data of SRGN protein and/or mRNA in a test sample of a subject;

Giant cell tumor of bone diagnosis-giant cell tumor of bone module, the giant cell tumor of bone diagnosis-giant cell tumor of bone module is configured to: based on the concentration data of the SRGN protein and/or mRNA, the Carry out diagnostic analysis on whether the subject has giant cell tumor of bone, and/or perform typing and typing of giant cell tumor of bone, and obtain diagnostic analysis results and/or typing analysis results;

An output module, the output module is configured to output the diagnostic analysis results and/or typing analysis results.

In another preferred example, the output module includes: a printer, a display, a screen, a mobile phone, a PAD, or a combination thereof.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, we will not repeat them here.

Description of drawings

Figure 1 shows that inhibition of CD44 reduces osteoclast formation in vitro. Among them, (A) to (D) show that treatment of mouse primary bone marrow cells with hFOB1.19 conditioned medium (A-B) or SRGN recombinant protein (C-D) and CD44 neutralizing antibody (10ng/mL) induces osteoclasts Cell Differentiation. Osteoclast counts (A,C) and representative images (B,D). (E) to (H) show that treatment of RAW264.7 cells with hFOB1.19 conditioned medium (E and F) or SRGN recombinant protein (G and H) and CD44 neutralizing antibody (10 ng/mL) induces osteoclasts Cell Differentiation. Counts of osteoclasts (E,G) and representative images (F,H). (I) shows protein level validation of Cd44 knockdown. (J) to (M) show that treatment of Cd44 knockout RAW264.7 cells with hFOB1.19 conditioned medium (J-K) or SRGN recombinant protein (L-M) induces osteoclast differentiation. Osteoclast count (J,L) and representative images (K,M).

Figure 2 shows that CD44 neutralizing antibody inhibits the formation of multinucleated giant cells and the growth of giant cell tumor of bone in mice. Among them, (A) to (D) show the representative images of in vivo imaging, ex vivo imaging of leg bone and CT after GCTB-19 cell tibial injection, CD44 neutralizing antibody treatment group and control group (A), indicated by the arrow site of bone injury. Fluorescence statistical value of mouse in vivo imaging (B), fluorescence value statistics of mouse leg bone in vitro imaging (C), relative bone volume statistics of mouse leg bone (D). (E) H&E and TRAP staining of mouse leg bone sections are shown. (F) shows the statistics of the number of TRAP+ multinucleated giant cells. (G) shows the body weight statistics of the mice in the CD44 neutralizing antibody group and the control group. (H) shows the blood cell statistics of mice in CD44 neutralizing antibody group and control group. In the figure, WBC is white blood cells and RBC is red blood cells.

Detailed ways

After extensive and in-depth research, the present inventors unexpectedly found for the first time that the serum SRGN protein level of patients with giant cell tumor of bone was significantly higher than that of the control group, so SRGN can be used as a new type of biomarker for predicting giant cell tumor of bone. For the diagnosis, classification and/or treatment of giant cell tumor of bone. In addition, SRGN secreted by spindle stromal cells interacts with the CD44 receptor on monocytes, thereby promoting the differentiation of monocytes into multinucleated giant cells. Therefore, CD44 can be used as a target for clinical treatment of giant cell tumor of bone. On this basis, the present inventors have completed the present invention.

Specifically, experiments have shown that SRGN is specifically highly expressed in the spindle-shaped stromal cells of giant cell tumor of bone, and through the interaction with its receptor CD44, promotes the formation of multinucleated giant cells, thereby helping the growth of giant cell tumor of bone in vivo . Therefore, CD44 and/or SRGN can be used as therapeutic targets for giant cell tumor of bone.

the term

sample

The term "sample" or "specimen" as used herein refers to material specifically associated with a subject from which specific information relating to the subject can be determined, calculated or inferred. A sample may consist in whole or in part of biological material from a subject. A sample can also be a material that has been in contact with a subject in such a way that tests performed on the sample can provide information about the subject. A sample may also be a material that has been in contact with other material that is not the subject's but enables the first material to be subsequently tested to determine information about the subject, for example the sample could be a probe or an anatomical Knife cleaning solution. The sample may be a source of biological material other than the contacting subject, so long as a person skilled in the art is still able to determine information about the subject from the sample.

Express

As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of protein encoded by RNA or a gene or gene portion, and also includes the appearance of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (eg, antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic moieties of the binding ligand are all included within the scope of the term "expression". Thus, an increase in half-spot density on an immunoblot such as a western blot is also within the scope of the term "expression" on a biological molecule basis.

Reference value

As used herein, the term "reference value" refers to a value that is statistically related to a particular result when compared to an assay result. In preferred embodiments, the reference value is determined based on statistical analysis of studies comparing SRGN protein expression with known clinical outcomes. Some of these studies are shown in the Examples section herein. However, studies from the literature and user experience with the methods disclosed herein can also be used to generate or adjust reference values. Reference values can also be determined by taking into account circumstances and outcomes particularly relevant to the patient's medical history, genetics, age, and other factors.

In the present invention, the reference value refers to a cut-off value (cut-off value), preferably 0.8 ng/ml and 0.9 ng/ml (the concentration of SRGN in the serum of giant cell tumor of bone patients).

giant cell tumor of bone

As used herein, the term "giant cell tumor of bone" refers to a tumor arising from giant cell tumor of bone.

There are three main cell types in giant cell tumor of bone tissue: spindle-shaped stromal cells, multinucleated giant cells, and monocytes. Multinucleated giant cells, ^which are highly similar in shape and function to osteoclasts, are considered to be the main cause of osteolysis in giant cell tumors of bone, while spindle-shaped stromal cells are the main malignant component5-7. Spindle stromal cells can not only proliferate malignantly, but also secrete various cytokines to recruit monocytes and induce monocytes to differentiate into multinucleated giant cells.

SRGN proteins and polynucleotides

In the present invention, the terms "protein of the present invention", "SRGN protein", and "SRGN polypeptide" are used interchangeably, and all refer to a protein or polypeptide having an SRGN amino acid sequence. They include SRGN proteins with or without an initial methionine. In addition, the term also includes full-length SRGN and fragments thereof. The SRGN protein referred to in the present invention includes its complete amino acid sequence, its secreted protein, its mutant and its functionally active fragments.

Sericin (SRGN) is one of the earliest proteoglycans discovered. It is mainly composed of 17.6kDa core protein and glycosaminoglycan ^chains17 .

SRGN can be stored intracellularly or secreted into the extracellular matrix. SRGN is mainly expressed in cells of the hematopoietic lineage, such as neutrophils, lymphocytes, macrophages, platelets, etc. ^18-22 . Some studies have reported that SRGN is related to the occurrence and development of some tumors ^23-26 .

The full length of human SRGN protein is 158 amino acids (accession number is P10124). The amino acid sequence is as follows: MMQKLLKCSRLVLALALILVLESSVQGYPTRRARYQWVRCNPDSNSANCLEEKGPMFELLPGESNKIPRLRTDLFPKTRIQDLNRIFPLSEDYSGSGFGSGSGSGSGSGSGFLTEMEQDYQLVDESDAFHDNLRSLDRNLPSDSQDLGQHGLEEDFML (SEQ ID No: 1)

The SRGN protein is encoded by the SRGN gene located on chromosome 10q22.1, with a total length of 158 amino acids and consists of three regions: signal peptide (composed of amino acid residues 1-27), N-terminal (composed of amino acid residues 28-76 ) and the C-terminus (composed of amino acid residues 77-158), the C-terminus has multiple serine/glycine repeat regions, mainly combined with GAG, and the N-terminus has two cysteine residues.

In the present invention, the terms "SRGN gene" and "SRGN polynucleotide" are used interchangeably, and both refer to a nucleic acid sequence having an SRGN nucleotide sequence.

The full genome length of the human SRGN gene is 47279bp (NCBI GenBank accession number is 5552), and the full length of its transcript mRNA sequence is 1217bp (NCBI GenBank accession number is NM_002727.4).

It is understood that substitutions of nucleotides in codons are acceptable when encoding the same amino acid. It should also be understood that where conservative amino acid substitutions result from nucleotide substitutions, nucleotide changes are also acceptable.

When the amino acid fragment of SRGN is obtained, a nucleic acid sequence encoding it can be constructed based on it, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the SRGN nucleotide sequence disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, related sequences can also be synthesized by artificial synthesis, especially when the fragment length is relatively short. Often, fragments with very long sequences are obtained by synthesizing multiple small fragments and then ligating them.

At present, the DNA sequence encoding the protein of the present invention (or its fragments and derivatives) can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (such as vectors) and cells known in the art.

The polynucleotide sequences of the present invention can be used to express or produce recombinant SRGN polypeptides by conventional recombinant DNA techniques. Generally speaking, there are the following steps:

(1). Transform or transduce a suitable host cell with the polynucleotide (or variant) encoding the human SRGN polypeptide of the present invention, or with a recombinant expression vector containing the polynucleotide;

(2). Host cells cultured in a suitable medium;

(3). Isolate and purify protein from culture medium or cells.

In the present invention, the SRGN polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.

Methods well known to those skilled in the art can be used to construct expression vectors containing the SRGN-encoding DNA sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins.

The host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells; animal cells and the like.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as E. coli, competent cells capable of taking up DNA can be harvested after the exponential growth phase and treated with the _CaCl2 method using procedures well known in the art. Another method is to use _MgCl2 . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

The obtained transformant can be cultured by conventional methods to express the polypeptide encoded by the gene of the present invention. The medium used in the culture can be selected from various conventional media according to the host cells used. The culture is carried out under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method can be expressed inside the cell, or on the cell membrane, or secreted outside the cell. The recombinant protein can be isolated and purified by various separation methods by taking advantage of its physical, chemical and other properties, if desired. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic disruption, supertreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

CD44 protein and polynucleotide

CD44 is a transmembrane glycoprotein involved in a variety of cellular processes including cell division, survival, migration and adhesion ¹⁴ .

The CD44 gene is located on chromosome 11p13 and has 20 exons in total, ten of which are regulated by alternative splicing, and these exons are called alternative exons ¹⁵ . Through alternative splicing and post-translational modification, the CD44 gene can express different CD44 protein isoforms. The molecular mass of these isoforms ranges from 85kDa to 250kDa, and the expression also has some tissue specificity.

The functions of CD44 protein mainly include: interacting with soluble extracellular components or extracellular matrix as a receptor; cooperating with other receptors to activate downstream signaling pathways; regulating actin cytoskeleton ¹⁶ .

The full length of human CD44 protein is 361 amino acids (accession number is P16070), and the amino acid sequence is as follows: MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSISRTEADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVN RDGTRYVQKGEYRTNPEDIYPSNPTDDDVSSGSSSERSTSGGYIFYTFSTVHPIPDEDSPWITDSTDRIPATRDQDTFHPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPEWLIILASLLALALILAVCIAVNSRRRCGQKKKLVINSGNGAVEDRKPSGLNGEASKSQEMVHLVNKESSETPDQFMTADETRN LQNVDMKIGV (SEQ ID No: 4).

In the present invention, the terms "CD44 gene" and "CD44 polynucleotide" are used interchangeably, and both refer to a nucleic acid sequence having a CD44 nucleotide sequence.

The genome length of the human CD44 gene is 93232bp (NCBI GenBank accession number is 960), and its transcript mRNA sequence is 4288bp in length (NCBI GenBank accession number is NM_001001391.2).

Correlation between SRGN and giant cell tumor of bone

The inventors unexpectedly found that the serum SRGN protein level of patients with giant cell tumor of bone was significantly higher than that of the control group, so SRGN can be used as a new type of biomarker for predicting giant cell tumor of bone for the diagnosis of giant cell tumor of bone , typing and/or treatment.

Experiments have shown that SRGN is specifically highly expressed in the spindle-shaped stromal cells of giant cell tumor of bone, and through the interaction with its receptor CD44, it promotes the formation of multinucleated giant cells, thereby helping the growth of giant cell tumor of bone in vivo. Therefore, SRGN can be used as a diagnostic indicator and therapeutic target for giant cell tumor of bone.

The experimental results of the present invention further suggest that different treatment options should be selected according to the concentration level of SRGN in the serum of patients with giant cell tumor of bone. For example, when the concentration of serum SRGN in a patient with giant cell tumor of bone is ≥0.8 ng/ml, the treatment plan of SRGN inhibitor, the treatment plan of CD44 inhibitor or the combined treatment of SRGN inhibitor and CD44 inhibitor are selected. When the concentration of serum SRGN in patients with giant cell tumor of bone is less than 0.8ng/ml, conventional treatment options for giant cell tumor of bone can be used, such as bisphosphonates and RANKL monoclonal antibody (such as denosumab), surgical treatment, etc. . Of course, SRGN inhibitors and/or CD44 inhibitors can also be additionally administered while using these conventional therapeutic agents or treatment regimens.

In addition, the inventors specifically knocked down SRGN through shRNA interference in giant cell tumor cells of bone (the shRNA used is ShSRGN-2: CCAGGACTTGAATCGTATCTT, SEQ ID No: 2; and ShSRGN-5: ACATGGATTAGAAGAGGATTT SEQ ID No: 3). The results showed that after SRGN was knocked down, the ability of the conditioned medium of giant cell tumor of bone to induce the differentiation of primary mouse bone marrow cells or RAW264.7 cells into osteoclasts in vitro was significantly inhibited (the experimental results can also be found in The applicant submitted a Chinese application titled "A Diagnostic Method for Giant Cell Tumor of Bone" on the same day as July 20, 2021).

Interaction of SRGN and CD44

The present inventors have discovered for the first time that in giant cell tumor of bone, spindle-shaped stromal cells secrete sericin (SRGN), and that SRGN interacts with the CD44 receptor on monocytes to promote monocyte activation. Cells differentiate into multinucleated giant cells. This suggests that the interaction between SRGN and CD44 on monocytes can lead to the occurrence of giant cell tumor of bone and promote the progression of giant cell tumor of bone. Therefore, SRGN inhibitors and/or CD44 inhibitors can be used in the clinical treatment of giant cell tumor of bone .

CD44 inhibitors and SRGN inhibitors

As used herein, the term "inhibitor of the invention" includes CD44 inhibitors, SRGN inhibitors or combinations thereof.

As used herein, the terms "CD44 inhibitor of the present invention" and "CD44 targeting inhibitor of the present invention" are used interchangeably, and both can be used for inhibitors that inhibit CD44, including inhibitors that inhibit the expression level and activity of CD44, and Inhibitors that inhibit the interaction of CD44 with SGRN.

As used herein, the terms "SRGN inhibitor of the present invention" and "SRGN targeting inhibitor of the present invention" are used interchangeably, and both can be used for inhibitors of SRGN, including inhibitors of SRGN expression level, activity, and Inhibitors that inhibit the interaction of SRGN with CD44.

In the present invention, the types of inhibitors of the present invention include (but are not limited to): small molecule drugs, specific antibodies, nucleic acid drugs, gene editing drugs or combinations thereof.

In another preferred example, the nucleic acid drug includes antisense RNA and microRNA.

In another preferred embodiment, the inhibitor of the present invention is a blocking inhibitor (such as a blocking antibody) that blocks the binding or interaction between SRGN and CD44.

Typically, the CD44 inhibitors include (but are not limited to): small molecule drugs that inhibit CD44, specific antibodies against CD44, nucleic acid drugs, gene editing drugs, or combinations thereof.

In another preferred example, the CD44 inhibitor blocks the binding or interaction between SRGN and CD44.

Particularly preferred anti-CD44 antibodies include (but are not limited to): IM7, bivatuzumab, RG-7356, H90, PF-3475952, RO5429083, or combinations thereof.

In another preferred example, the CD44 inhibitors include (but not limited to): hyaluronan-cisplatinconjugate, Angstrom6, Verbascoside, AMC303.

Typically, the SRGN inhibitors include: small molecule drugs that inhibit SRGN, specific antibodies against SRGN, nucleic acid drugs, gene editing drugs or combinations thereof. Preferably, the nucleic acid drug includes antisense RNA and microRNA.

In another preferred example, the SRGN inhibitor blocks the binding or interaction between SRGN and CD44.

specific antibody

In the present invention, the term "antibody of the present invention" includes "specific antibody against CD44" and/or "specific antibody against SRGN".

In one aspect, the invention includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for human CD44 polypeptide. Here, "specificity" means that the antibody can bind to human CD44 gene product or fragment. Preferably, it refers to those antibodies that can bind to human CD44 gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules capable of binding and inhibiting human CD44 protein, as well as those antibodies that do not affect the function of human CD44 protein. Also included in the invention are antibodies that bind to modified or unmodified forms of the human CD44 gene product.

On the other hand, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, specific for human SRGN polypeptide. Here, "specificity" means that the antibody can bind to human SRGN gene product or fragment. Preferably, it refers to those antibodies that can bind to human SRGN gene products or fragments but do not recognize and bind to other irrelevant antigen molecules. Antibodies in the present invention include those molecules that can bind to and inhibit human SRGN protein, and those that do not affect the function of human SRGN protein. The invention also includes antibodies that bind to modified or unmodified forms of the human SRGN gene product.

In the present invention, the preferred antibody is a blocking antibody, that is, an antibody that blocks the binding or interaction between SRGN and CD44.

The present invention includes not only complete monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules ( Ladner et al., US Patent No. 4,946,778); or chimeric antibodies, such as antibodies that have the binding specificity of a murine antibody but retain portions of the antibody from humans.

Antibodies of the present invention can be prepared by various techniques known to those skilled in the art. For example, purified human CD44 gene product, or antigenic fragments thereof, can be administered to animals to induce polyclonal antibody production. Similarly, cells expressing human CD44 protein or antigenic fragments thereof can be used to immunize animals to produce antibodies. Antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur.J. Immunol. 6: 511, 1976; Kohler et al., Eur.J.Immunol . 6:292, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas , Elsevier, NY, 1981). The antibodies of the present invention include antibodies capable of blocking the function of human CD44 protein and antibodies that do not affect the function of human CD44 protein. Various antibodies of the present invention can be obtained by conventional immunization techniques using fragments or functional regions of human CD44 gene products. These fragments or functional regions can be prepared using recombinant methods or synthesized using a polypeptide synthesizer. Antibodies that bind to unmodified forms of the human CD44 gene product can be produced by immunizing animals with gene products produced in prokaryotic cells (e.g., E. coli); antibodies that bind to post-translationally modified forms (such as glycosylated or phosphorylated Proteins or polypeptides), which can be obtained by immunizing animals with gene products produced in eukaryotic cells (such as yeast or insect cells).

Similarly, the SRGN antibody of the present invention can also be prepared by various techniques known to those skilled in the art. Since SRGN proteins are secreted and enter the blood, these secreted SRGNs can be the target of serum detection.

Detection method

Utilizing that SRGN exists in the serum or blood of patients with giant cell tumor of bone and specifically exists in the spindle-shaped stromal cells of giant cell tumor of bone, and is related to the occurrence and progression of giant cell tumor of bone, the present invention also provides a method for detecting bone approach to giant cell tumors.

In a preferred embodiment of the present invention, the present invention provides a detection method based on SRGN protein, such as by detecting the level of SRGN in serum, such as by ELISA and time-resolved immunofluorescence (TRFIA). In addition, the expression level of SRGN protein in giant cell tumor of bone tissue or spindle stromal cells can also be detected.

In a preferred example of the present invention, the present invention provides a detection method based on SRGN mRNA, for example, by detecting the mRNA level of SRGN in giant cell tumor tissue or spindle-shaped stromal cells, such as by ddPCR or fluorescent PCR.

Detection methods, typing methods and detection kits

Based on the correlation between SRGN and giant cell tumor of bone, and the concentration of SRGN is different, the type of giant cell tumor of bone is different, so SRGN can be used as a diagnostic marker and typing marker for giant cell tumor of bone.

The invention provides a diagnostic method (including typing method) for quantitatively or qualitatively detecting human SRGN protein level or mRNA level. In the present invention, the detected human SRGN protein or mRNA level can be used for diagnosis (including auxiliary diagnosis) and type of giant cell tumor of bone.

A method for detecting whether there is SRGN protein in a sample is to use a specific antibody for SRGN protein to detect, which includes: contacting the sample with an SRGN protein specific antibody; observing whether an antibody complex is formed, and the formation of an antibody complex indicates that the sample SRGN protein is present in

In the present invention, preferred samples are blood, serum, giant cell tumor tissue of bone or spindle stromal cells thereof.

The SRGN protein or its polynucleotide can be used in the diagnosis and treatment of SRGN protein-related diseases. A part or all of the polynucleotides of the present invention can be fixed on microarrays or DNA chips as probes for analysis of differential expression of genes in tissues and gene diagnosis. The anti-SRGN antibody can be immobilized on the protein chip to detect the SRGN protein in the sample.

The present invention also provides a kit for detecting giant cell tumor of bone or typing giant cell tumor of bone, which contains detection reagents for detecting SRGN gene, mRNA, cDNA, or protein; and labels or instructions, the labels or The instructions indicate that the kit is used for detecting giant cell tumor of bone or typing giant cell tumor of bone;

Among them, the label or instructions indicate the following:

(i) When the serum SRGN concentration of the subject (subject) is ≥0.8ng/ml (preferably, ≥0.9ng/ml), it indicates that the subject has a high risk of developing giant cell tumor of bone, and/or the subject The test object is classified as SRGN-positive giant cell tumor of bone (or SRGN-type giant cell tumor of bone);

(ii) When the serum SRGN concentration of the subject (subject) is <0.8ng/ml (preferably, <0.6ng/ml), it indicates that the subject has a low risk of giant cell tumor of bone, and/or the subject The type of test object is SRGN-negative giant cell tumor of bone (or non-SRGN giant cell tumor of bone).

In a preferred embodiment, the present invention also provides a diagnostic kit for SRGN, including: SRGN mRNA diagnostic kit or SRGN enzyme-linked immunosorbent immunoassay (ELISA) detection kit.

Pharmaceutical composition and method of administration

The pharmaceutical composition containing the inhibitor of the present invention and its pharmaceutically acceptable inorganic or organic salt as the main active ingredient can be used for treating giant cell tumor of bone.

The pharmaceutical composition of the present invention comprises the inhibitor of the present invention (especially the CD44 inhibitor) or a pharmaceutically acceptable salt thereof within a safe and effective amount range and a pharmaceutically acceptable excipient or carrier. Wherein, "safe and effective dose" refers to: the amount of the compound is sufficient to obviously improve the condition without causing severe side effects. Usually, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-100 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.

"Pharmaceutically acceptable carrier" refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and low enough toxicity. "Compatibility" herein means that the components of the composition can be blended with the compound of the present invention and with each other without significantly reducing the efficacy of the compound. Examples of pharmaceutically acceptable carrier parts include cellulose and derivatives thereof (such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid , magnesium stearate), calcium sulfate, vegetable oil (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting Agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The administration method of the compound or pharmaceutical composition of the present invention is not particularly limited, and representative administration methods include (but not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is admixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or extenders, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow agents, such as paraffin; (f) Absorption accelerators such as quaternary ammonium compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared with coatings and shell materials, such as enteric coatings and others well known in the art. They may contain opacifying agents and, in such compositions, the release of the active compound or compounds may be in a certain part of the alimentary canal in a delayed manner. Examples of usable embedding components are polymeric substances and waxy substances. The active compounds can also be in microencapsulated form, if desired, with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, etc.

Besides such inert diluents, the compositions can also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols, and suitable mixtures thereof.

For antibody inhibitors, the dosage form of the pharmaceutical composition of the present invention is preferably an injection.

The CD44 inhibitors of the present invention can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, including SRGN inhibitors or other treatments.

When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage when administered, for a person with a body weight of 60kg, the daily The dosage is usually 1-2000 mg, preferably 5-100 mg. Of course, factors such as the route of administration and the health status of the patient should also be considered for the specific dosage, which are within the skill of skilled physicians.

The main advantages of the present invention include:

(1) The present invention finds for the first time that the serum SRGN protein level of patients with giant cell tumor of bone is significantly higher than that of the control group, which shows that SRGN is a new class of biological markers that can be used to detect giant cell tumor of bone and can be used to treat giant cell tumor of bone. Classification of cell tumors.

(2) The present invention finds for the first time that patients with giant cell tumor of bone can be classified according to the serum SRGN protein level, which provides a reference for formulating more targeted treatment strategies.

(3) The present invention finds for the first time that SRGN is highly specifically expressed in spindle-shaped stromal cells, and the highly-expressed SRGN in spindle-shaped stromal cells interacts with CD44 on monocytes, thereby promoting the differentiation of monocytes into multinucleated giant cells. Therefore, targeting CD44 can be used for targeted therapy of giant cell tumor of bone.

(4) The present invention finds for the first time that the use of CD44 inhibitors and/or SRGN inhibitors can be used to treat giant cell tumors of bone, especially when used in combination, it can treat giant cell tumors of bone more effectively and synergistically, providing a basis for the clinical treatment of giant cell tumors of bone Tumors provide new treatment options.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as Sambrook et al., molecular cloning: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's suggested conditions. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise specified, the materials and reagents used in the examples of the present invention are all commercially available products.

Material

GCTB-1, GCTB-2 and GCTB-3 cells are giant cell tumor cell lines derived from different patients.

U2OS cells: human osteosarcoma cells

MG63 cells: human osteosarcoma cells

general method

(1) Detection of patient serum samples: Immediately freeze and store patient serum samples at -80°C after obtaining them. Before the test, the serum samples were thawed on ice, centrifuged at 3000 rpm for 5 minutes at 4°C, the supernatant was aspirated and diluted according to the SRGN ELISA kit (purchased from USCN Company) and the SRGN concentration of the patient serum samples was detected.

Example 1

Inhibition of CD44 reduces osteoclast formation in vitro

Firstly, the conditioned medium of SRGN-overexpressed hFOB1.19 cells and SRGN recombinant protein were used to induce the formation of osteoclasts in vitro.

The results showed that SRGN could significantly promote the formation of osteoclasts no matter using mouse primary bone marrow cells or RAW264.7 cell line. However, when a neutralizing antibody to CD44 was further added, the formation of osteoclasts was significantly inhibited (Figure 1A-H).

In addition, the Cd44 gene was knocked out in RAW264.7 cells using CRISPR-Cas9 technology (Fig. 1I).

The results showed that after Cd44 was knocked out, neither the conditioned medium of SRGN-overexpressed hFOB1.19 cells nor the SRGN recombinant protein could continue to promote the formation of osteoclasts (J-M of Figure 1).

These results demonstrate that inhibition of CD44 reduces osteoclast formation in vitro.

Example 2

CD44 neutralizing antibody inhibits the formation of multinucleated giant cells and the growth of giant cell tumor of bone in mice

In this example, firstly, GCTB-19 giant cell tumor cells of bone were injected into immunodeficient NOD/SCID mice by way of tibial injection. One week after cell injection, mice were injected with CD44 neutralizing antibody or IgG control every two days. Since tumor cells are pre-labeled with luciferase, tumor cell growth can be tracked in vivo by injecting mice with a luciferase substrate for live imaging.

The results showed that in mice injected with CD44 neutralizing antibody, the growth of giant cell tumor of bone was significantly inhibited (Figure 2A-C), and compared with the control group, these mice injected with CD44 neutralizing antibody had bone damage The condition improved markedly, and the relative bone volume recovered to some extent (Fig. 2, A and D).

In addition, mouse leg bone sections were stained and analyzed for tartrate-resistant acid phosphatase (TRAP). The results showed that the number of TRAP+ multinucleated giant cells was significantly reduced after injection of CD44 neutralizing antibody (Fig. 2 E-F).

Example 3

Safety of CD44-targeted therapy

In this example, the safety of CD44-targeted therapy (CD44 neutralizing antibody) was further verified. The method is as follows: the immune-normal Balb/c mice were injected with CD44 neutralizing antibody, and the body weight and blood cells of the mice were detected.

The results showed that, compared with the control group, the body weight of mice injected with CD44 neutralizing antibody had no significant change (Fig. 2G); in addition to the decrease in the number of platelets in blood cells, the number of other red blood cells and white blood cells did not change significantly (Fig. 2H ).

These results suggest that CD44-targeted therapy has high safety, and CD44 can be used as a new target for clinical treatment of giant cell tumor of bone. Giant cell tumor of bone can be treated with CD44 inhibitors (such as small molecules, shRNA, antibodies, etc.).

discuss

The research of the present invention finds that targeted inhibition of CD44 can inhibit the progression of giant cell tumor of bone in vitro and in vivo, indicating that CD44 can be used as a target for clinical treatment of giant cell tumor of bone.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

references

1. Balke, M. et al. Giant cell tumor of bone: treatment and outcome of 214 cases. J Cancer Res Clin Oncol 134, 969-978 (2008).

2. Mendenhall, W.M., Zlotecki, R.A., Scarborough, M.T., Gibbs, C.P. & Mendenhall, N.P. Giant cell tumor of bone. American journal of clinical oncology 29, 96-99 (2006).

3. Alberghini, M. et al. Morphological and immunophenotypic features of primary and metastatic giant cell tumor of bone. Virchows Arch 456, 97-103 (2010).

4.Liao,T.S.et al.Recruitment of osteoclast precursors by stromal cell derived factor-1(SDF-1)in giant cell tumor of bone.Journal of orthopedic research: official publication of the Orthopedic Research Society 23,203- 209 (2005).

5. Goldring, S.R., Roelke, M.S., Petrison, K.K. & Bhan, A.K. Human giant cell tumors of bone identification and characterization of cell types. J ClinInvest 79, 483-491 (1987).

6. Wülling, M., Delling, G. & Kaiser, E. The origin of the neoplastic stromal cell in giant cell tumor of bone. Human pathology 34, 983-993 (2003).

7. Zheng, M.H. et al. The histogenesis of giant cell tumor of bone: a model of interaction between neoplastic cells and osteoclasts. Histology and histopathology 16, 297-307 (2001).

8. van der Heijden, L. et al. The clinical approach toward giant cell tumor of bone. Oncologist 19, 550-561 (2014).

9.Balke,M.et al.Bisphosphonate treatment of aggressive primary,recurrent and metastatic Giant Cell Tumor of Bone.BMC Cancer 10,462(2010).

10. Dufresne, A. et al. Giant-cell tumor of bone, anti-RANKL therapy. Bonekey Rep 1, 149 (2012).

11. Chawla, S. et al. Safety and efficacy of denosumab for adults and skeletally mature adolescents with giant cell tumor of bone: interim analysis of an open-label, parallel-group, phase 2 study. The Lancet Oncology 14,901- 908(2013).

12. Tralongo, P. et al. Safety of long-term administration of bisphosphonates in elderly cancer patients. Oncology 67, 112-116 (2004).

13. Chandran, M. & Zeng, W. Severe Oral Mucosal Ulceration Associated with Oral Bisphosphonate Use: The Importance of Imparting Proper Instructions on Medication Administration and Intake. Case Rep Med 2021, 6620489 (2021).

M.&Yip,G.W.Heparanase,hyaluronan,and CD44 in cancers：abreast carcinoma perspective.Cancer research 66,10233-10237(2006).14.

M. & Yip, GW Heparanase, hyaluronan, and CD44 in cancers: abreast carcinoma perspective. Cancer research 66, 10233-10237 (2006).

15. Prochazka, L., Tesarik, R. & Turanek, J. Regulation of alternative splicing of CD44 in cancer. Cellular signaling 26, 2234-2239 (2014).

16. Ponta, H., Sherman, L. & Herrlich, P.A. CD44: from adhesion molecules to signaling regulators. Nat Rev Mol Cell Biol 4, 33-45 (2003).

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Claims (9)

1. Use of a CD44 inhibitor for the manufacture of a medicament for the treatment of bone giant cell tumor, wherein the CD44 inhibitor is a specific antibody.

2. The use of claim 1, wherein the CD44 inhibitor is a blocking inhibitor that blocks the binding or interaction of SRGN and CD 44.

3. The use of claim 1, wherein said CD44 specific antibody is selected from the group consisting of: IM7, bivatuzumab, RG-7356, H90, PF-3475952, RO5429083.

4. The use of claim 1, wherein the medicament further comprises an SRGN inhibitor, or wherein the medicament is used in combination with an SRGN inhibitor, wherein the SRGN inhibitor is a specific antibody against SRGN.

5. Use of a pharmaceutical composition for the manufacture of a medicament for the treatment of bone giant cell tumor, wherein the pharmaceutical composition comprises (a) an antibody specific for CD44, (b) an antibody specific for SRGN and (c) a pharmaceutically acceptable carrier.

6. Use of a kit for the manufacture of a medicament for the treatment of a bone giant cell tumor, the kit comprising:

(i) A first pharmaceutical composition comprising a CD44 specific antibody and a pharmaceutically acceptable carrier;

(ii) A second pharmaceutical composition comprising an anti-SRGN-specific antibody and a pharmaceutically acceptable carrier.

7. The use according to claim 6, wherein the pharmaceutical composition is an oral or non-oral formulation.

8. Use of a kit for the manufacture of a medicament for the treatment of a bone giant cell tumor, said kit comprising:

(Z1) a diagnostic reagent for detecting or typing of a bone giant cell tumor, said diagnostic reagent being selected from the group consisting of: detection reagents for SRGN genes, mRNA, cDNA, or proteins; and

(Z2) an active ingredient for the treatment of bone giant cell tumor, wherein the active ingredient is selected from the group consisting of: a CD44 specific antibody, or a combination of a CD44 specific antibody and an anti-SRGN specific antibody.

9. A method of non-therapeutically inhibiting bone giant cell tumor cells in vitro comprising the steps of: culturing said bone giant cell tumor cells in a culture system comprising an effective amount of a CD44 specific antibody or a combination of a CD44 specific antibody and an anti-SRGN specific antibody, thereby inhibiting bone giant cell tumor cells.

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