CN113398270B

CN113398270B - Method for treating bone giant cell tumor

Info

Publication number: CN113398270B
Application number: CN202110819908.5A
Authority: CN
Inventors: 胡国宏; 贺云飞
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2021-07-20
Filing date: 2021-07-20
Publication date: 2023-04-25
Anticipated expiration: 2041-07-20
Also published as: CN113398270A

Abstract

The present invention relates to a method for treating bone giant cell tumor. In particular, the invention provides the use of a CD44 inhibitor for the manufacture of a medicament for the treatment of bone giant cell neoplasms. The experimental result of the invention shows that; SRGN can be used as a novel biological marker for predicting bone giant cell tumor and is used for diagnosing, typing and/or treating the bone giant cell tumor. In addition, in bone giant cell tumor tissue, spindle-shaped stromal cells secrete glycoproteins of the glycosaminoglycan (SRGN) and interact with CD44 receptors on monocytes, thereby promoting differentiation of monocytes into polynuclear giant cells. Thus, CD44 can be a target for clinical treatment of bone giant cell tumor.

Description

Method for treating bone giant cell tumor

Technical Field

The present invention relates to the field of oncology and diagnostics, in particular to a method for treating bone giant cell neoplasms.

Background

Bone giant cell tumor is a common primary bone tumor ¹ Typically occurs at the diaphyseal end of the long limb, including the distal femur, proximal femur and proximal tibia ² . Although bone giant cell tumor is generally considered to be a benign tumor and rarely metastasizes, it is locally invasive and often leads to severe bone destruction ^3,4 。

Currently, there are very limited means for diagnosis and treatment of bone giant cell tumor. Diagnosis of bone giant cell tumor is mainly by histopathological and radiological evaluation, as well as detection of H3.3G34W mutation.

Means for the treatment of bone giant cell tumor are very limited at present. In clinical treatment, surgery is the most common treatment, but 27% -65% of patients will relapse or metastasis after surgery ⁸ . In addition to surgery, bisphosphonates ⁹ And RANKL monoclonal antibody dirofySemai (Chinese character) ¹⁰ Also used for treating bone giant cell tumor. However, both drugs have a series of side effects ^11-13 And patients are at risk of relapse after withdrawal. Therefore, finding more therapeutic targets is helpful for the clinical treatment of bone giant cell tumor.

In view of the above, it is urgent to find new markers and therapeutic targets of bone giant cell tumor and develop targeted drugs. Thus, there is an urgent need in the art to develop targeted drugs that can be used to treat bone giant cell tumors.

Disclosure of Invention

The invention aims to provide a targeting drug for treating bone giant cell tumor and application thereof.

In a first aspect of the invention there is provided the use of a CD44 inhibitor for the manufacture of a medicament for the treatment of bone giant cell tumours.

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 embodiment, the nucleic acid drug comprises antisense RNA, microRNA.

In another preferred embodiment, the CD44 inhibitor is a blocking inhibitor that blocks the binding or interaction of SRGN and CD 44.

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

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

In another preferred embodiment, the CD44 inhibitor is selected from the group consisting of: hyaluronan-cisplatin conjugate, angstrom6, verbascoside, AMC.

In another preferred embodiment, the medicament further comprises an SRGN inhibitor, or the medicament 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 SRGN inhibitors include: a small molecule drug that inhibits SRGN, an antibody specific for SRGN, a nucleic acid drug, a gene editing drug, or a combination thereof. Preferably, the nucleic acid drug comprises antisense RNA and microRNA.

In another preferred embodiment, the SRGN inhibitor blocks binding or interaction of SRGN and CD 44.

In a second aspect of the invention, there is provided a pharmaceutical composition for use in the treatment of bone giant cell tumor, the pharmaceutical composition comprising (a) a CD44 inhibitor, (b) an SRGN inhibitor and (c) a pharmaceutically acceptable carrier.

In a third aspect of the invention, there is provided a kit comprising:

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

(ii) A second pharmaceutical composition comprising an SRGN inhibitor and a pharmaceutically acceptable carrier.

In another preferred embodiment, the first and second pharmaceutical compositions are different or independent from each other.

In another preferred embodiment, the pharmaceutical composition is an oral formulation or a non-oral formulation (e.g., an injection).

In a fourth aspect of the invention, there is provided a 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: CD44 inhibitors, SRGN inhibitors, or combinations thereof.

In a fifth aspect of the invention, there is provided 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 inhibitor and/or an SRGN inhibitor, thereby inhibiting bone giant cell tumor cells.

In another preferred embodiment, the monocytes of the bone giant cell tumor.

In another preferred embodiment, the inhibiting bone giant cell tumor cell comprises: inhibit the differentiation of monocytes into multinucleated megacytes.

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

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

In another preferred embodiment, the subject is a patient positive for SRGN.

In a seventh aspect of the invention, there is provided the use of an SRGN gene, mRNA, cDNA, or protein, or a detection reagent thereof, as (a) a marker for detecting a risk of a bone giant cell tumor or a bone giant cell tumor; (b) a marker for typing bone giant cell tumor; and/or (c) for the preparation of a diagnostic reagent or kit for detecting bone giant cell tumor or a risk of bone giant cell tumor, or for typing bone giant cell tumor.

In another preferred embodiment, the diagnostic reagent comprises an antibody, a primer, a probe, a sequencing library, a nucleic acid chip (e.g., a DNA chip), or a protein chip.

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

In another preferred embodiment, the SRGN gene, mRNA, cDNA, or protein is derived from a mammal, preferably from a human or non-human mammal (e.g., a primate), and more preferably from a patient diagnosed with a bone giant cell tumor.

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

In another preferred embodiment, the SRGN protein is human SRGN, preferably having the amino acid sequence set forth in SEQ ID No: 1.

In another preferred embodiment, the detection comprises serum detection, detection of a tissue or cell sample.

In another preferred embodiment, the tissue comprises isolated bone giant cell tumor tissue.

In another preferred embodiment, the cells comprise spindle stromal cells of a bone giant cell tumor.

In another preferred embodiment, the assay is a blood sample assay and/or a serum sample assay.

In another preferred embodiment, the detection reagent comprises an antibody specific for SRGN, a specific binding molecule for SRGN, a specific amplification primer, a probe, or a chip.

In another preferred embodiment, the SRGN protein or specific antibody or specific binding molecule thereof is conjugated or otherwise provided with 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 embodiment, the specific antibody for SRGN is a monoclonal antibody or a polyclonal antibody.

In an eighth aspect of the present invention, there is provided a diagnostic kit for detecting or typing of a bone giant cell tumor, the kit comprising a container containing a detection reagent for detecting an SRGN gene, mRNA, cDNA, or protein;

and a label or instructions that prescribe use of the kit for detecting a bone giant cell tumor or a bone giant cell tumor risk; or for typing bone giant cell tumors.

In another preferred embodiment, the detection includes a pre-judgment (prediction), a typing detection, and a prognosis detection.

In another preferred embodiment, the typing comprises classifying the bone giant cell tumor into an SRGN type bone giant cell tumor (i.e., an SRGN positive bone giant cell tumor) and a non-SRGN type bone giant cell tumor (i.e., an SRGN negative bone giant cell tumor).

In another preferred embodiment, the detection reagent for detecting SRGN gene, mRNA, cDNA, or protein comprises:

(a) Specific antibodies against SRGN proteins; and/or

(b) Specific primers for specific amplification of mRNA or cDNA of SRGN.

In another preferred embodiment, the label or description refers to the following:

(i) When the concentration of serum SRGN of a test object (subject) is more than or equal to 0.8ng/ml (preferably more than or equal to 0.9 ng/ml), the test object is indicated to have high risk of developing bone giant cell tumor, and/or the test object is typed as SRGN positive bone giant cell tumor (or SRGN type bone giant cell tumor); and

(ii) When the concentration of serum SRGN of the test subject is < 0.8ng/ml (preferably, < 0.6 ng/ml), the test subject is indicated to have a low risk of developing bone giant cell tumor and/or the test subject is typed as SRGN negative bone giant cell tumor (or non-SRGN type bone giant cell tumor).

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

a) Providing a test sample from a subject;

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

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

wherein a lower concentration of SRGN protein in the sample than the control, as compared to the control, indicates that the subject is likely to have or have a higher probability of developing bone giant cell tumor than in the normal population.

In another preferred embodiment, the test sample comprises a blood sample and/or a serum sample from a patient with a bone giant cell tumor.

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

In another preferred embodiment, the reference value is a cut-off value.

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

a) Providing a test sample from a subject;

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

c) A treatment regimen is determined based on the concentration of SRGN protein or mRNA in the sample.

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

In another preferred example, when the serum SRGN concentration of a patient with the bone giant cell tumor is more than or equal to 0.8ng/ml, a treatment scheme of the SRGN inhibitor, a treatment scheme of the CD44 inhibitor or a combined treatment scheme of the SRGN inhibitor and the CD44 inhibitor is selected; when the serum SRGN concentration of the patient with the bone giant cell tumor is less than 0.8ng/ml, the conventional bone giant cell tumor treatment scheme can be selected.

In another preferred embodiment, the SRGN inhibitor comprises: small molecule drugs for inhibiting SRGN, specific antibodies for resisting SRGN and nucleic acid drugs.

In another preferred embodiment, the CD44 inhibitor comprises: small molecule drugs for inhibiting CD44, specific antibodies for resisting CD44, and nucleic acid drugs.

In another preferred embodiment, the CD44 inhibitor blocks binding or interaction of SRGN and CD 44.

In another preferred embodiment, the conventional treatment regimen for bone giant cell tumor is selected from the group consisting of: bisphosphonates, RANKL monoclonal antibodies (e.g., diels semanteme), surgical treatment, radiation therapy, or combinations thereof.

In another preferred embodiment, the SRGN inhibitor is selected from the group consisting of:

ShSRGN-2 has the sequence: CCAGGACTTGAATCGTATCTT (SEQ ID No: 2) or

ShSRGN-5 has the sequence: ACATGGATTAGAAGAGGATTT (SEQ ID No: 3).

In an eleventh aspect of the present invention, there is provided a diagnostic apparatus comprising:

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

a bone giant cell tumor diagnosis-bone giant cell tumor typing module configured to: based on the concentration data of the SRGN protein and/or mRNA, performing diagnostic analysis on whether the subject suffers from the bone giant cell tumor, and/or performing typing on the bone giant cell tumor, and obtaining a diagnostic analysis result and/or a typing analysis result;

and the output module is configured to output the diagnosis analysis result and/or the typing analysis result.

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

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

Figure 1 shows that inhibition of CD44 can reduce osteoclast formation in vitro. Wherein, (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 (10 ng/mL) induces osteoclast differentiation. Osteoclast number statistics (a, C) and representative images (B, D). (E) To (H) shows that RAW264.7 cells were treated with hFOB1.19 conditioned medium (E and F) or SRGN recombinant proteins (G and H) and CD44 neutralizing antibody (10 ng/mL) to induce osteoclast differentiation. Osteoclast number statistics (E, G) and representative images (F, H). (I) shows that protein level verifies Cd44 knockout. (J) To (M) shows that treatment of Cd44 knockdown RAW264.7 cells with hFOB1.19 conditioned medium (J-K) or SRGN recombinant protein (L-M) induced osteoclast differentiation. Osteoclast number statistics (J, L) and representative images (K, M).

Figure 2 shows that CD44 neutralizing antibodies inhibit the formation of multinucleated giant cells and the growth of bone giant cell tumors in mice. Wherein, (a) to (D) show representative images (a) of in vivo imaging of mice, in the CD44 neutralizing antibody treatment group and the control group, in vitro imaging of the leg bones, and CT after GCTB-19 cell tibial injection, and the arrows indicate the bone injury sites. In vivo imaging fluorescence statistics (B) of mice, in vitro imaging fluorescence statistics (C) of the leg bones of mice, and relative bone volume statistics (D) of the leg bones of mice. (E) shows the staining of mice leg bone sections H & E and TRAP. (F) shows TRAP+ multi-core giant cell count statistics. (G) The body weight statistics of mice from the CD44 neutralizing antibody group and control group are shown. (H) Mouse blood cell statistics for the CD44 neutralizing antibody group and control group are shown. In the figure, WBCs are white blood cells and RBCs are red blood cells.

Detailed Description

The inventors have conducted extensive and intensive studies and have unexpectedly found for the first time that serum SRGN protein levels in patients with bone giant cell tumors are significantly higher than those in control groups, and therefore SRGN can be used as a novel biological marker for predicting bone giant cell tumors, and for diagnosis, typing and/or treatment of bone giant cell tumors. In addition, SRGN secreted by spindle stromal cells interacts with CD44 receptors on monocytes, thereby promoting differentiation of monocytes into multinucleated megacytes. Thus, CD44 can be a target for clinical treatment of bone giant cell tumors. On this basis, the present inventors have completed the present invention.

In particular, experiments have shown that SRGN is specifically highly expressed in spindle stromal cells of bone giant cell tumors and promotes the formation of multinucleated giant cells through interaction with its receptor CD44, thereby helping bone giant cell tumor growth in vivo. Thus, CD44 and/or SRGN may be a therapeutic target for bone giant cell tumor.

Terminology

Sample of

The term "sample" or "specimen" as used herein refers to a material that is specifically associated with a subject from which particular information about the subject can be determined, calculated, or inferred. The sample may be composed in whole or in part of biological material from the subject. The sample may also be a material that has been contacted with the subject in a manner that allows the test performed on the sample to provide information about the subject. The sample may also be a material that has been contacted with another material that is not the subject, but that enables the first material to be subsequently tested to determine information about the subject, e.g., the sample may be a cleaning solution for a probe or scalpel. The sample may be a source of biological material other than that contacting the subject, so long as one skilled in the art is still able to determine information about the subject from the sample.

Expression of

As used herein, the term "expression" includes the production of mRNA from a gene or gene portion, and includes the production of a protein encoded by RNA or gene portion, and also includes the presence of a detection substance associated with expression. For example, cDNA, binding of a binding ligand (e.g., an antibody) to a gene or other oligonucleotide, protein or protein fragment, and chromogenic portions of the binding ligand are included within the term "expressed". Thus, an increase in half-pel density in immunoblots, such as western blots, is also within the term "expression" based on biological molecules.

Reference value

As used herein, the term "reference value" refers to a value that is statistically relevant to a particular result when compared to the result of an analysis. In a preferred embodiment, the reference value is determined based on statistical analysis performed in comparison to studies of expression of SRGN protein with known clinical results. Some of these studies are shown in the examples section herein. However, the studies from the literature and the user experience of the methods disclosed herein can also be used to produce or adjust the reference value. Reference values may also be determined by considering conditions and results that are 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, preferably 0.8ng/ml and 0.9ng/ml (concentration of SRGN in serum of a patient with bone giant cell tumor).

Giant cell tumor of bone

As used herein, the term "bone giant cell tumor" refers to a tumor caused by bone giant cell tumor.

Bone giant cell tumor tissue has three main cell types, namely spindle-shaped stromal cells, polynuclear giant cells and monocytes. Polynuclear megacells are highly similar in morphology and function to osteoclasts, and are thought to be the primary cause of osteolysis by osteocarcinoma, whereas spindle stromal cells are the primary malignant component thereof ^5-7 . Spindle-shaped stromal cells not only can proliferate maliciously, but also can 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", "SRGN polypeptide" are used interchangeably and refer to a protein or polypeptide having an amino acid sequence of SRGN. They include SRGN proteins with or without an initiating methionine. In addition, the term also includes full length SRGN and fragments thereof. The SRGN proteins referred to herein include their complete amino acid sequences, their secreted proteins, their mutants, and functionally active fragments thereof.

Silk-glycylglycosylated (SRGN) is one of the earliest proteoglycans found. It is mainly composed of 17.6kDa core protein and glycosaminoglycan chains ¹⁷ 。

SRGN can be either stored intracellularly or secreted into the extracellular matrix. SRGN is mainly expressed in cells of the hematopoietic lineage, e.g., neutrophils, lymphocytes, macrophages, platelets, and the like ^18-22 . Several studies have reported that SRGN is involved in the development and progression of some tumors ^23-26 。

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

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

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

The genome of the human SRGN gene is 47279bp (NCBI GenBank accession No. 5552), and the mRNA sequence of the transcription product is 1217bp (NCBI GenBank accession No. NM-002727.4).

It is understood that substitution of nucleotides in the codon is acceptable when encoding the same amino acid. It is further understood that nucleotide substitutions are also acceptable when conservative amino acid substitutions are made by the nucleotide substitutions.

In the case of the resulting amino acid fragment of SRGN, a nucleic acid sequence encoding it can be constructed therefrom, and specific probes can be designed based on the nucleotide sequence. The full-length nucleotide sequence or a fragment thereof can be obtained by PCR amplification, recombinant methods or artificial synthesis. For PCR amplification, primers can be designed based on the disclosed SRGN nucleotide sequences, particularly open reading frame sequences, and amplified to obtain the relevant sequences using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is longer, it is often necessary to perform two or more PCR amplifications, and then splice the amplified fragments together in the correct order.

Once the relevant sequences are obtained, recombinant methods can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

Furthermore, the sequences concerned, in particular fragments of short length, can also be synthesized by artificial synthesis. In general, fragments of very long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

At present, it is entirely possible to obtain the DNA sequences encoding the proteins of the invention (or fragments, derivatives thereof) by chemical synthesis. The DNA sequence may then be introduced into a variety of existing DNA molecules (e.g., vectors) and cells known in the art.

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

(1) Transforming or transducing a suitable host cell with a polynucleotide (or variant) encoding a human SRGN polypeptide of the present invention, or with a recombinant expression vector comprising the polynucleotide;

(2) Host cells cultured in a suitable medium;

(3) Isolating and purifying the protein from the culture medium or the cells.

In the present invention, the SRGN polynucleotide sequence may be inserted into a recombinant expression vector. In general, any plasmid or vector can be used as long as it replicates and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene 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 sequences and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The DNA sequence may be operably linked to an appropriate promoter in an 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 comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance and Green Fluorescent Protein (GFP) for eukaryotic cell culture, or tetracycline or ampicillin resistance for E.coli.

Vectors comprising the appropriate DNA sequences as described above, as well as appropriate promoter or control sequences, may be used to transform appropriate host cells to enable expression of the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; 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 prokaryote such as E.coli, competent cells, which can take up DNA, can be obtained after the exponential growth phase and then treated with CaCl ₂ The process is carried out using procedures well known in the art. Another approach is to use MgCl ₂ . Transformation can also be performed by electroporation, if desired. When the host is eukaryotic, the following DNA transfection methods may be used: calcium phosphate co-precipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The transformant obtained can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culture is carried out under conditions suitable for the growth of the host cell. After the host cells have grown to the appropriate cell density, the selected promoters are induced by suitable means (e.g., temperature switching or chemical induction) and the cells are cultured for an additional period of time.

The recombinant polypeptide in the above method may be expressed in a cell, or on a cell membrane, or secreted outside the cell. If desired, the recombinant proteins can be isolated and purified by various separation methods using their physical, chemical and other properties. Such methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic sterilization, super-treatment, super-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques and combinations of these methods.

CD44 proteins and polynucleotides

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 the 11p13 chromosome and has a total of 20 exons, ten of which are regulated by alternative splicing, these exons being termed variable exons ¹⁵ . The CD44 gene is capable of expressing different CD44 protein subtypes through alternative splicing and post-translational modification. The molecular masses of these subtypes vary from 85kDa to 250kDa, and expression also has a certain tissue specificity.

The functions of CD44 protein are mainly: as receptors for interaction with soluble extracellular components or extracellular matrix; as a co-receptor with other receptors to activate downstream signaling pathways; modulating actin cytoskeleton ¹⁶ 。

The human CD44 protein is 361 amino acids in length (accession number P16070) and has the following amino acid sequence: MDKFWWHAAWGLCLVPLSLAQIDLNITCRFAGVFHVEKNGRYSISRTEAADLCKAFNSTLPTMAQMEKALSIGFETCRYGFIEGHVVIPRIHPNSICAANNTGVYILTSNTSQYDTYCFNASAPPEEDCTSVTDLPNAFDGPITITIVNRDGTRYVQKGEYRTNPEDIYPSNPTDDDVSSGSSSERSSTSGGYIFYTFSTVHPIPDEDSPWITDSTDRIPATRDQDTFHPSGGSHTTHGSESDGHSHGSQEGGANTTSGPIRTPQIPEWLIILASLLALALILAVCIAVNSRRRCGQKKKLVINSGNGAVEDRKPSGLNGEASKSQEMVHLVNKESSETPDQFMTADETRNLQNVDMKIGV (SEQ ID No: 4).

In the present invention, the terms "CD44 gene", "CD44 polynucleotide" are used interchangeably and refer to a nucleic acid sequence having the nucleotide sequence of CD 44.

The genome of the human CD44 gene is 93232bp (NCBI GenBank accession number 960) and the mRNA sequence of the transcription product is 4288bp (NCBI GenBank accession number NM-001001391.2).

Correlation of SRGN and bone giant cell tumor

The inventors have unexpectedly found that serum SRGN protein levels in patients with bone giant cell tumors are significantly higher than in control groups, and thus SRGN can be used as a novel class of biological markers for predicting bone giant cell tumors for diagnosis, typing and/or treatment of bone giant cell tumors.

Experiments show that SRGN is specifically highly expressed in spindle stromal cells of bone giant cell tumor and promotes the formation of multinucleated giant cells through interaction with its receptor CD44, thereby helping the growth of bone giant cell tumor in vivo. Therefore, SRGN can be used as a diagnosis index and a treatment target point of the bone giant cell tumor.

The experimental results of the present invention further suggest that different treatment regimens are selected based on the concentration levels of SRGN in serum of patients with bone giant cell neoplasms. For example, when the serum SRGN concentration of a patient with a bone giant cell tumor is more than or equal to 0.8ng/ml, a treatment regimen of an SRGN inhibitor, a treatment regimen of a CD44 inhibitor or a combination treatment regimen of an SRGN inhibitor and a CD44 inhibitor is selected. When the serum SRGN concentration of the patient with the bone giant cell tumor is less than 0.8ng/ml, the conventional bone giant cell tumor treatment scheme, such as bisphosphonate and RANKL monoclonal antibody (such as Dinozaki), surgical treatment and the like, can be selected. Of course, the SRGN inhibitor and/or CD44 inhibitor may also be administered in addition to the use of these conventional therapeutic agents or treatment regimens.

In addition, the inventors have specifically knocked down SRGN in bone giant cell tumor cells by way of shRNA interference (shRNA employed is ShSRGN-2:CCAGGACTTGAATCGTATCTT,SEQ ID No:2; and ShSRGN-5:ACATGGATTAGAAGAGGATTTSEQ ID No:3). The results show that the ability of the conditioned medium of bone giant cell tumor to induce differentiation of mouse primary bone marrow cells or RAW264.7 cells to osteoclasts in vitro is significantly inhibited after SRGN is knocked down (see also the chinese application filed by the applicant 2021, day 7, and 20, entitled "a diagnostic method of bone giant cell tumor").

Interaction of SRGN and CD44

The inventors have unexpectedly found for the first time that: in bone giant cell tumor tissue, spindle stromal cells secrete glypican (SRGN), and SRGN interacts with CD44 receptors on monocytes, thereby promoting differentiation of monocytes into multinucleated giant cells. This suggests that the interaction of SRGN with CD44 on monocytes may lead to the development and promotion of bone giant cell tumor progression, and thus, SRGN inhibitors and/or CD44 inhibitors may be useful in the clinical treatment of bone giant cell tumors.

CD44 inhibitors and SRGN inhibitors

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

As used herein, the terms "CD 44 inhibitors of the invention", "CD 44 targeted inhibitors of the invention" are used interchangeably and are intended to be inhibitors of CD44, including inhibitors that inhibit CD44 expression levels, activity, and inhibitors that inhibit CD44 interaction with SGRN.

As used herein, the terms "SRGN inhibitors of the present invention", "SRGN targeted inhibitors of the present invention" are used interchangeably and are used to inhibit inhibitors of SRGN, including inhibitors that inhibit the level of expression, activity, and inhibitors that inhibit the interaction of SRGN with CD 44.

In the present invention, the classes 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 embodiment, the inhibitors of the invention are blocking inhibitors (e.g., blocking antibodies) that block the binding or interaction of SRGN and CD 44.

Typically, the CD44 inhibitor includes (but is not limited to): small molecule drugs that inhibit CD44, specific antibodies to CD44, nucleic acid drugs, gene editing drugs, or combinations thereof.

Particularly preferred anti-CD 44 antibodies include (but are not limited to): IM7, bivatuzumab, RG-7356, H90, PF-3475952, RO5429083, or a combination thereof.

In another preferred embodiment, the CD44 inhibitor includes (but is not limited to): hyaluronan-cisplatin conjugate, angstrom6, verbascoside, AMC.

Typically, the SRGN inhibitors comprise: a small molecule drug that inhibits SRGN, an antibody specific for SRGN, a nucleic acid drug, a gene editing drug, or a combination thereof. Preferably, the nucleic acid drug comprises antisense RNA and microRNA.

Specific antibodies

In the present invention, the term "antibody of the present invention" includes "specific antibody against CD 44" and/or "specific antibody against SRGN".

In one aspect, the invention includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, specific for a human CD44 polypeptide. As used herein, "specific" refers to antibodies that bind to a human CD44 gene product or fragment. Preferably, those antibodies that bind to human CD44 gene products or fragments but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the human CD44 protein, as well as those that do not affect the function of the human CD44 protein. The invention also includes antibodies that bind to the modified or unmodified form of the human CD44 gene product.

In another aspect, the invention also includes polyclonal and monoclonal antibodies, particularly monoclonal antibodies, that are specific for human SRGN polypeptides. Here, "specific" refers to antibodies that bind to human SRGN gene products or fragments. Preferably, those antibodies that bind to human SRGN gene products or fragments but do not recognize and bind to other unrelated antigenic molecules. Antibodies of the invention include those molecules that bind to and inhibit the human SRGN protein, as well as those that do not affect the function of the human SRGN protein. The invention also includes antibodies that bind to the modified or unmodified form of the human SRGN gene product.

In the present invention, preferred antibodies are blocking antibodies, i.e., antibodies that block the binding or interaction of SRGN with CD 44.

The invention includes not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab) ₂ Fragments;antibody heavy chain; an antibody light chain; genetically engineered single chain Fv molecules (Ladner et al, U.S. Pat. No.4,946,778); or chimeric antibodies, such as antibodies having murine antibody binding specificity but retaining antibody portions derived from humans.

Antibodies of the invention may be prepared by various techniques known to those skilled in the art. For example, a purified human CD44 gene product, or an antigenic fragment thereof, may be administered to an animal to induce the production of polyclonal antibodies. Similarly, cells expressing the human CD44 protein or antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibodies of the invention may also be monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al, Nature256;495,1975; the composition of Kohler et al,Eur.J.Immunol.6:511,1976; the composition of Kohler et al,Eur.J.Immunol.6:292,1976; hammerling et al,In Monoclonal Antibodies and T Cell Hybridomaselsevier, n.y., 1981). Antibodies of the invention include antibodies that block human CD44 protein function and do not affect human CD44 protein function. The various antibodies of the invention may be obtained by conventional immunization techniques using fragments or functional regions of the human CD44 gene product. These fragments or functional regions may be prepared by recombinant methods or synthesized by a polypeptide synthesizer. Antibodies that bind to an unmodified form of the human CD44 gene product can be produced by immunizing an animal with the gene product produced in a prokaryotic cell (e.g., e.coli); antibodies (e.g., glycosylated or phosphorylated proteins or polypeptides) that bind to post-translational modifications can be obtained by immunizing an animal with a gene product produced in a eukaryotic cell (e.g., a yeast or insect cell).

Similarly, the SRGN antibodies of the present invention may 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 targets for serum detection.

Detection method

The present invention also provides methods for detecting bone giant cell tumors utilizing the presence of SRGN in serum or blood of a patient with bone giant cell tumor and specifically in spindle stromal cells of bone giant cell tumor and in association with the occurrence and progression of bone giant cell tumor.

In a preferred embodiment of the invention, the invention provides a method of detection based on SRGN proteins, for example by detecting SRGN levels in serum, for example by ELISA methods and time resolved immunofluorescence (TRFIA) methods. In addition, the expression level of SRGN protein in the bone giant tumor tissue or spindle stromal cells can be detected.

In a preferred embodiment of the invention, the invention provides a method of detection based on SRGN mRNA, for example by detecting the mRNA level of SRGN in a giant bone tumor tissue or spindle stromal cell, for example by ddPCR or fluorescent PCR, etc.

Detection method, typing method and detection kit

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

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 levels may be used to diagnose (including aid in diagnosis of) and to type bone giant cell tumors.

One method of detecting the presence or absence of an SRGN protein in a sample is by using an antibody specific for the SRGN protein, which comprises: contacting the sample with an SRGN protein specific antibody; whether an antibody complex is formed is observed, and the formation of the antibody complex indicates the presence of SRGN protein in the sample.

In the present invention, the preferred sample is blood, serum, bone giant cell tumor tissue or spindle stromal cells thereof.

The SRGN proteins or polynucleotides thereof may be used for diagnosis and treatment of SRGN protein-related disorders. A part or all of the polynucleotides of the present invention can be immobilized as probes on a microarray or DNA chip for analysis of differential expression of genes in tissues and diagnosis of genes. Antibodies against SRGN can be immobilized on a protein chip for detection of SRGN proteins in a sample.

The invention also provides a kit for detecting or typing the bone giant cell tumor, which contains a detection reagent for detecting SRGN genes, mRNA, cDNA or protein; and a label or instruction stating that the kit is for detecting or typing bone giant cell tumor;

wherein the label or the instruction notes the following:

(i) When the concentration of serum SRGN of a test object (subject) is more than or equal to 0.8ng/ml (preferably more than or equal to 0.9 ng/ml), the test object is indicated to have high risk of developing bone giant cell tumor, and/or the test object is typed as SRGN positive bone giant cell tumor (or SRGN type bone giant cell tumor);

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

Pharmaceutical compositions and methods of administration

Pharmaceutical compositions containing the inhibitors of the present invention and pharmaceutically acceptable inorganic or organic salts thereof as the main active ingredient are useful for the treatment of bone giant cell tumor.

The pharmaceutical compositions of the present invention comprise a safe and effective amount of an inhibitor of the present invention (particularly a CD44 inhibitor) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Wherein "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical compositions contain 1-2000mg of the compound of the invention per dose, more preferably 5-100mg of the compound of the invention per dose. Preferably, the "one dose" is a capsule or tablet.

"pharmaceutically acceptable carrier" means: one or more compatible solid or liquid filler or gel materials which are suitable for human use and must be of sufficient purity and sufficiently low toxicity. "compatible" as used herein means that the components of the composition are capable of blending with and between the compounds of the present invention without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties are cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifying agents (e.g., tween), wetting agents (e.g., sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are 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 the following ingredients: (a) Fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, e.g., glycerin; (d) Disintegrants, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) a slow solvent, such as paraffin; (f) an absorption accelerator, e.g., a quaternary amine compound; (g) Wetting agents, such as cetyl alcohol and glycerol monostearate; (h) an adsorbent, for example, kaolin; and (i) a lubricant, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills and granules can be prepared with coatings and shells, such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. The active compound may also be in the form of microcapsules with one or more of the above excipients, if desired.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of these substances and the like.

In addition to these inert diluents, the compositions can also include 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 as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar-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 nonaqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

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

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

When a pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dose at the time of administration is a pharmaceutically effective dose, and the daily dose is usually 1 to 2000mg, preferably 5 to 100mg, for a human having a body weight of 60 kg. Of course, the particular dosage should also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

The main advantages of the invention include:

(1) The invention discovers for the first time that the serum SRGN protein level of a patient with the bone giant cell tumor is obviously higher than that of a control group, which indicates that the SRGN is a novel biological marker which can be used for detecting the bone giant cell tumor and can be used for typing the bone giant cell tumor.

(2) The invention discovers for the first time that the invention can type the patient with the bone giant cell tumor according to the SRGN protein level of serum and provides a reference basis for formulating a more targeted treatment strategy.

(3) The invention discovers for the first time that the specific high-expression SRGN in the fusiform stroma cells and the SRGN which is highly expressed by the fusiform stroma cells acts on CD44 on monocytes, thereby promoting the differentiation of the monocytes into polynuclear giant cells. Thus, targeted therapy for bone giant cell tumor can be employed against the CD44 target.

(4) The invention discovers for the first time that the CD44 inhibitor and/or the SRGN inhibitor can be used for treating the giant cell tumor, especially the giant cell tumor can be effectively and synergistically treated when combined, and a new treatment scheme is provided for clinically treating the giant cell tumor.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

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

Material

The cells of GCTB-1, GCTB-2, GCTB-3 and the like are bone giant cell tumor cell lines from different patients.

U2OS cells: human osteosarcoma cell

MG63 cells: human osteosarcoma cell

General method

(1) Detection of patient serum samples: after obtaining a patient serum sample, the sample was immediately frozen at-80 ℃. Prior to testing, serum samples were thawed on ice, centrifuged at 3000 rpm for 5 minutes at 4℃and the supernatant was aspirated and diluted according to the SRGN ELISA kit (available from USCN) and the patient serum samples were tested for SRGN concentration.

Example 1

Inhibition of CD44 reduces osteoclast formation in vitro

First, the formation of osteoclasts was induced in vitro using a conditioned medium of hfob1.19 cells over-expressed by SRGN and SRGN recombinant protein.

The results indicate that SRGN significantly promoted the formation of osteoclasts, whether using mouse primary bone marrow cells or RAW264.7 cell line. However, when further neutralizing antibodies to CD44 were added, osteoclast formation was significantly inhibited (FIG. 1A-H).

In addition, cd44 gene was knocked out in RAW264.7 cells using CRISPR-Cas9 technology (fig. 1I).

The results indicate that neither the conditioned medium of SRGN overexpressed hfob1.19 cells nor the SRGN recombinant protein continued to promote osteoclast formation after Cd44 knockout (J-M of fig. 1).

These results demonstrate that osteoclast formation can be reduced after in vitro inhibition of CD 44.

Example 2

CD44 neutralizing antibodies inhibit the formation of multinucleated giant cells and the growth of bone giant cell tumors in mice

In this example, bone giant cell tumor cells GCTB-19 were first injected into immunodeficient NOD/SCID mice by tibial injection. After one week of cell injection, mice were injected with CD44 neutralizing antibodies or IgG controls at a frequency of once every two days. Since tumor cells are pre-labeled with luciferase, growth of tumor cells in vivo can be tracked by injecting a luciferase substrate into mice for in vivo imaging.

The results showed that bone giant cell tumor growth was significantly inhibited in mice injected with CD44 neutralizing antibodies (a-C of fig. 2), and that these mice injected with CD44 neutralizing antibodies had significantly reduced bone damage compared to the control group, and also had some recovery of relative bone volume (a and D of fig. 2).

In addition, mouse leg bone sections were stained and analyzed for tartrate-resistant acid phosphatase (TRAP). The results showed a significant decrease in the number of multinucleated giant cells of trap+ after injection of CD44 neutralizing antibodies (E-F of fig. 2).

Example 3

Safety of CD44 targeted therapies

In this example, the safety of CD44 targeted therapies (CD 44 neutralizing antibodies) was further validated. The method comprises the following steps: immunized Balb/c mice were injected with CD44 neutralizing antibodies and the body weight of the mice, as well as blood cells, were examined.

The results showed that there was no significant change in body weight of mice injected with CD44 neutralizing antibodies compared to the control group (fig. 2G); there was no significant change in the number of red and white blood cells except for a decrease in the number of platelets in the blood cells (fig. 2H).

These results suggest that CD44 targeted therapy has high safety, and CD44 can be a new target for clinical treatment of bone giant cell tumor. Bone giant cell tumor can be treated by using CD44 inhibitor (such as small molecule, shRNA, antibody, etc.).

Discussion of the invention

The research of the invention discovers that the targeted inhibition of CD44 can inhibit the development of the bone giant cell tumor in vitro and in vivo, and shows that the CD44 can be used as a target for clinically treating the bone giant cell tumor.

All documents mentioned in this application are incorporated by reference as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the claims appended hereto.

Reference to the literature

1.Balke,M.et al.Giant cell tumor of bone：treatment and outcome of214cases.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 tumour 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 orthopaedic research：official publication of the Orthopaedic 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 Clin Invest 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 tumour 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 Tumour 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 tumour of bone：interim analysis of an open-label,parallel-group,phase 2study.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).

14.

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

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

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

Sequence listing

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Claims

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:

(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.