CN111205370B

CN111205370B - Application of anti-PTN antibody in inhibiting leukemia stem cells and treating chronic granulocytic leukemia

Info

Publication number: CN111205370B
Application number: CN202010329134.3A
Authority: CN
Inventors: 彭菲; 顾超
Original assignee: Bison Holdings Group Ltd
Current assignee: Bishen Holdings Ltd
Priority date: 2020-04-23
Filing date: 2020-04-23
Publication date: 2021-01-01
Anticipated expiration: 2040-04-23
Also published as: CN111205370A

Abstract

The invention relates to an application of an anti-PTN antibody in inhibiting leukemia stem cells and treating chronic granulocytic leukemia, wherein the anti-PTN antibody can be combined with PAI-1 protein with higher affinity, can inhibit implantation of CML stem cells, can inhibit growth of CML cells, can be used for preventing or treating chronic granulocytic leukemia, and has wide application prospect.

Description

Application of anti-PTN antibody in inhibiting leukemia stem cells and treating chronic granulocytic leukemia

Technical Field

The invention relates to the field of biological medicine, in particular to application of an anti-PTN antibody in inhibiting leukemia stem cells and treating chronic granulocytic leukemia.

Background

Chronic Myelocytic Leukemia (CML) is a malignant tumor formed by abnormal proliferation of bone marrow hematopoietic stem cells. The disease is slow, the spleen is enlarged, peripheral blood granulocytes are obviously increased, translocation of chromosome 9 and chromosome 22 forms Philadelphia chromosome (Philadelphia chromosome, Ph), and a BCR-ABL1 fusion gene is a special biological marker, and finally Leukemia Stem Cells (LSCs) are formed [1-3 ]. The BCR-ABL1 oncogenic protein has the tyrosine kinase activity which is continuously activated, can induce the uncontrolled proliferation and apoptosis inhibition of leukemia cells, and causes the occurrence and development of chronic granulocytic leukemia [4 ].

CML is currently treated primarily by Tyrosine Kinase Inhibitors (TKIs). However, once treatment with TKIs is discontinued, the disease often recurs, suggesting that TKIs only inhibit but fail to cure the disease [5-7 ]. Subsequent studies demonstrated that recurrence after CML withdrawal is associated with the persistence of leukemic stem cells in the patient. Various studies have demonstrated that TKIs only kill leukemic cells in a dividing proliferative state and do not clear the SLL stem cells in a quiescent state [8-11], which is also a key cause of CML recurrence. Therefore, the elimination of leukemic stem cells becomes the key point for complete cure of chronic myelogenous leukemia and is also the hot spot in current chronic myelogenous leukemia research [12 ].

Studies have shown that Pleiotrophin (PTN) plays an important role in the growth and survival of chronic myelogenous leukemia stem cells and in the development of chronic myelogenous leukemia disease [13 ]. Pleiotropic growth factor is a protein isolated and purified by Bohlen et al from adult bovine brain in the 80's 20 th century, and is called heparin-binding growth factor because it can bind heparin. And then renamed pleiotrophin for its multiple functions. Experimental studies show that PTN is related to functions such as cell proliferation and migration [14], tumor growth [15], cell differentiation [16, 17] and the like. In addition, studies have shown that PTN expression is upregulated and secretion is increased in mouse CML stem cells [18 ]; PTN expression was upregulated in mouse CML stem cells after TKIs treatment and PTN induced CML stem cell growth and survival in a manner dependent on c-Jun and UPR gene activation, suggesting that PTN gene expression may be responsible for CML stem cell survival after TKIs treatment [13 ]. Therefore, the aims of eliminating CML stem cells and completely curing chronic granulocytic leukemia can be achieved by targeting PTN.

To date, PTN antibodies have been mentioned in US20040234519, but on the one hand their binding capacity to PTN is not very ideal and on the other hand it has not been used for the treatment of chronic granulocytic leukemia.

Disclosure of Invention

In order to overcome the above-mentioned drawbacks of the prior art, the object of the present invention is to provide a new anti-PTN antibody with higher PTN binding capacity, offering a new possibility for curing chronic myeloid leukemia.

The invention provides the following technical scheme:

a PTN antibody consisting of a heavy chain and a light chain comprising a variable region and a constant region, respectively, wherein the heavy chain variable region comprises 3 complementarity determining regions designated VH-CDR1, VH-CDR2, VH-CDR3, respectively; similarly, the light chain variable region also contains 3 complementarity determining regions, designated VL-CDR1, VL-VDR2, VL-CDR 3. The heavy chain of the PTN antibody comprises a VH-CDR1 amino acid sequence shown as SEQ ID NO. 1, a VH-CDR2 amino acid sequence shown as SEQ ID NO. 2, and a VH-CDR3 amino acid sequence shown as SEQ ID NO. 3; the light chain of the PTN antibody comprises a VL-CDR1 amino acid sequence shown as SEQ ID NO. 4, and a VL-CDR2 amino acid sequence shown as SEQ ID NO. 5, and a VL-CDR3 amino acid sequence shown as SEQ ID NO. 6.

The PTN antibody of the present invention comprises a heavy chain variable region shown by SEQ ID NO. 7 and a light chain variable region shown by SEQ ID NO. 8.

The PTN antibody of the invention comprises both a heavy chain variable region and a light chain variable region comprising framework regions, said antibody light chain variable region further comprising light chain framework regions of human kappa, lambda chains or variants thereof. The antibody heavy chain variable region further comprises heavy chain framework regions of human IgG1, IgG2a, IgG2b, or IgG3, or IgG4, or variants thereof.

The PTN antibody of the invention comprises a constant region, and the constant region is humanized, comprising a heavy chain constant region selected from IgG1, IgG2, IgG3 or IgG4 and comprising a light chain constant region selected from a kappa or Lambda subtype.

In some embodiments, the PTN antibody binds to PTN with an EC50 value of 37.2 ± 5.3 ng/ml.

In some embodiments, the PTN antibody is capable of inhibiting the engraftment of CML stem cells.

In some embodiments, the PTN antibody is capable of inhibiting CML cell growth.

The invention provides a method for treating chronic granulocytic leukemia, which is characterized by comprising the following steps: the anti-PTN antibodies of the present invention are used or a combination of TKI inhibitors with the anti-PTN antibodies of the present invention.

The PTN antibody can be applied to the preparation of medicaments for treating chronic granulocytic leukemia, and is preferably combined with TKI inhibitors.

The term "antibody" as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, and encompasses full-length antibodies (e.g., IgG1 or IgG4 antibodies), various functional fragments thereof (e.g., may comprise only antigen binding portions, such as Fab, Fab ', F (ab') 2). Examples of antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, Fab ', F (ab') 2 fragments, and the like.

The term "monoclonal antibody" refers to an antibody derived from a single clonal cell line, not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained by recombination using, for example, hybridoma technology, recombinant technology, phage display technology, synthetic techniques (e.g., CDR-grafting), or other known techniques. A Fab fragment "consists of one light and one heavy chain of CH1 and the variable region. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab ' fragment" contains the VH and CH1 domains of one light and one heavy chain and the constant region portion between the CH1 and CH2 domains, whereby an interchain disulfide bond can be formed between the two heavy chains of the two Fab ' fragments to form the F (ab ') 2 molecule. An "F (ab') 2 fragment" contains the VH and CH1 domains of two light and two heavy chains and a constant region portion between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. Thus, a F (ab ') 2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

The term "hypervariable region" or "CDR region" or "complementarity determining region" as used herein refers to the amino acid residues of an antibody which are responsible for antigen binding. CDR region sequences can be defined by Kabat, Chothia method definition or the field of any known CDR region sequence determination method and identification of the variable region within amino acid residues. The methods used in the present invention may utilize or be defined according to CDRs defined by any of these methods, including but not limited to any of the Kabat definitions, Chothia definitions. In particular, the CDR sequences provided herein are according to the Kabat definition.

The term "humanized antibody" refers to antibodies having a framework, hinge and constant regions of human origin that are identical or substantially identical (substantially human) to the framework and constant regions from human genomic sequences. Fully human framework, hinge and constant regions are those of human germline sequences and sequences with naturally occurring somatic mutations. Engineered antibodies of human origin may comprise a framework, hinge or constant region from a fully human framework, hinge or constant region and comprise one or more amino acid substitutions, deletions or additions therein. In general, engineered antibodies of human origin are preferably substantially non-immunogenic in nature. In a preferred embodiment of the present invention, the antibody light chain variable region of said PTN humanized antibody further comprises a light chain FR region of a human kappa, lambda chain or a variant thereof. The antibody heavy chain variable region of the PTN humanized antibody further comprises a heavy chain FR region of human IgG1, IgG2a, IgG2b or IgG3 or IgG4 or variants thereof. The constant region of the humanized antibody may be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising an IgG1 heavy chain constant region of human origin. The humanized antibody further comprises a light chain constant region selected from the group consisting of kappa and Lambda subtypes.

Advantageous effects

The PTN antibodies of the invention have the following properties: 1) capable of binding PTN proteins with higher affinity; 2) can inhibit the engraftment of CML stem cells; 3) can inhibit CML cell growth; 4) can be used for preventing or treating chronic granulocytic leukemia.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

FIG. 1 shows the results of the engraftment experiments for the inhibition of CML stem cells by the h5D7-2 antibody.

FIG. 2a shows that the h5D7-2 antibody inhibits CML cell growth; FIG. 2b shows that the combination of the h5D7-2 antibody and IM inhibited CML cell growth, and the effect was superior to that of IM alone treatment.

Detailed Description

The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.

Example 1 preparation of PTN antigens

The PTN cDNA was inserted into pET-43.1b (+) vector to construct a PTN prokaryotic expression plasmid, which was designated as pET-43.1b (+) -PTN. The above plasmid was transformed into E.coli competent BL21 (DE 3), and the competence was added to 2mL LB medium containing ampicillin resistance and cultured overnight, and the next day, 20 mL LB medium containing ampicillin resistance was inoculated with an inoculum size of 1%, followed by shaking culture at 37 ℃ for 3 hours, and then IPTG was added to a final concentration of 0.5 mmol/L, followed by induction culture at 30 ℃ for 6 hours. And centrifugally collecting thalli, adding lysate, carrying out ultrasonic crushing, centrifugally collecting supernatant, further purifying by a nickel column to obtain soluble recombinant antigen protein, carrying out enzyme digestion by enterokinase, removing the tag protein carried on the plasmid, and finally obtaining the PTN recombinant protein. Because PTN protein genes are highly conserved, PTN proteins are cross-linked to Keyhole Limpet Hemocyanin (KLH) to increase immunogenicity. Adopting bifunctional coupling agent glutaraldehyde for coupling, dissolving the recombinant expression PTN protein and KLH (purchased from Sigma) in PBS, uniformly mixing in a coupling bottle, slowly dropwise adding 0.25% glutaraldehyde solution into the mixed solution, reacting for 4h at 4 ℃, and then placing the reaction solution at 4 ℃ for dialyzing the PBS to finally obtain the chemically coupled PTN-KLH.

Example 2 anti-PTN antibody preparation

The PTN-KLH antigen prepared in example 1 was injected intraperitoneally into female Balb/c mice together with Freund's complete adjuvant which elicited an immune response, and the primary immunization was performed, followed by injecting the same amount of antigen intraperitoneally into the female Balb/c mice every 2 weeks, and the immunization was continued. And finally, carrying out final immunization 3 days before the fusion with myeloma cells, mixing and emulsifying the PTN-KLH antigen and an immunologic adjuvant, and carrying out intraperitoneal injection for the final immunization. Spleen cells of immunized Balb/c mice were fused with a myeloma Sp2/0 cell line, and the fused cells were diluted to an appropriate concentration in Iscove's medium (0.1 mM hypoxanthine, 0.4. mu.M aminopterin and 16. mu.M thymidine) containing 10% serum, and then cultured in a 96-well plate. After 10 days, cell supernatants were removed and primary cultures showing a positive reaction with PTN in the supernatants were examined by high throughput ELISA. And diluting the hybridoma cells in the hole for subcloning, and screening by an ELISA method to finally obtain the positive hybridoma cell strain 5D 7-2.

Example 2 Gene cloning and humanization of anti-PTN antibodies

The cultured hybridoma cell line 5D7-2 was lysed with TRNzol-A +, and then placed in a centrifuge tube, 200. mu.l of chloroform was added to each ml of TRNzol-A +, and the mixture was vortexed for 15 seconds and left for 3 minutes. 13000 rpm, 4 ℃ for 10 minutes, Trizol-A + cell solution is divided into three layers: transferring the water phase dissolved with the RNA into a centrifuge tube, adding isopropanol with the same volume into the water phase, uniformly mixing, and standing at room temperature for 25 minutes. 13000 rpm, 4 ℃ for 10 minutes, discard the waste liquid to get the bottom RNA precipitation. After washing the RNA pellet twice with 75% ethanol, the RNA was dissolved in PEDC water and stored at-80 ℃. Preparing cDNA encoding antibody gene by using a reverse transcription kit (purchased from Beijing Quanzijin biotechnology limited); using cDNA as template, amplifying DNA product containing antibody heavy chain variable region and light chain variable region by PCR, separating and recovering purified target fragment containing antibody heavy chain variable region and light chain variable region by agarose gel electrophoresis. Cloning the DNA into pGEM-T vector, screening positive clone sequencing, and further obtaining the amino acid sequence corresponding to the variable region gene according to the sequencing result.

According to the variable region sequence of the antibody secreted by the hybridoma cell strain 5D7-2, the humanized modification is carried out by adopting a CDR grafting antibody humanized modification method, and the CDR region sequence is defined by adopting a kabat method. Firstly, comparing a VH region and a VK region of a hybridoma cell strain 5D7-2 with existing human antibody sequences in an NCBI database by using an online sequence comparison method for Ig sequences provided in NCBI, and selecting a human embryonic line framework sequence; and then, building a mouse source antibody variable region three-dimensional structure by using a SwissModel, determining key amino acids for maintaining the binding affinity and the space framework by calculating electrostatic force, van der Waals force, hydrophilicity and hydrophobicity and entropy value, and grafting the key amino acids back to the selected human embryonic system antibody framework. Cloning the obtained humanized and modified antibody variable region gene into a eukaryotic expression vector pCMV-163 containing a human IgG constant region gene to construct a full antibody expression vector. Each component of the eukaryotic expression vector pCMV-163 is a component known in the art, and is recombined in the order shown.

Finally, the humanized and transformed antibody is named as h5D7-2, and the sequencing result shows that the amino acid sequences of VH-CDR1-3 of the antibody h5D7-2 are respectively shown as SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3; the amino acid sequence of the antibody VH is shown as SEQ ID NO. 7; the amino acid sequences of VL-CDR1-3 of h5D7-2 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6; the amino acid sequence of antibody VL is as shown in SEQ ID NO: shown in fig. 8.

Example 3 determination of the binding Capacity of anti-PTN antibodies to PTN

The antibody plasmid obtained in example 2 was transfected into CHO-K1 cells to select high expression clones, which were then cultured in serum-free medium and humanized antibody h5D7-2 was isolated and purified using Protein A affinity column. The purified antibody was dialyzed against PBS to obtain a highly pure humanized antibody h5D7-2 of PTN. The sequence of anti-PTN antibody 3B10 (see fig. 1B, 1C of the patent specification for its light and heavy chain sequences) obtained by US20040234519 patent and antibody 3B10 obtained by the method described above were used as control antibodies. The binding affinity of the humanized anti-PTN antibody h5D7-2, the control antibody 3B10 obtained above to the PTN protein at 50% effective concentration (EC50) was evaluated by ELISA assay. Recombinant human PTN protein was diluted 1000ng/mL with PBS, added to 96 plates at 100. mu.L per well, and coated overnight at 4 ℃. The solid phase was removed by reverse centrifugation, and 200. mu.L of a blocking agent was added to 1 well, and the mixture was allowed to stand at room temperature for 30 minutes. The above PTN-immobilized plate was washed with TBS, and then PTN antibody was added in an amount of 100. mu.l per well. PTN antibody h5D7-2 and control antibody 3B10 were diluted in culture medium to a concentration gradient of 1000ng/ml, 250ng/ml, 62.5ng/ml, 15.63ng/ml, 3.91ng/ml, 0.98ng/ml, incubated at 4 ℃ for 2h, the medium aspirated, glutaraldehyde added and incubated at room temperature for 10min, washed 3 times with PBS, 100ul of anti-human IgG antibody added and incubated at room temperature for 2 h. Washing with PBS for 3 times, adding 50 μ l of color development solution, incubating at room temperature in dark place for 15min, adding stop solution to stop color development reaction, detecting absorbance value of sample at 450nm wavelength with microplate reader, and correcting with absorbance value at 620nm wavelength. The data were processed to finally determine the EC50 value for each sample, the results are shown in table one. The results indicate that the humanized anti-PTN antibody h5D7-2 exhibits greater binding capacity than the control antibody 3B 10.

TABLE I determination of the binding Capacity of anti-PTN antibodies to PTN

Name (R)	EC50（ng/ml）
		h5D7-2	37.2±5.3
Control antibody 3B10	93.4±11.2

Example 4 anti-PTN antibodies inhibit CML Stem cell function

Taking a CML patient peripheral blood sample, diluting cells to a proper concentration by PBS (containing 2mM EDTA, Invitrogen), slowly dripping the diluted cells into a 15ml centrifuge tube containing a Ficoll solution (GE Healthcare), centrifuging for 30min at 20 ℃ at a volume ratio of Ficoll to the diluted peripheral blood of 1:1, 400g, sucking a mononuclear cell layer of a junction of serum and Ficoll by a sterile plastic dropper, transferring the mononuclear cell layer into the 15ml centrifuge tube, adding 3 times of volume of PBS solution for elution, centrifuging for 10min at 400g, removing supernatant, and precipitating to obtain mononuclear cells. Monocyte suspension was prepared using human CD34+ isolation kit (Miltenyi Biotec), the column was fixed in a MACS magnetic field, the cell suspension was passed slowly through the column, and the eluted fraction was CD34^-A cell. Removing magnetic field from the column, adding buffer solution to elute CD34⁺A cell. CD34+ cells isolated from bone marrow of a CML patient were co-cultured with anti-PTN antibody h5D7-2, control antibody anti-PTN antibody 3B10, and IgG (not having the ability to bind PTN) antibody, respectively, and after 72h the cells were harvested and injected into 300-cGy irradiated NOD/SCID IL-2-receptor gamma-Null (NSG) mice. CML Stem cell namely CD34⁺After 16 weeks of transplantation, mice were sacrificed, and bone marrow cells of the mice were taken and analyzed for human CD45 in the bone marrow cells by flow cytometry⁺The content of cells. The results are shown in FIG. 1:CD45 in the anti-PTN antibody h5D7-2 and anti-PTN antibody 3B 10-treated groups⁺The cell content was significantly lower than the IgG treated group and the anti-PTN antibody h5D7-2 was superior to the anti-PTN antibody 3B 10. The anti-PTN antibody treatment proves that the implantation of the CML stem cells can be obviously inhibited.

Example 5 anti-PTN antibodies inhibit CML cell growth

Culturing chronic myelogenous leukemia cell line K562, and injecting it into tibia bone of mouse

The medullary cavity. Injection of 5X10 per mouse⁷And K562 cells. Mice injected with K562 cells were randomly divided into 3 groups, treated with anti-PTN antibody h5D7-2, control antibody anti-PTN antibody 3B10 or IgG (not having the ability to bind PTN) antibody, respectively, and injected intraperitoneally every two days at 2mg/kg for 2 weeks. After two weeks, the solid tumor at the tibia was removed and its size was measured. As shown in FIG. 2a, the CML graft tumor size was significantly reduced and the anti-PTN antibody h5D7-2 was superior to the anti-PTN antibody 3B10 in the CML graft tumor model after treatment with the anti-PTN antibody h5D7-2 and the anti-PTN antibody 3B 10. The effect of the PTN antibody in combination with IM (imatinib) was further investigated. After the chronic myelocytic leukemia cell strain K562 and IM are co-cultured for 72h, the cells are collected and injected into the tibial medullary cavity of the mouse. Injection of 5X10 per mouse⁷And K562 cells. Mice injected with K562 cells were randomly divided into 3 groups, treated with anti-PTN antibody h5D7-2, control anti-PTN antibody 3B10 or IgG antibody, and injected intraperitoneally every two days at 2mg/kg for a total of 2 weeks. After two weeks, the solid tumor at the tibia was removed and its size was measured. The results are shown in FIG. 2B, where the CML transplantable tumor size was significantly reduced and the anti-PTN antibody h5D7-2 was superior to the anti-PTN antibody 3B10 after co-treatment with IM in the CML transplantable tumor model. The results show that the anti-PTN antibody can inhibit the growth of CML cells, and the effect of the anti-PTN antibody is better when the anti-PTN antibody is combined with IM.

Reference to the literature

[1]JF A. - Chronic myeloid leukaemia [J]. Lancet, 2015, 385(9976): 1447-59.

[2]RUBIN H. Chronic neutrophilic leukemia [J]. Ann Intern Med, 1966, 65(1): 93-100.

[3]BENNETT J M, CATOVSKY D, DANIEL M T, et al. The chronic myeloid leukaemias: guidelines for distinguishing chronic granulocytic, atypical chronic myeloid, and chronic myelomonocytic leukaemia. Proposals by the French-American-British Cooperative Leukaemia Group [J]. Br J Haematol, 1994, 87(4): 746-54.

[4]JM G, JV M. - Chronic myeloid leukemia--advances in biology and new approaches to treatment [J]. N Engl J Med, 2003, 349(15): 1451-64.

[5]FX M, D R, J G, et al. - Discontinuation of imatinib in patients with chronic myeloid leukaemia who have [J]. Lancet Oncol, 2010, 11(11): 1029-35.

[6]N T, B V D H, JJ C, et al. - Imatinib discontinuation in chronic phase myeloid leukaemia patients in sustained [J]. Eur J Cancer, 2013, 49(15): 3242-6.

[7]DM R, S B, JF S, et al. - Safety and efficacy of imatinib cessation for CML patients with stable [J]. Blood, 2013, 122(4): 515-22.

[8]LA C, CH J. - Selective elimination of leukemia stem cells: hitting a moving target [J]. Cancer Lett, 2013, 338(1): 15-22.

[9]S S, J R, A H, et al. - The concept of treatment-free remission in chronic myeloid leukemia [J]. Leukemia, 2016, 30(8): 1638-47.

[10]JC C, AG T. - Chronic myeloid leukemia stem cells in the era of targeted therapies: resistance [J]. Oncotarget, 2011, 2(9): 713-27.

[11]JC C, ML B, N S, et al. - Leukemic stem cell persistence in chronic myeloid leukemia patients in deep [J]. Oncotarget, 2016, 7(23): 35293-301.

[12]H Z, R X. - Leukemia stem cells: the root of chronic myeloid leukemia [J]. Protein Cell, 2015, 6(6): 403-12.

[13]HIMBURG H A, ROOS M, FANG T, et al. Chronic myeloid leukemia stem cells require cell-autonomous pleiotrophin signaling [J]. J Clin Invest, 2020, 130(1): 315-28.

[14]ELAHOUEL R, BLANC C, CARPENTIER G, et al. Pleiotrophin exerts its migration and invasion effect through the neuropilin-1 pathway [J]. Neoplasia, 2015, 17(8): 613-24.

[15]ZHA L, HE L, XIE W, et al. Therapeutic silence of pleiotrophin by targeted delivery of siRNA and its effect on the inhibition of tumor growth and metastasis [J]. PLoS One, 2017, 12(5): e0177964.

[16]JIN K, MAO X O, BATTEUR S, et al. Induction of neuronal markers in bone marrow cells: differential effects of growth factors and patterns of intracellular expression [J]. Exp Neurol, 2003, 184(1): 78-89.

[17]FURUTA M, SHIRAISHI T, OKAMOTO H, et al. Identification of pleiotrophin in conditioned medium secreted from neural stem cells by SELDI-TOF and SELDI-tandem mass spectrometry [J]. Brain Res Dev Brain Res, 2004, 152(2): 189-97.

[18]HIMBURG H A, TERMINI C M, SCHLUSSEL L, et al. Distinct Bone Marrow Sources of Pleiotrophin Control Hematopoietic Stem Cell Maintenance and Regeneration [J]. Cell Stem Cell, 2018, 23(3): 370-81 e5.

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Claims

1. An anti-PTN antibody, consisting of a heavy chain and a light chain, the heavy chain and the light chain respectively comprising a variable region and a constant region, wherein the sequences of VH-CDR1-3 in the variable region of the heavy chain are respectively shown as SEQ ID NO 1-3; the sequences of VL-CDR1-3 in the variable region of the light chain are shown in SEQ ID NOS: 4-6, respectively.

2. anti-PTN antibody according to claim 1, characterized in that: the heavy chain variable region sequence is shown as SEQ ID No. 7; the light chain variable region sequence is shown in SEQ ID No. 8.

3. anti-PTN antibody according to any one of claims 1-2, characterized in that: the antibody is a full-length antibody.

4. anti-PTN antibody according to any one of claims 1-2, characterized in that: the antibody further comprises a heavy chain constant region selected from IgG1, IgG2, IgG3, or IgG4 and a light chain constant region selected from a kappa or Lambda subtype.

5. anti-PTN antibody according to claim 4, characterized in that: the antibody heavy chain constant region is IgG1 and the antibody light chain constant region is of the kappa subtype.

6. An expression vector for replication in a prokaryotic or eukaryotic cell line, characterized in that: encoding an antibody according to any one of claims 1-2.

7. Use of an anti-PTN antibody according to any one of claims 1 to 5 for the preparation of a medicament for the treatment of chronic myeloid leukemia.

8. Use of an anti-PTN antibody according to any one of claims 1 to 5 for the preparation of a formulation for the detection of PTN proteins.

9. Use of an anti-PTN antibody according to any one of claims 1-5 in the manufacture of a medicament for inhibiting leukemic stem cells.

10. A pharmaceutical composition for treating chronic myeloid leukemia, comprising: comprising the antibody of any one of claims 1-5 and imatinib.