CN107118276A

CN107118276A - A kind of monoclonal antibody for targetting human tumour stem cell and its application

Info

Publication number: CN107118276A
Application number: CN201710485302.6A
Authority: CN
Inventors: 沈敏
Original assignee: Suzhou Bojuhua Biology Medicine Technology Co ltd
Current assignee: Suzhou Bojuhua Biology Medicine Technology Co ltd
Priority date: 2017-06-23
Filing date: 2017-06-23
Publication date: 2017-09-01
Anticipated expiration: 2037-06-23
Also published as: CN107118276B

Abstract

The present invention relates to biomedicine field.Specifically, the present invention relates to a kind of mouse monoclonal antibody or its antigen-binding fragment for human tumour stem cell, and the hybridoma of the antibody is produced.The invention further relates to purposes of the antibody in oncotherapy and diagnosis.

Description

A kind of monoclonal antibody for targetting human tumour stem cell and its application

Technical field

The present invention relates to biomedicine field.Specifically, the present invention relates to a kind of mouse for human tumour stem cell Monoclonal antibody or its antigen-binding fragment, and produce the hybridoma of the antibody.The invention further relates to the antibody Purposes in oncotherapy and diagnosis.

Background technology

Malignant tumour (cancer) turns into threatens the people of the world's life and " number one killer " of health.Swell every year in the whole world The tumor patient that the number of the infected of knurl is increased newly in China every year more than 14,000,000, only is more than 3,000,000.

The basic reason for causing cancer high mortality be cancer cell diffusion, transfer and treatment after most of patients easily recurrence and Resistance.Clinically existing treatment means, operation, radiotherapy, chemotherapy are little to cancer metastasis, recurrence, resistance curative effect, or only Short term effect, can not change the long-term survival condition of patient.At present, surgery excision is imitated to about 10-20% early stage patient It is really good but nearly unavailable to having occurred the patient of diffusion transfer.Radiotherapy can only treat local lesion, frequently as preoperative, postoperative The radical treatment of auxiliary treatment and a few species cancer.Chemotherapy can be used for the patient for having occurred diffusion transfer, but because of malicious secondary work With big, recent or at a specified future date resistance is easily produced, thus there can only be obvious short term effect to about 20-30% patient.I.e. Make using operation, the complex treatment measure of radiation and chemotherapy use in conjunction, the late result of its 5-year Survival is hovered always for many years 20-30%, about 70-80% the patient death in 5 years because of transfer, recurrence and resistance after the treatment.Even go to a doctor when without turn The earlier stage cancer patients of shifting, also or some occurs transfer and relapse and dead after the treatment.The tumour that developed recently gets up Novel targeted medicine, including polypeptide, small molecule, protein factor, gene therapy and antibody drug are logical with chemotherapeutic use in conjunction Often also can only existing treatment means extension patient survival 3~9 months relatively, 5 years survival rates to patient at a specified future date are not notable Raising.The immunotherapy of tumors means newly risen for nearest 2 years, for example the PD-1 monoclonal antibody medicines for immunologic test point and CAR-T cell therapies, although shown that the sign of some encouraging late results can be obtained, but it is overall to cancer trouble The effective percentage of person is only capable of reaching 20-30% or so, still have substantial amounts of cancer patient cannot real effective medicine treatment. Therefore it is that exploitation suppresses metastases, recurrence, the novel medicine of resistance to improve tumor patient late result, extend the key of life cycle Thing.

Summary of the invention

In a first aspect, the invention provides a kind of monoclonal antibody or its antigen-binding fragment, the monoclonal antibody It is common due to preserving number CGMCC No.12251 being preserved in China Committee for Culture Collection of Microorganisms on March 16th, 2016 The mouse hybridoma cell at microorganism center is produced.

In second aspect, the invention provides a kind of hybridoma, it is with preserving number CGMCC No.12251 in 2016 On March 16, in is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.

In the third aspect, the invention provides a kind of pharmaceutical composition, its monoclonal antibody comprising the present invention or it is anti- Former binding fragment and pharmaceutically acceptable carrier.

In some embodiments, the monoclonal antibody or its antigen-binding fragment are with being selected from cytotoxin, radioactivity The therapeutic moieties of isotope or biological activity protein are conjugated.

In fourth aspect, the invention provides a kind of malignant tumour for the treatment of in patients, prevention and/or treatment malignant tumour Transfer or the method for recurrence, methods described include the monoclonal antibody of the invention or its antigen that effective dose is applied to the patient The pharmaceutical composition of binding fragment or the present invention.

In some embodiments, the malignant tumour be selected from breast cancer, colorectal cancer, cancer of pancreas, prostate cancer, liver cancer, Lung cancer and stomach cancer.

In some embodiments, methods described also includes applying other antineoplaston means to the patient, for example Using chemotherapeutics, the antibody of the other tumour specific antigens of targeting or radiotherapy.

At the 5th aspect, the invention provides the monoclonal antibody of the present invention or its antigen-binding fragment or the medicine of the present invention Compositions are preparing the purposes in being used to treat malignant tumour, prevention and/or treatment Malignant tumor of bonal metastasis or the medicine of recurrence.

At the 6th aspect, the invention provides a kind of method for detecting that tumor stem cell is present in biological sample, including：

A) biological sample is made to be contacted with the monoclonal antibody or its antigen-binding fragment of the present invention；

B) monoclonal antibody or its antigen-binding fragment and the target antigen in the biological sample of the present invention is detected With reference to there is tumor stem cell wherein detecting the combination and representing in the biological sample.

At the 7th aspect, the present invention also provides a kind of method for separating tumor stem cell, and methods described includes：

(a) the doubtful cell mass for including tumor stem cell is provided；

(b) subgroup of the cell is identified, it combines the monoclonal antibody or its antigen-binding fragment of the present invention；With

(c) subgroup is separated.

In some embodiments of the 6th and the 7th aspect, the tumor stem cell is selected from breast carcinoma stem cell, large intestine Cancer stem cell, pancreas cancer stem cell, prostate cancer stem cells, liver-cancer stem cell, lung cancer stem cell and stomach cancer stem cell.

In eighth aspect, present invention also offers a kind of method for detecting the presence of malignant tumour in patient, including：

A) biological sample for being derived from the patient is made to be connect with monoclonal antibody or its antigen-binding fragment of the invention Touch；

B) monoclonal antibody or its antigen-binding fragment and the target antigen in the biological sample of the present invention is detected With reference to wherein detection represents in the patient and there is malignant tumour.

At the 9th aspect, the present invention also provides a kind of method for Malignant Tumor Recurrence or progress in prognosis patients, institute The method of stating includes：

(a) separation includes the biological sample of circulating cells from the patient；

(b) biological sample comprising circulating cells and the monoclonal antibody or its antigen-binding fragment of the present invention are made Contact；With

(c) identification combines the presence of the monoclonal antibody of the present invention or the circulating cells of its antigen-binding fragment,

So as to the recurrence of malignant tumour or progress in patient described in prognosis.

In some embodiments, the progress of the malignant tumour includes the transfer malignant tumour in patients.

In some embodiments of the 8th and the 9th aspect, the biological sample includes blood sample, lymph sample Or its component.In some embodiments, the malignant tumour be selected from breast cancer, colorectal cancer, cancer of pancreas, prostate cancer, liver cancer, Lung cancer and stomach cancer.

Brief description of the drawings

Fig. 1 living cells immunofluorescence technique detects monoclonal antibody Hetumomab target antigens in kinds of tumor cells liver cell surface Expression (part typical positive result).

Fig. 2 SABCs detect the special high expression in human liver cancer, lung cancer, stomach organization of monoclonal antibody Hetumomab target antigens (part typical positive result).

The cancer cell of streaming fluoroscopic examination monoclonal antibody Hetumomab identifications is immunized in people's kinds of tumor cells system in Fig. 3 Equal significant enrichment (the typical streaming fluorescence pattern in part) in sphere culture cells.

A variety of human tumor cells (such as liver cancer, lung cancer, stomach cancer) of Fig. 4 .CCK8 methods detection monoclonal antibody Hetumomab identifications The resistance ability (IC50) of Hetumomab+ cells.

Fig. 5 monoclonal antibodies Hetumomab significantly inhibits self of the tumor stem cell of kinds of tumors (such as liver cancer, lung cancer, stomach cancer) Updating ability (balling-up).

Fig. 6 monoclonal antibodies Hetumomab significantly inhibits the invasion and attack of the tumor stem cell of kinds of tumors (such as liver cancer, lung cancer, stomach cancer) Ability.

Fig. 7 monoclonal antibodies Hetumomab significantly inhibits the invasive ability of the tumor stem cell of kinds of tumors.

Fig. 8 monoclonal antibodies Hetumomab and combined chemotherapy drug therapy human liver cancer transplantable tumor Bel7402-V13 in-vivo tumour Growth curve.

Fig. 9 monoclonal antibodies Hetumomab and combined chemotherapy drug therapy human liver cancer transplantable tumor Bel7402-V13 in-vivo tumour Volume inhibiting rate (during drug withdrawal).

Figure 10 monoclonal antibodies Hetumomab and combined chemotherapy drug therapy human liver cancer transplantable tumor Bel7402-V13 in-vivo tumour Volume inhibiting rate (after being discontinued one month).

The life of Figure 11 monoclonal antibodies Hetumomab and combined chemotherapy drug therapy human liver cancer transplantable tumor Bel7402-V13 mouse Deposit curve.

Figure 12 monoclonal antibodies Hetumomab treatment human lung cancer transplantable tumors SPCA-1 tumor growth in vivo curve.

Figure 13 monoclonal antibodies Hetumomab and combined chemotherapy drug therapy Human gastric cancer xenografts SNU-5 tumor growth in vivo are bent Line.

Detailed description of the invention

First, define

In the present invention, unless otherwise stated, Science and Technology noun used herein has art technology The implication that personnel are generally understood that.Also, protein used herein and nucleic acid chemistry, molecular biology, cell and tissue Culture, microbiology, immunology relational language and laboratory operation step be in corresponding field widely used term and often Advise step.Meanwhile, for a better understanding of the present invention, the definition and explanation of relational language is provided below.

As used herein, " antibody " refers to immunoglobulin and immunoglobulin fragment, no matter natural or partly or complete Portion's synthesis (for example recombinating) is produced, including the reservation total length of its part variable region for comprising at least immunoglobulin molecules is immunized Any fragment of the binding specificity ability of globulin.Therefore, antibody includes having and immunoglobulin antigen-binding domains Any albumen of (antibody combining site) homologous or substantially homologous binding structural domain.Antibody includes antibody fragment, for example, resist Tumor stem cell antibody fragment.As used herein, therefore term antibody includes synthetic antibody, restructuring is produced antibody, how special Property antibody (such as bispecific antibody), human antibody, non-human antibody, humanized antibody, chimeric antibody, intracellular antibody and antibody Fragment, such as, but not limited to Fab fragments, Fab' fragments, F (ab ')₂Fragment, Fv fragments, Fv (dsFv), the Fd of disulfide bond Fragment, Fd ' fragments, scFv (scFv), single chain Fab (scFab), double antibody, antiidiotype (anti-Id) antibody or above-mentioned The antigen-binding fragment of what antibody.Antibody provided in this article include any immunoglobulin class (for example, IgG, IgM, IgD, IgE, IgA and IgY), any classification (such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass (for example, IgG2a and IgG2b member).

As used herein, " antibody fragment " or " antigen-binding fragment " of antibody refers to any part of full length antibody, and its is few In total length, but it is (such as one or more CDR and/or one including at least the part variable region of the antibody with reference to antigen Or multiple antibody combining sites), and therefore retain at least part specificity knot of binding specificity and the full length antibody Conjunction ability.Therefore, antigen-binding fragment refers to comprising the antigen-binding portion thereof with the antibody binding same antigen of derivative antibody fragment Antibody fragment.Antibody fragment includes passing through the antibody derivatives produced by enzymatic treatment full length antibody, and synthetically produced Derivative, for example, recombinate the derivative of generation.Antibody includes antibody fragment.The example of antibody fragment include but is not limited to Fab, Fab'、F(ab’)₂, scFv (scFv), Fv, dsFv, double antibody, Fd and Fd ' fragments and other fragments, include the piece of modification Section is (see, e.g., Methods in Molecular Biology, Vol 207:Recombinant Antibodies for Cancer Therapy Methods and Protocols(2003)；Chapter 1；p 3-25,Kipriyanov).It is described Fragment can include a plurality of chain that links together, such as by disulfide bond and/or pass through peptide linker.Antibody fragment is generally comprised At least or about 50 amino acid, and typical case at least or about 200 amino acid.Antigen-binding fragment includes any antibody fragment, Its when being inserted into antibody framework (such as by replacing respective regions) adaptive immune specifically combine (show at least or At least about 10⁷-10⁸M^-1Ka) antigen antibody.

As used herein, " monoclonal antibody " refers to the colony of same antibody, represents each list in monoclonal antibody colony Only antibody molecule is identical with other antibody molecules.The characteristic of the polyclonal population of this characteristic and antibody is on the contrary, the antibody Polyclonal population include have a variety of not homotactic antibody.Monoclonal antibody can be prepared by many known methods (Smith et al.(2004)J.Clin.Pathol.57,912-917；With Nelson et al., J Clin Pathol (2000),53,111-117).For example, monoclonal antibody can be prepared by immortalised B-cell, for example by with myeloma Cell fusion with produce hybridoma cell line or by using such as EBV virus infection B cell.Recombinant technique can also be used to Carry out gram from host cell by using the plasmid conversion host cell of the artificial sequence for the nucleotides for carrying encoding antibody in vitro Grand colony prepares antibody.

As used herein, term " hybridoma " or " hybridoma ", which refer to, is produced the lymphocyte of antibody by fusion and is not produced The cancer cell of antibody and the cell or cell line (be usually myeloma or lymphoma cell) produced.Such as ordinary skill people Known to member, hybridoma, which can breed and continue supply, produces specific monoclonal antibody.Method for producing hybridoma is ability (see for example, Harlow＆Lane, 1988) known to domain.When referring to term " hybridoma " or " hybridoma ", it also includes The subclone and progeny cell of hybridoma.

As used herein, term " epitope " refers to any antigenic determinant on the antigen of the paratope combination of antibody.Epitope Determinant generally comprises the chemically reactive surface parting of molecule, such as amino acid or sugared side chain, and generally has specific three Tie up architectural feature and specific charge characteristic.

As used herein, " specific binding " or " immunospecifically with reference to " on antibody or its antigen-binding fragment It is used interchangeably herein, and refers to antibody or antigen-binding fragment and passes through between antibody and the antibody combining site of antigen Noncovalent interaction forms the ability of one or more non-covalent bonds with isoantigen.The antigen can be the antigen of separation Or it is present in tumour cell.Generally, with reference to the antibody of (or specific binding) antigen it is immunospecifically with about or 1 × 10⁷M^-1Or 1x 10⁸M^-1Or bigger affinity costant Ka (or 1x 10^-7M or 1 × 10^-8M or lower dissociation constant (K_d)) combine The antigen.Affinity costant can be determined by the Standard kinetic method of antibody response, for example, immunoassays, surface etc. Ion resonance (SPR) (Rich and Myszka (2000) Curr.Opin.Biotechnol 11:54；Englebienne (1998)Analyst.123:1599), identical titration calorimetry (ITC) or other dynamical inleractions known in the art are surveyed Fixed (see, e.g., Paul, ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages 332-336(1989)；Referring further to description for the exemplary SPR and ITC methods of the binding affinity of calculating antibody U.S. Patent No. 7,229,619).Instrument and method for detecting in real time and monitor association rate are known, and can It is commercially available (referring to BiaCore 2000, Biacore AB, Upsala, Sweden and GE Healthcare Life Sciences；Malmqvist(2000)Biochem.Soc.Trans.27:335).

As used herein, the term " competition " on antibody refers to first antibody or its antigen-binding fragment with second to resist Body or its antigen-binding fragment similar mode enough combine an epitope, and thus first antibody is associated with the combination knot of epitope Fruit under conditions of there is secondary antibody under conditions of in the absence of secondary antibody compared with detectably reduce.Or, second The combination of antibody and its epitope under the conditions of there is first antibody also detectably reduce in the case of, can with but need not this The situation of kind.That is, first antibody can suppress secondary antibody and the combination of its epitope, and suppress first without secondary antibody The combination of antibody and its respective epitope.It is associated with epitope however, detectably suppressing another antibody in each antibody or matches somebody with somebody In the case of the combination of body, either identical, higher or lower degree, the antibody is referred to as " cross competition " each other and combines it Respective epitope.Competition and cross-competing antibodies are encompassed by the present invention.No matter it is this competition or cross competition occur machine System how (such as steric hindrance, conformational change combine common epitope or its fragment), those skilled in the art are carried based on the present invention The teaching of confession will recognize that this competition and/or cross-competing antibodies are covered in the present invention and available for the side of the invention disclosed In method.

As used herein, " polypeptide " refers to two or more amino acid of covalent attachment.Term " polypeptide " and " protein " It is used interchangeably herein.

" protein of separation ", " polypeptide of separation " or " antibody of separation " refers to the protein, polypeptide or antibody (1) no Associate with its native state with its natural Related Component, (2) without the other oroteins from same species, (3) by Cell expression from different plant species, or (4) do not occur in natural.Therefore, polypeptide through chemical synthesis or different from many The polypeptide synthesized in the cell system of the natural origin cell of peptide will be with its natural Related Component " separation ".Separation can also be passed through So that protein is substantially free of natural Related Component, i.e., using purified technology of protein well-known in the art.

As used herein, the individual of " treatment " with disease or disease condition represents that the individual symptom is part or all of Alleviate, or keep after the treatment constant.Therefore, treatment includes prevention, treatment and/or cured.Prevention, which refers to, prevents potential disease And/or prevent symptom from deteriorating or disease development.Treat any antibody or its antigen-binding fragment and this for also including being provided Any pharmaceutical use for the composition that text is provided.

As used herein, " curative effect " represents the effect caused by the treatment of individual, and it changes, generally improves or improve disease The symptom of disease or disease condition, or cure disease or disease condition.

As used herein, " therapeutically effective amount " or " treatment effective dose " refers to be applied to after object and is at least enough to produce treatment The material of effect, compound, the amount of the composition of material or inclusion compound.Therefore, it is to prevent, cure, improving, blocking or portion Divide amount necessary to the symptom of retardance disease or illness.

As used herein, " prevention effective dose " or " prevention effective dose " refer to when being applied to object can have it is expected pre- The material of anti-effect, compound, the amount of the composition of material or inclusion compound, for example, preventing or postponing disease or symptom Occur or recur, reduce the possibility that disease or symptom occur or recurred.Complete prevention effective dose is without going through using one Dosage occurs, and only can occur after a series of dosage are applied.Therefore, prevention effective dose can be applied one or many Applied with middle.

As used herein, term " patient " refers to mammal, such as people.

The sub-fraction that " tumor stem cell " refers to be present in tumor tissues has the cancer cell of dryness feature, its with it is general Logical cancer cell is compared with self-renewal capacity, strong invasive ability, tolerance chemotherapeutics ability and strong tumorigenesis ability.

2nd, for the monoclonal antibody of tumor stem cell

In the present invention, classical hybridoma is passed through as immunogen immune mouse using human tumour multipotential stem cell system Integration technology obtains monoclonal antibody Hetumomab.Produce monoclonal antibody Hetumomab mouse hybridoma cell strain Hetumomab is preserved in Chinese microorganism strain preservation management committee on March 16th, 2016 with preserving number CGMCC No.12251 Member can common micro-organisms center.(embodiment 1)

The inventors discovered that, monoclonal antibody Hetumomab of the invention target antigen is expressed in a variety of human tumor cells Liver cell surface, and the specificity overexpression (positive rate 79%-94%) in kinds of tumors tissue.The present invention's Hetumomab monoclonal antibodies can be enriched with the sphere culture cells of kinds of tumor cells and can recognize the tumours such as ESA, CD90 The tumour cell of stem cell markers, prompting Hetumomab monoclonal antibodies are that the monoclonal antibody of specific for cancer stem cell is (real Apply example 2).

The further studies have shown that based on Hetumomab target antigen positive tumor cells is relative to parent's tumour cell With Hetumomab target antigen negative tumor cells, Hetumomab target antigen positive tumor cells have stronger self-renewing, Invasion and attack, resistance and internal tumorigenesis ability, further prove the selectively targeted tumor stem cell of Hetumomab monoclonal antibodies (embodiment 3).

The selectively targeted tumor stem cell of Hetumomab monoclonal antibodies of the present invention include but is not limited to breast carcinoma stem cell, Large intestine cancer stem cell, pancreas cancer stem cell, prostate cancer stem cells, liver-cancer stem cell, lung cancer stem cell and stomach cancer stem cell.

Therefore, the invention provides a kind of monoclonal antibody or its antigen-binding fragment, the monoclonal antibody due to On March 16th, 2016 is preserved in the common micro- life of China Committee for Culture Collection of Microorganisms with preserving number CGMCC No.12251 The mouse hybridoma cell at thing center is produced.

Present invention also contemplates that the monoclonal antibody or its antigen-binding fragment of separation, it is swollen with Hetumomab competition bindings Knurl stem cell.

Present invention also contemplates that the monoclonal antibody or its antigen-binding fragment of separation, itself and Hetumomab combination Tumor Stems Same epitope on cell.

3rd, disease treatment

Tumor stem cell is the cancer cell that the sub-fraction being present in tumor tissues has dryness feature, with following life Thing feature：Can self-renewing, replicate, non-directional differentiation, high oncogenicity, height invasion and attack diffusion transfer ability, it is equal to chemicotherapy It is insensitive.Due to there is tumor stem cell so that the continuous fast-growth of tumour, diffusion, transfer, recurrence.More seriously swell Knurl stem cell is equal to almost all of classic chemotherapy medicine, radiotherapy and the targeted drug (including antibody target medicine) listed in recent years Resistance.The G0 phases that tumor stem cell is in the cell cycle do not grow, not vegetative state.Chemicotherapy is only in the fast fast-growing of height The cancer cell of long propagation has effect, and can not kill the tumor stem cell in the G0 phases.And when chemicotherapy will a large amount of fast fast-growing After long cancer cell is killed, the tumor stem cell of resistance chemicotherapy is screened enrichment on the contrary, and ratio is greatly improved.Due to Tumor Stem Cell has extremely strong the of self-replication capacity and a diffusion transfer ability, these tumor stem cells can quick differentiation and proliferation, growth is simultaneously Diffusion, is transferred to the new metastatic lesion of each orga- nogenesis of whole body, and the cancer cell of this metastatic lesion resists chemicotherapy, in breast Have been proven that Tumor Stem is thin in the Several Kinds of Malignancy such as gland cancer, colorectal cancer, cancer of pancreas, prostate cancer, liver cancer, lung cancer, stomach cancer The presence of born of the same parents.The higher cancer of grade malignancy, tumor stem cell is more, and the higher cancer patient of tumor stem cell ratio turns Move, recur more, life cycle is shorter.

The inventors discovered that, the Hetumomab monoclonal antibodies of specific recognition tumor stem cell of the invention can show in vitro Write self-renewing, invasion and attack and the resistance ability (embodiment 4) for suppressing kinds of tumors stem cell.Further experiment shows, Hetumomab monoclonal antibodies can suppress the growth, transfer and resistance (embodiment 5) of kinds of tumors transplantable tumor in animal model.Cause This, Hetumomab monoclonal antibodies of the invention can be used to treat malignant tumour by targeting tumor stem cells, prevent or/or control Treat Malignant tumor of bonal metastasis or recurrence.

Thus, the invention provides a kind of malignant tumour for the treatment of in patients, prevention or/or treatment Malignant tumor of bonal metastasis or The method of recurrence, methods described include to the patient apply effective dose it is of the invention for the antibody of tumor stem cell or its Antigen-binding fragment.The malignant tumour that can be treated by the method for the present invention includes but is not limited to breast cancer, colorectal cancer, pancreas Cancer, prostate cancer, liver cancer, lung cancer and stomach cancer.

4th, pharmaceutical composition

Present invention also offers a kind of pharmaceutical composition, its comprising the present invention for the antibody of tumor stem cell or its resist Former binding fragment and pharmaceutically acceptable carrier.Described pharmaceutical composition is used for treatment malignant tumour, prevention in patients Or/or treatment Malignant tumor of bonal metastasis or recurrence.

" pharmaceutically acceptable carrier " used herein includes any and all solvent of physiological compatible, scattered Jie Matter, coating, antibacterial agent and antifungal agent, isotonic agent and absorption delaying agent etc..Preferably, the carrier is suitable for intravenous, flesh Interior, subcutaneous, parenteral, backbone or epidermis are applied (such as by injecting or being transfused).According to route of administration, can be by reactive compound Antibody molecule, immunoconjugates are wrapped in a kind of material, with protect the compound from can make its inactivation acid and The effect of other natural endowments.

The pharmaceutical composition of the present invention can also contain pharmaceutically acceptable antioxidant.It is pharmaceutically acceptable anti-oxidant The example of agent includes：(1) water soluble antioxidant, such as ascorbic acid, cysteine hydrochloride, niter cake, sodium pyrosulfite, it is sub- Sodium sulphate etc.；(2) oil-soluble inhibitor, such as ascorbyl palmitate, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), ovum Phosphatide, propylgallate, alpha-tocopherol etc.；(3) metal-chelator, such as citric acid, ethylenediamine tetra-acetic acid (EDTA), sorb Sugar alcohol, tartaric acid, phosphoric acid etc..

These compositions can also contain adjuvant, such as preservative, wetting agent, emulsifying agent and dispersant.

Can be by sterilizing program or by each comprising p-hydroxybenzoate, chlorobutanol, phenol sorbic acid etc. Planting antibacterial agent and antifungal agent ensures to prevent there is microorganism.Under many circumstances, isotonic agent, example are preferably comprised in composition Such as, sugar, polyhydric alcohols such as mannitol, D-sorbite or sodium oxide molybdena.By adding delay absorbent in the composition, for example singly Stearate and gelatin, can be achieved the absorption of injection-type protracted drug.

Pharmaceutically acceptable carrier or including aseptic aqueous solution or dispersion liquid and for extemporaneous preparation of sterile parenteral solution divide The powder agent of dispersion liquid.These uses for being used for the medium and reagent of pharmaceutically active substances are well known in the art.Conventional media or Reagent, all may be in the pharmaceutical composition of the present invention in addition to any scope incompatible with reactive compound.Can also be to The reactive compound of supplement is mixed in composition.

Therapeutic composition typically must be sterile and stablized under preparation and storage requirement.Can be by composition It is configured to the ordered structure of solution, micro emulsion, liposome or other suitable high drug concentrations.Carrier can be containing for example Water, ethanol, the solvent or scattered of polyalcohol (for example, glycerine, propane diols and liquid polyethylene glycol etc.) and its suitable mixture Agent.For example, by using coating, such as lecithin, passing through the granular size needed for holding, Yi Jitong in the case of dispersion liquid Cross and use surfactant, appropriate mobility can be kept.

It is mixed into suitable solvent, and is added as needed listed above by the amount for needing reactive compound One kind or its combination in composition, then sterile microfiltration, can prepare aseptic parenteral solution.Generally, by the way that reactive compound is mixed Enter and prepare dispersant into the sterile carrier containing basic decentralized medium and other required compositions listed above.For for preparing The aseptic powdery agent of aseptic parenteral solution, preparation method preferably is vacuum drying and is freeze-dried (lyophilized), sterile in advance by it The solution of filtering obtains the powder of active component plus any additional desired ingredient.

The amount for the active component for preparing ingle dose form can be combined with carrier material according to the object and spy treated Determine administering mode and different.The amount that the active component for preparing ingle dose form can be combined with carrier material is usually that generation is controlled The amount of the composition of therapeutic effect.Generally, in terms of 100%, the scope of this amount be about 0.01% to about 99% activity into Point, preferably approximately 0.1% to about 70%, the active component of most preferably from about 1% to about 30% is and pharmaceutically acceptable Carrier is combined.

Optimal desired reaction (for example, therapeutic response) can be provided with regulating dosage scheme.For example, can apply single One is injected, and dosage separated several times can be applied with the time, or according to needed for the emergency for the treatment of situation, can be in proportion Dosage is reduced or increased.Parenteral composition is particularly advantageously configured to the dosage unit of easily administration and dose uniformity Form.Dosage unit form used herein refer to be suitable as unit dose be used for treated object physics it is discontinuously single Position；Each unit contains the reactive compound of scheduled volume, the reactive compound for being computed the scheduled volume and the pharmaceutical carrier needed Combine the therapeutic effect needed for generation.(a) work is defined in and depends directly on to illustrating for dosage unit form of the present invention Property compound unique property and the particular treatment effect to be reached, and (b) this area in it is intrinsic for preparing this be used for Treat the limitation of the reactive compound of individual sensitivity.

For the administration of antibody molecule, dosage range be about 0.0001 to 100mg/kg, more typically 0.01 to 20mg/kg receptor's body weight.For example, dosage can be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20mg/kg.Exemplary therapeutic scheme needs weekly administration one It is secondary, once every two weeks, per once in three weeks, every four weeks once, monthly, every 3 months once, per 3-6 month once or starting Slightly short (as weekly to per once in three weeks) the later stage dosing interval of dosing interval is lengthened (as monthly to every 3-6 months one It is secondary).

Or, it can also be administered for the antibody molecule of tumor stem cell as extended release preparation, in this case Need the administration that frequency is relatively low.Dosage and frequency according to antibody molecule half-life period in patients and it is different.Generally, human antibody table Reveal most long half-life period, be humanized antibody, chimeric antibody and non-human antibody afterwards.Dosage and frequency are according to processing It is preventative or curative and different.In prophylactic use, phase is given in a long time with interval less frequently To relatively low dosage.Some patients lasting receiving processing in the remaining years.In therapeutic application, it is sometimes desirable to shorter interval Higher dosage is given, until the progress of disease mitigates or stops, disease symptomses are shown as partially or completely preferably up to patient Improve.Afterwards, it can be administered with Prevention scheme to patient.

The actual dose level of active component may change in pharmaceutical composition of the present invention, can effectively be realized to spy with obtaining Determine the required therapeutic response of patient, composition and administering mode, and to the amount of the avirulent active component of patient.The dosage of selection Level depends on multi-medicament dynamic metabolism factor, includes the activity of the particular composition of the present invention of application, method of administration is given Medicine time, the discharge rate of the specific compound of application, the duration for the treatment of, with the particular composition use in conjunction of application Other drugs, compound and/or material, age, sex, body weight, situation, general health and the disease of patient receiving treatment Known similar factor in history, and medical domain.

The antibody of the invention or its antigen-binding fragment of " effective dose " preferably result in the seriousness reduction of disease symptomses, The frequency of disease asymptomatic stage and duration increase, or prevent damage or disability because of caused by disease pain.For example, right In the treatment of tumour, relative to the object for not receiving treatment, the antibody of the invention or its antigen-binding fragment of " effective dose " are excellent Selection of land is by cell growth or Tumor growth inhibition at least about 10%, and preferably at least about 20%, more preferably at least about 30%, more preferably At least about 40%, more preferably at least about 50%, more preferably at least about 60%, more preferably at least about 70%, more preferably at least about 80%.Suppressing the ability of tumour growth can evaluate in animal model system of the prediction to the curative effect of human tumor.Or, It can be evaluated by checking cytostatic ability, this suppression can be by the way that well known to a person skilled in the art experiment Determine in vitro.The antibody of the invention or its antigen-binding fragment of effective dose can reduce tumor size, or with its other party Formula alleviates the symptom of object as prevented and/or treating transfer or recur.Those skilled in the art can determine according to following factor The size of this amount, such as object, the seriousness of object symptom and the particular composition of selection or method of administration.

The antibody or its antigen-binding fragment of the present invention or the pharmaceutical composition of the present invention can utilize well known in the art One or more methods are administered by one or more methods of administration.It will be appreciated by those skilled in the art that method of administration and/or Mode is different according to desired result.The antibody preferred route of administering of the present invention includes intravenous, intramuscular, intracutaneous, peritonaeum Interior, subcutaneous, backbone or other parenteral route of administration, for example, inject or be transfused.Phrase " parenteral " used herein is Refer to the mode of administration in addition to intestines and local administration, typically inject, including but not limited to intravenous, intramuscular, intra-arterial, sheath Interior, intracapsular, socket of the eye is interior, intracardiac, intracutaneous, intraperitoneal, transtracheal, under subcutaneous, epidermis, under intra-articular, capsule, under arachnoid, in backbone, Epidural cavity and breastbone inner injection and infusion.

Or, the antibody or its antigen-binding fragment or the pharmaceutical composition of the present invention for tumor stem cell of the invention It can also be administered by non-parenteral routes, such as local, epidermis or mucosal route administration, for example, intranasal, oral, vagina, straight It is intestines, sublingual or local.

Reactive compound can be prepared together with protecting the carrier that compound is not fast released, such as controlled release preparation, bag Include implant, transdermal patch and microcapsules delivery system.Biodegradable, bio-compatible polymer can be used, for example Ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, poe and PLA.Prepare many methods of such preparation By patent protection or generally known to those skilled in the art.See, e.g., Sustainedand controlled Release Drug Delivery Systems, J.R.Robinson, ed., Marcel Dekker, Inc., New York, 1978。

Therapeutic composition can be using medical treatment device well known in the art administration.For example, in a preferred embodiment, The therapeutic combination of the present invention can be administered with needlefree injection device, such as in United States Patent (USP) No.5,399,163；5,383, 851；5,312,335；5,064,413；4,941,880；4,790,824；Or the device disclosed in 4,596,556.Available for this The known implant of invention and the example of module include：United States Patent (USP) No.4,487,603, which disclose for by Rate controlling rate disperses the implantable micro infusion pump of medicine；United States Patent (USP) No.4,486,194, it which disclose for passing through skin The therapeutic system of skin administration；United States Patent (USP) No.4,447,233, it which disclose for delivering medicine with accurate infusion rates The medical infusion pump of thing；United States Patent (USP) No.4,447,224, it which disclose implantable for the unsteady flow that continuously delivers medicine Infusion device；United States Patent (USP) No.4,439,196, it which disclose the osmotic drug delivery system with multi-cavity compartment：And U.S. State patent No.4,475,196, it which disclose a kind of osmotic drug delivery system.These patents are incorporated herein by reference. Many other such implant, delivery system and modules as well known to those skilled in the art.

In certain embodiments, the antibody for tumor stem cell of the invention can be formulated as ensuring in vivo correct Distribution.For example, blood brain barrier (BBB) prevents many highly hydrophilic compounds.In order to ensure therapeuticization of the present invention Compound can prepare them in such as liposome across BBB (if desired).As for the method for preparing liposome, ginseng See, for example, United States Patent (USP) 4,522,811；5,374,548 and 5,399,331.Liposome is included and can optionally transported into spy Cell or intraorganic one or more targeting moieties are determined, so as to strengthen targeted delivery of drugs (see, e.g., V.V.Ranade (1989)J.Clin.Pharmacol.29：685).The example of targeting moiety includes folic acid or biotin (see, e.g., Low etc. United States Patent (USP) 5,416,016)；Mannoside (Umezawa etc. (1988) Biochem.Biophys.Res.Commun.153： 1038)；Antibody (P.G.Bloeman etc. (1995) FEBS Lett.357:140；M.Owais etc. (1995) Antimicrob.Agents Chemother.39:180)；Surfactant proteins A acceptors (Briscoe etc. (1995) Am.J.Physiol.1233:134)；P120 (Schreier etc. (1994) J.Biol.Chem.269:9090)；Referring also to K.Keinanen；M.L.Laukkanen(1994)FEBS Lett.346:123；J.J.Killion；I.J.Fidler(1994) Immunomethods 4：273.

The antibody or its antigen-binding fragment for tumor stem cell of the invention can also be with described pharmaceutical composition The therapeutic moieties such as cytotoxin, radio isotope or biological activity protein are conjugated.

Cytotoxin includes being harmful to cell any reagent of (for example killing cell).Example includes：Taxol, cell pine Relax element B, Gramicidin D, Ethidum Eremide, ipecine, mitomycin, etioposide, teniposide, Vincristine, vincaleukoblastinum, colchicine, adriamycin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, radiance are mould Element, actinomycin D, 1- dehydrogenations testosterone, glucocorticoid, procaine, totokaine, lidocaine, Propranolol and purine are mould Element and their analog or homologue.

Also include available for conjugated therapeutic agent, for example：Antimetabolite is (for example, methotrexate, Ismipur, 6- sulphur For guanine, cytarabine, 5 FU 5 fluorouracil, decarbazine), alkylating agent is (for example, mustargen, Chlorambucil, phenylalanine nitrogen Mustard, BCNU (BSNU) and lomustine (CCNU), endoxan, busulfan, dibromo mannitol, streptozocin, mitogen Mycin C and cis- dichlorodiamine close platinum (II) (DDP) cis-platinum), anthramycin class is (for example, daunorubicin (is formerly referred to as promise mould Element) and adriamycin), antibiotic is (for example, actinomycin D (being formerly referred to as D actinomycin D), bleomycin, mithramycin and peace are bent Mycin (AMC)), and antimitotic agent (for example, vincristine and vincaleukoblastinum).

Can with the present invention the antibody conjugate for tumor stem cell therapeutic cells toxin other preference attached bags Include a times carcinomycin, calicheamycin, maytansine, Ali's statin and their derivative.

The joint technique that this area be used can be utilized by cytotoxin and the antibody for tumor stem cell of the present invention It is conjugated.Have been used to include but is not limited to cytotoxin and the example of the joint categories of the antibody conjugate for tumor stem cell Hydrazone, thioether, ester, disulphide and the joint containing peptide.It can select, for example, easily being cut in lysosomal compartment by low pH or easily The joint cut by protease, the protease is, for example, the protease that precedence table reaches in tumor tissues, such as cathepsin (example Such as cathepsin B, C, D).

Joint and method on cytotoxic type, for therapeutic agent and antibody to be conjugated it is discussed further, referring to Saito, G. etc. (2003) Adv.Drug Deliv.Rev.55：199-215；Trail, P.A. etc. (2003) Cancer.Immunol.Immunother.52：328-337；Payne, G. (2003) Cancer Cell 3：207-212； Allen, T.M. (2002) Nat.Rev.Cancer 2：750-763；Pastan, I. and Kreitman, R.J. (2002) Curr.Opin.Investig.Drugs 3：1089-1091；Senter, P.D. and Springer, C.J. (2001) Adv.Drug Deliv.Rev.53：247-264.

The antibody for tumor stem cell of the present invention can also be conjugated with radio isotope, produces cytotoxicity radiation Property medicine, also referred to as radioactive antibody conjugate.Can be with diagnosis or the same position of radioactivity of the therapeutic antibody conjugate used The example of element includes but is not limited to iodine 131, indium 111, Y90 and lutetium 177.The method of radioactive antibody conjugate is prepared in ability Had built up in domain.

The antibody for tumor stem cell of the present invention can also be with having the protein-conjugate of bioactivity in need, can For modifying specific biologically.Such biological activity protein includes, for example：With the toxin of enzymatic activity or its work Property fragment, such as abrin, ricin A, Pseudomonas exotoxin or diphtheria toxin；Protein, such as neoplasm necrosis The factor or interferon-γ；Or biologically instrumentality, such as lymphokine, il-1 (" IL-1 "), proleulzin (" IL- 2 "), interleukin-6 (" IL-6 "), interleukin-10 (" IL-10 "), granulocyte macrophage colony stimulating factor (" GM-CSF "), Granulocyte colony stimulating factor (" G-CSF ") or other immune factors such as IFN etc..

The technology that this therapeutic moieties and antibody molecule are conjugated be it is well known that see, e.g., Arnon etc., " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", (ed.), pp.243-56 (Alan such as Monoclonal Antibodies And Cancer Therapy, Reisfeld R.Liss, Inc.1985)；Hellstrom etc., " Antibodies For Drug Delivery ", Controlled Drug Delivery (2nd Ed.), Robinson etc. (ed.), pp.623-53 (Marcel Dekker, Inc.1987)；Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy：A Review ", Monoclonal Antibodies’84：(ed.), pp.475-506 such as Biological And Clinical Applications, Pinchera (1985)；" Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ", Monoclonal Antibodies For Cancer (ed.) such as Detection And Therapy, Baldwin, pp.303-16 (Academic Press 1985), and Thorpe Deng " The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates, " Immunol.Rev., 62：119-58(1982).

5th, therapeutic alliance

The antibody or pharmaceutical composition for tumor stem cell of the present invention can be with chemotherapeutics or to target other tumours anti- Former antibody combined administration.The Hetumomab monoclonal antibodies that the embodiment of the present application 5 demonstrates the present invention are applied with chemotherapeutic agent There is synergy in oncotherapy.It is without wishing to be bound by any theory, it is believed that resisting for tumor stem cell of the invention Body can suppress tumor drug resistance function, so as to chemotherapeutics or target the Antibody Combinations of other tumour antigens using when take Obtain synergy.

Can be with the antibody of the present invention or the chemotherapeutics that be applied in combination of pharmaceutical composition of the present invention or targetting other tumours The antibody of antigen is not particularly limited.The example of the antibody of other tumour antigens of the chemotherapeutics and targeting includes but is not limited to： Ifosfamide, endoxan, Dacarbazine, Temozolomide, Nimustine, busulfan, melphalan, enalinbin, Ka Peita Shore, Carmofur, Cladribine, gemcitabine, cytarabine, Tegafur, UFT, TS-1, doxifluridine, how Draw shore, hydroxycarbamide, fluorouracil, fludarabine, pemetrexed, Pentostatin, mercaptopurine, methotrexate (MTX), Irinotecan, support Moor glycosides, Ai Libulin, Sobuzoxane, docetaxel, taxol, vinorelbine, vincristine, eldisine, vincaleukoblastinum, unwrapping wire The soft ratio of rhzomorph D, Aclarubicin, Amrubicin, idarubicin, epirubicin, Zinostatin ester, daunorubicin, Doxorubicin, pyrrole Star, bleomycin, Peplomycin, mitomycin C, mitoxantrone, oxaliplatin, carboplatin, cis-platinum, Nedaplatin, Anastrozole, according to Xi Meitan, ethinyloestradiol, chlormadinone, Goserelin, TAM, dexamethasone, Bicalutamide, Toremifene, Flutamide, sprinkle Ni Songlong, Fosfestrol, mitotane, methyltestosterone, Leuprorelin, Letrozole, methyl medroxyproges-terone acetate, ibritumomab tiuxetan, she horse replaces Buddhist nun, everolimus, Tarceva, Gefitinib, Sutent, Cetuximab, Sorafenib, Dasatinib, his meter Ba Luo Spit of fland, Herceptin, vitamin A acid, the wooden monoclonal antibody of handkerchief, bevacizumab, bortezomib and Lapatinib.In a specific embodiment party In formula, the chemotherapeutics is the chemotherapeutics of platiniferous, such as cis-platinum.

The antibody and the chemotherapeutics of the present invention or target other tumour antigens antibody can all in applied once or Separate administration.When separate administration (using in the case of mutually different application program), they can be with continuous administration without in Break or apply at predetermined intervals.

The antibody and the chemotherapeutics of the present invention or pharmaceutical composition of the antibody in the present invention for targetting other tumour antigens In unitized dose be not particularly limited.As described above, the dosage of the antibody of the present invention can be by reference to when the antibody list Solely dosage when using is determined.What the antibody of other tumour antigens of the chemotherapeutics and targeting can be indicated according to respective medicine Dosage uses or can reduce and (consider the combined effect with the antibody of the present invention).

The present invention antibody or the present invention pharmaceutical composition can also and chemotherapy combined radiotherapy, for example including to patient apply electricity From radiation, its earlier than, during it and/or be later than the antibody of the present invention or the application of pharmaceutical composition.

6th, tumor stem cell detection and purifying

Hetumomab monoclonal antibody specificities identification tumor stem cell as described herein, of the invention.Therefore, this hair The bright method that tumor stem cell presence in a kind of detection biological sample is also provided, including：

B) monoclonal antibody or its antigen-binding fragment and the target antigen in the biological sample of the present invention is detected With reference to wherein detection represents in the biological sample and there is tumor stem cell.

In some embodiments, the tumor stem cell is selected from breast carcinoma stem cell, large intestine cancer stem cell, cancer of pancreas and done Cell, prostate cancer stem cells, liver-cancer stem cell, lung cancer stem cell and stomach cancer stem cell.

In some embodiments of above-mentioned detection method of the invention, monoclonal antibody of the invention or its antigen binding fragment Section is also conjugated with available for detection or fluorescent dye, chemical substance, polypeptide, enzyme, isotope, the mark that can be detected by other reagents Label etc..

For detecting that the method for antibody-antigen binding is known in the art, such as ELISA.

The present invention also provides a kind of method for separating tumor stem cell, and methods described includes：

(a) the doubtful cell mass for including tumor stem cell is provided；

(c) subgroup is separated.

For example, liver-cancer stem cell can be separated by flow cytometry.

7th, diagnosis and prognosis

As described herein, monoclonal antibody Hetumomab of the invention target antigen is expressed in a variety of human tumor cells Liver cell surface, and the specificity overexpression (positive rate 79%-94%) in kinds of tumors tissue.

Therefore, present invention also offers a kind of method for detecting the presence of malignant tumour in patient, including：

The present invention also provides a kind of method for Malignant Tumor Recurrence or progress in prognosis patients, and methods described includes：

Identify with reference to described in the presence prompting of the circulating cells of monoclonal antibody or its antigen-binding fragment of the invention The excessive risk of Malignant Tumor Recurrence or progress in patient.

In some embodiments, the biological sample includes blood sample, lymph sample or its component.

In some embodiments of the above method of the present invention, monoclonal antibody of the invention or its antigen-binding fragment are also It is conjugated with available for detection or fluorescent dye, chemical substance, polypeptide, enzyme, isotope, the label that can be detected by other reagents Deng.

8th, kit

Also include the kit of the method for the present invention in the scope of the present invention, the kit includes the Dan Ke of the present invention Grand antibody or its antigen-binding fragment, and operation instruction.The kit may further include at least one other detection Reagent, it is used for the presence for detecting the monoclonal antibody of the present invention.Kit generally comprises the expection for showing Kit Contents The label of purposes and/or application method.Term tag be included on kit or provided together with kit or with its other party Any written or record the material that formula is provided with kit.

Embodiment

Further understanding of the invention can be obtained by reference to some specific embodiments given herein, these implementations Example is merely to illustrate the present invention, and it has no intention to make the scope of the present invention any limitation.Obviously, the present invention can be made many Kind of the essence changed and changed without departing from the present invention, therefore, these change and change it is same this application claims model In enclosing.

The mouse monoclonal antibody Hetumomab of embodiment 1 preparation

First, the preparation in human tumor's multipotential stem cell mouse monoclonal antibody storehouse

The present embodiment is used as immunogene (Liu H, et al.Cell using a kind of human tumour multipotential stem cell system T3A-A3 Death and Disease.2013,4:e857).The liver cancer group that the human tumor stem cell line is cut off from primary hepatic carcinoma corrective surgery Middle separation is knitted to obtain, being capable of Long Term Passages culture in vitro..The cell line has been passed on more than 100 times, and cell still grows fast Speed, and keep stem cell properties.The cell line had both expressed the mark of a variety of stem cells, the self-renewing energy with stem cell Power, with the potential to different tumour cell directed differentiations；Also there is tumour property simultaneously, with one-tenth knurl ability and transfer energy Power.The cell line in vitro long-term cultivation and retention properties is constant, and in Immune deficient mice body one-tenth knurl ability and transfer energy Power is strong.

The liver-cancer stem cell (human tumour multipotential stem cell) that will be enlarged by culture acquisition is that T3A-A3 is fixed using paraformaldehyde, Common Balb/c mouse are immunized, 2-4 weeks is once, every time about 1 × 10⁷Cell.Permanent immunity, until using regular growth immunochemistry Method measure immune serum anti-human liver cancer stem cell (human tumour multipotential stem cell) is T3A-A3 titre more than 1: 50000.Take mouse boosting cell and methods of the murine myeloma cell SP2/0 using conventional PEG mediated fusions, fusion formation point Secrete the hybridoma of mouse monoclonal antibody.Hybridoma monoclonal is prepared using conventional methylcellulose flat band method, each list is treated Picking is distinguished after clonal growth to continue to cultivate to 96 orifice plates, is derived from containing a large amount of anti-human liver cancer stem cell (human tumours Multipotential stem cell) be T3A-A3 monoclonal antibody hybridoma clone library.Collect each hybridoma clone in 96 orifice plates Culture supernatant, measure and screening process for next step.

2nd, human tumor's multipotential stem cell mouse monoclonal antibody Hetumomab screening

Serum free suspension culture medium is using EGF containing 20ng/mL, 20ng/mL bFGF, by 1:50 ratios addition B27, 10ng/mL LIF, 2mmol/mL glutamine, 1 μ/mL Heparin DMEM/F12 (1:1) nutrient solution.Trained using serum-free Support and 1 cell is washed before base culture.By human liver cancer stem cell (human tumour multipotential stem cell) system through serum free suspension culture T3A-A3 sphaerocysts, softly dispel into it is unicellular, with 2000 cells/wells be inoculated with 96 orifice plates.With serum free medium culture 24h Afterwards, cell is washed with the PBS containing 1%BSA, the μ L of culture supernatant 100 of a hybridoma clone is separately added into every hole, room temperature is incubated Educate 2 hours；Washed with the PBS containing 1%BSA after 5 times, add the anti-mouse secondary antibody room temperature of biotin labeling, reacted 30 minutes；Again with containing After 1%BSA PBS is washed 5 times, the Avidin of Cy3 marks is added, is reacted at room temperature 30 minutes；PBS containing 1%BSA is washed 5 times Afterwards, fluorescence microscopy is carried out under fluorescence microscope, each monoclonal antibody hybridoma supematant in monoclonal antibody storehouse and human liver cancer stem cell is judged (human tumour multipotential stem cell) is T3A-A3 reaction.It is observed that there is part cell fluorescent staining to be judged as the positive, just Step screening acquisition can be the murine monoclonal that T3A-A3 film surface antigens are combined with human liver cancer stem cell (human tumour multipotential stem cell) Antibody.

Further, Bel7402 (Yan Li, the Zhao-You Tang, Sheng- of serum free suspension culture are selected Long Ye,Yin-Kun Liu,Jie CHEN,Qiong Xue,Jun Chen,Dong-Mei Gao,Wei-Hua Bao.Establishment of cell clones with different metastatic potential from the Metastatic hepatocellular carcinoma cell line MHCC97. worlds Gastroenterology (English Version) .2001,7 (05):Sphaerocyst 630-636.), equally carries out procedure above, and fluorescence microscopy judges monoclonal antibody storehouse In hybridoma Mab supernatant and human liver cancer stem cell (MHCC97L sphaerocysts are rich in liver-cancer stem cell) reaction, obtain energy The mouse monoclonal antibody combined with liver-cancer stem cell film surface antigen.

1 plant of mouse monoclonal antibody Hetumomab is filtered out from human tumor's multipotential stem cell mouse monoclonal antibody storehouse, It is not only combined with human tumour multipotential stem cell system T3A-A3 film surface antigens, also simultaneously with human liver cancer MHCC97L cell lines Stem cell (MHCC97L sphaerocysts) film surface antigen is combined, it was demonstrated that mouse monoclonal antibody Hetumomab can recognize difference The human liver cancer stem cell in source.Mouse monoclonal antibody Hetumomab is selected to carry out further identification and drug efficacy study.

The mouse hybridoma cell of secretion Hetumomab monoclonal antibodies is with preserving number CGMCC No.12251 March 16 in 2016 It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) (the Chaoyang District, Beijing City North Star west day No. 3 Institute of Microorganism, Academia Sinica of institute of road 1).

Hetumomab hybridomas are enlarged culture, collects and contains antibody supernatant.Using Southern Biotech The class and subclass and the ELISA secondary antibodies of Sigma companies of the monoclonal antibody subclass detection kit identification monoclonal antibody of company detect supernatant Antibody production.Experimental result shows that monoclonal antibody Hetumomab is IgG1 types heavy chain, κ type light chains.

Secretion monoclonal antibody Hetumomab hybridoma expands culture in vitro, treats that cell length, to 80%, changes serum-free Culture medium, continues after cultivating 4-5 days, and collecting secretion has the serum-free supernatant of antibody.Purified using anti-Protein G purification columns Monoclonal antibody Hetumomab.Again the pure of the Hetumomab monoclonal antibodies that identification is isolated and purified is dyed with 10%SDS-PAGE through Coomassie brilliant blue Degree.As a result show, Hetumomab heavy chain molecule amount about in 47kDa, light chain molecule amount about in 26kDa, this with it is general in theory The molecular weight of IgG antibody heavy chain and light chain is consistent.The purity of scanning analysis electrophoresis purpose band more than 95%, purifying Hetumomab monoclonal antibody purity meets the requirement of subsequent experimental.

Embodiment 2Hetumomab target antigens specifically expressing in kinds of tumor cells and tissue

First, Hetumomab target antigens are expressed in a variety of liver cancer of people, lung cancer, gastric carcinoma cell lines and can be expressed in living cells Surface.

Monoclonal antibody Hetumomab target antigens are thin in people's kinds of tumors using conventional living cells immunofluorescence dyeing technology for detection Born of the same parents system such as the expression in liver cancer, lung cancer, gastric carcinoma cell lines in liver cell surface.Particular technique method approximately as：Cell 96 well culture plates (4 × 10 of creep plate or inoculation³Individual/hole), when cell grows to 60%~70% completely, serum-free medium is washed 2 times, PBS is washed 1 time.Add primary antibody (hybridoma supematant or antibody purification), mouse anti alpha-tublin antibody (dilution factors 1:1000) as thin The whether penetrating control of born of the same parents, normal mice IgG, SP2/0 supernatant, PBS are incubated at room temperature 1h as negative control；Living cells washing lotion (contains 1%BSA PBS) wash 5 times, each 5min；4% paraformaldehyde fixes 15min at room temperature；Fixed cell washing lotion (contains 0.1% BSA, 0.05%tween-20 PBS) wash 5 times, each 5min；Add 100 μ l secondary antibody, lucifuge incubation at room temperature 30min；Gu Determine cell wash liquid 5 times, each 5min；PBS closings of the 50 μ l containing 10 μ g/ml DAPI, 50% glycerine；Under fluorescence microscope Observation.To observe that part cell film fluorescent staining occurs as positive findings.

As a result find, monoclonal antibody Hetumomab target antigens can be expressed in these following human liver cancers, lung cancer, gastric carcinoma cell lines (purchase From Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell resource center) surface of living cells：

Bel7402：MHCC97L、Bel7402-V13；

Human lung cancer cell line：A549、SPCA-1；

Human gastric cancer cell line：SNU-5、BGC-823.

The typical positive findings photo in part is as shown in Figure 1.

It these results suggest that monoclonal antibody Hetumomab target antigens in people's kinds of tumors, such as liver cancer, lung cancer, gastric carcinoma cell lines In have expression and can be expressed in the film surface of cancer cell living.

2nd, Hetumomab target antigens special high expression in human liver cancer, lung cancer, stomach organization.

Using traditional Immunohistochemistry technology, using Hetumomab as primary antibody, using anti-mouse antibody secondary antibody, inspection Expression of the monoclonal antibody Hetumomab target antigens in many cases human liver cancer, lung cancer, patients with gastric cancer and its related hetero-organization is surveyed.

Particular technique method approximately as：Tissue slice, thin piece is routinely dewaxed；Citrate buffer solution (pH6.0) pours into antigen and repaiied Multiple box, is put into slice, thin piece, will repair box and is placed in boiling water, heating water bath 30min, room temperature natural cooling 2h；PBS washs 3min × 3 It is secondary, the water on slice, thin piece is dried, circle is drawn along tissue with groupization pen immediately；Drop endogenous peroxydase resistance that every piece of tissue Jia one Disconnected solution, is incubated at room temperature 20min, washing lotion is got rid of, plus one, drop Normal animal serum --- sheep blood serum is sealed in PBS washings 3min × 3 time Close, be incubated at room temperature 20min；Get rid of on closing serum, each interlacing point plus primary antibody, be put into moisture preservation box, 4 DEG C of night incubations；Get rid of Primary antibody, PBS washings 3min × 3 time, gets rid of washing lotion, plus secondary antibody biotin- anti-mouse antibody secondary antibodies, is incubated at room temperature 20min；PBS is washed 3min × 5 time are washed, washing lotion is got rid of, plus one and drips Avidin-HRP, 10min is incubated at room temperature；PBS washs 3min × 3 time, gets rid of and washes Liquid, the DAB for drop Fresh of plus one, micro- Microscopic observation, while strict timing, after tissue is positive, running water is rinsed eventually Only；Haematoxylin redyes 5min, and -75% alcohol color separation of 1% hydrochloric acid 2 seconds, running water is rinsed；Sealed after slice, thin piece dehydration with neutral gum Piece, micro- Microscopic observation.

Using monoclonal antibody Hetumomab as primary antibody, 120 human liver cancer tissues, 20 cancer beside organisms, 10 normal hepatocytes groups are have detected Knit, in 10 hepatitis tissues, 40 cirrhotic tissues monoclonal antibody Hetumomab target antigens expression.Experimental result (table 1) shows Show：Monoclonal antibody Hetumomab target antigen is specific expressed in the human liver cancer tissue of 79.17% (95/120), and the group by cancer Knit, be showed no its expression in normal liver tissue, hepatitis tissue and cirrhotic tissue, illustrate that monoclonal antibody Hetumomab target antigens exist Special high expression in human liver cancer tissue.

The result that the immunohistochemistry technique of table 1. detection monoclonal antibody Hetumomab target antigens are expressed in human liver cancer tissue

Using monoclonal antibody Hetumomab as primary antibody, 160 human lung cancer tissues, 32 cancer beside organisms, 3 normal lung groups are have detected Knit the expression of middle monoclonal antibody Hetumomab target antigens.Experimental result (table 2) is shown：Monoclonal antibody Hetumomab target antigen exists It is specific expressed in the human lung cancer tissue of 82.5% (132/160), and only expressed in 6.25% cancer beside organism, normal Have no that it is expressed in lung tissue, illustrate the special high expression in human lung cancer tissue of monoclonal antibody Hetumomab target antigens.

The immunohistochemistry technique of table 2. detects expression of the Hetumomab target antigens in human lung cancer tissue

Using monoclonal antibody Hetumomab as primary antibody, 110 Human Stomach Tissues, 20 cancer beside organisms, 3 normal gastric groups are have detected Knit the expression of middle monoclonal antibody Hetumomab target antigens.Experimental result (table 3) is shown：Monoclonal antibody Hetumomab target antigen exists It is specific expressed in the Human Stomach Tissue of 93.64% (103/110), and its table is showed no in cancer beside organism and normal gastric mucosa Reach, illustrate the special high expression in Human Stomach Tissue of monoclonal antibody Hetumomab target antigens.

The result that the immunohistochemistry technique of table 3. detection monoclonal antibody Hetumomab target antigens are expressed in Human Stomach Tissue

The typical positive findings photo in part of above SABC testing result is as shown in Figure 2.

The above result illustrates monoclonal antibody Hetumomab target antigens in people's kinds of tumors, for example liver cancer, lung cancer, stomach cancer There is special high expression in the cancerous tissue of most of patients.

The monoclonal antibody Hetumomab of embodiment 3 recognizes tumor stem cell

First, the cancer cell of monoclonal antibody Hetumomab identifications is enriched with the sphere culture cells of cancer cell.

Reported according to existing lot of documents, serum free suspension culture is that can be enriched with Tumor Stem thin for sphere (balling-up) cultures Born of the same parents (Reynolds, B.A.and S.Weiss, " Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell."Dev Biol, 1996.175(1):p.1-13.；Fang,N.,et al.,"pH responsive adhesion of phospholipid vesicle on poly(acrylic acid)cushion grafted to poly(ethylene terephthalate) surface."Colloids Surf B Biointerfaces,2005.42(3-4):p.245-52.；And Tirino, V., et al.,"The role of CD133in the identification and characterisation of tumour- initiating cells in non-small-cell lung cancer."Eur J Cardiothorac Surg, 2009.36(3):p.446-53.).So can the cancer cell that recognized according to monoclonal antibody Hetumomab obtain in sphere cultures Whether enrichment carrys out the preliminary cell for judging monoclonal antibody Hetumomab identifications related to tumor stem cell.

After having carried out free serum culture 5 days to Bel7402 Bel7402-V13 and MHCC97L cell, using living thin Born of the same parents' streaming fluorescence art cultivates Hetumomab in cell to parental cell and sphere⁺Cell is detected.Experimental result (table 4) Hetumomab is shown⁺Cell is 8.92% in the ratio of Bel7402-V13sphere cells, than it in parental cell Ratio 2.25% is enriched 3.96 times；Hetumomab⁺Ratio of the cell in MHCC97L sphere cells is 7.51%, than Its ratio 3.45% in parental cell is enriched 2.18 times.I.e. after free serum culture, liver cancer cell lines Hetumomab⁺Cell is enriched with.

Sphere of the liver cancer cells in liver cancer cells of streaming fluorescent technique detection monoclonal antibody Hetumomab identifications is immunized in table 4. The result being enriched with culture cell

It is glimmering using living cells streaming after having carried out free serum culture 5 days to human lung cancer cell line SPCA-1 and A549 cell Light art cultivates Hetumomab in cell to parental cell and sphere⁺Cell is detected.Experimental result (table 5) is shown Hetumomab⁺Cell is 7.34% in the ratio of SPCA-1 sphere cells, than its ratio 1.74% in parental cell It is enriched 4.22 times；Hetumomab⁺Ratio of the cell in A549sphere cells is 13.30%, than it in parental cell Ratio 7.23% be enriched 1.84 times.I.e. after free serum culture, the Hetumomab of lung cancer cell line⁺Cell is obtained Enrichment.

Sphere of the lung carcinoma cell in lung carcinoma cell of streaming fluorescent technique detection monoclonal antibody Hetumomab identifications is immunized in table 5. The result being enriched with culture cell

After having carried out free serum culture 5 days to human gastric cancer cell line SNU-5 and BGC-823 cell, using living cells streaming Fluorescence art cultivates Hetumomab in cell to parental cell and sphere⁺Cell is detected.Experimental result (table 6) is shown Hetumomab⁺Cell is 7.19% in the ratio of SNU-5sphere cells, richer than its ratio 3.38% in parental cell 2.13 times are collected；Hetumomab⁺Ratio of the cell in BGC-823sphere cells is 7.45%, than it in parental cell Ratio 2.95% be enriched 2.53 times.I.e. after free serum culture, the Hetumomab of gastric carcinoma cell lines⁺Cell is obtained Enrichment.

Sphere of the stomach cancer cell in stomach cancer cell of streaming fluorescent technique detection monoclonal antibody Hetumomab identifications is immunized in table 6. The result being enriched with culture cell

The typical collection of illustrative plates in part of immune streaming fluoroscopic examination result is as shown in Figure 3 above.

The above result illustrates the cancer cell of monoclonal antibody Hetumomab identifications in people's kinds of tumors, for example liver cancer, lung cancer, Equal significant enrichment in the sphere culture cells of gastric carcinoma cell lines.

2nd, monoclonal antibody Hetumomab recognizes the positive tumor stem cell of ESA, CD90.

It there is now many documents (Yamashita T, et al.EpCAMpositive hepatocellular carcinoma cells are tumor-initiating cells with stem/progenitor cell features.Gastroenterology.2009,136(3):1012-1024.；With Yang ZF.Identification of local and circulating cancer stem cells in human liver cancer.Hepatology.2008,47(3):919-928.) demonstrate the Tumor Stem that ESA and CD90 is some liver cancer cells thin Cellular surface mark.In order to detect that monoclonal antibody Hetumomab knows liver cancer cells simultaneously in human liver cancer cell Bel7402-V13 cells It is also the liver-cancer stem cell of ESA, CD90 positive markers, we use Two-color flow fluorescence art, in serum free medium The human liver cancer Bel7402-V13 cells of culture 5 days are dyed.

As a result (such as table 7) is shown, the cell proportion of monoclonal antibody Hetumomab identifications is 8.52%, ESA⁺Stem cell expression ratio Example is 9.02%, and both contaminate ratio and identify 40.2% ESA for 3.63%, i.e. monoclonal antibody Hetumomab altogether⁺Stem cell.To The human liver cancer MHCC97L cells cultivated 5 days in serum free medium are dyed, and as a result (such as table 7) is shown, monoclonal antibody The cell proportion of Hetumomab identifications is 2.38%, CD90⁺Stem cell expression ratio is 4.54%, and both contaminate ratio and are altogether 2.01%, i.e. monoclonal antibody Hetumomab identify 44.3% CD90⁺Stem cell.

The Two-color flow fluorescence art of table 7. detects monoclonal antibody Hetumomab with ESA, CD90 liver-cancer stem cell surface marker in liver The result dyed altogether in cancer cell

The above result illustrates the human tumour stem cell of the positive markers such as monoclonal antibody Hetumomab identifications ESA, CD90.

3rd, the cancer cell of monoclonal antibody Hetumomab identifications has stronger self-renewal capacity

Self-renewal capacity, strong invasive ability, tolerance chemotherapeutics ability and strong tumorigenesis ability are tumor stem cell differences In the important essential characteristic of common filial generation tumour cell.Therefore, in order to which the cell for further verifying monoclonal antibody Hetumomab identifications is It is no that there is tumor stem cell characteristic, sort the Hetumomab in a variety of human tumor cells⁺Cell, detects its self-renewing, invades Attack, resistance and internal tumorigenesis ability.

The form of expression that the self-renewal capacity of tumor stem cell is main is the balling-up ability in serum free medium, its When mode is referred to as the cell division of Asymmetric division, i.e., one into two daughter cells, one of daughter cell remains and parent For the identical feature of cell, and another daughter cell can continue division, form normal daughter cell.Therefore, may be used With by detecting Hetumomab⁺Balling-up ability of the cell in serum free medium determines its self-renewal capacity.

Isolated using flow sorting techniques from the human liver cancer Bel7402-V13sphere cells of culture 5 days Hetumomab⁺Cell, parental cell and Hetumomab^-Cell.The cell that sorting is obtained is inoculated in 500/hole to be contained 0.8% methylcellulose semisolid sphere culture mediums (semisolid sphere culture mediums be containing 0.8% methylcellulose, 20ng/mL EGF, 20ng/mL bFGF, by 1:50 ratios addition B27,10ng/mL LIF, 2mmol/mL glutamine, 1u/mL Heparin DMEM/F12 (1:1) nutrient solution) in, cultivated in 24 orifice plates of ultralow adhesion, observe each cell balling-up quantity.Knot Fruit shows (table 8), Hetumomab⁺Cell, parental cell and Hetumomab^-Balling-up of the cell under the conditions of serum free medium Number is respectively 262 ± 8.5,168 ± 5.6,98 ± 5.6, i.e. Hetumomab⁺The balling ratio of cell is thin apparently higher than other two kinds Born of the same parents (p<0.05).Therefore, Hetumomab⁺Cell is than parental cell and Hetumomab^-Cell has stronger self-renewing energy Power.

Hetumomab in the liver cancer cells of table 8⁺Cell, parental cell and Hetumomab^-The comparison of cell self-renewal ability

Using flow sorting techniques Hetumomab is isolated from human lung cancer SPCA-1 sphere cells⁺Cell, parent Cell and Hetumomab^-Cell.It is solid that the cell that sorting is obtained is inoculated in half containing 0.8% methylcellulose with 500/hole In state sphere culture mediums, cultivated in 24 orifice plate plates of ultralow adhesion, observe each cell balling-up quantity.As a result (table 8) is shown, Hetumomab⁺Cell, parental cell and Hetumomab^-Balling-up number of the cell under the conditions of serum free medium is respectively 165.7 ± 6.0,127 ± 5.6,83.7 ± 4.7, i.e. Hetumomab⁺The balling ratio of cell is apparently higher than other two kinds of cell (p< 0.05).Therefore, Hetumomab⁺Cell is than parental cell and Hetumomab^-Cell has stronger self-renewal capacity.

Hetumomab in the lung carcinoma cell of table 9⁺Cell, parental cell and Hetumomab^-The comparison of cell self-renewal ability

Using flow sorting techniques Hetumomab is isolated from human gastric cancer SNU-5sphere cells⁺Cell, Qin Benxi Born of the same parents and Hetumomab^-Cell.Obtained cell will be sorted and the semisolid containing 0.8% methylcellulose is inoculated in 500/hole In sphere culture mediums, cultivated in 24 orifice plate plates of ultralow adhesion, observe each cell balling-up quantity.As a result (table 10) is shown, Hetumomab⁺Cell, parental cell and Hetumomab^-Balling-up number of the cell under the conditions of serum free medium be respectively 24 ± 1.4th, 15.5 ± 0.7,11.5 ± 0.7, i.e. Hetumomab⁺The balling ratio of cell is apparently higher than other two kinds of cell (p<0.05). Therefore, Hetumomab⁺Cell is than parental cell and Hetumomab^-Cell has stronger self-renewal capacity.

Hetumomab in the stomach cancer cell of table 10⁺Cell, parental cell and Hetumomab^-The ratio of cell self-renewal ability Compared with

The above result illustrates that a variety of cancer cells (liver cancer, lung cancer, stomach cancer) of monoclonal antibody Hetumomab identifications are respectively provided with more Strong self-renewal capacity, i.e., with one of tumor stem cell principal character：High self-renewal capacity.

4th, the cancer cell of monoclonal antibody Hetumomab identifications has stronger invasive ability

Self-renewal capacity, strong invasive ability, tolerance chemotherapeutics ability and strong tumorigenesis ability are tumor stem cell differences In the important essential characteristic of common filial generation tumour cell.Therefore, in order to which the cell for further verifying monoclonal antibody Hetumomab identifications is It is no that there is tumor stem cell characteristic, therefore sorted the Hetumomab in a variety of human tumor cells⁺Cell, detect its self-renewing, Invasion and attack, resistance and internal tumorigenesis ability.

Isolated using flow sorting techniques from the human liver cancer Bel7402-V13sphere cells of culture 5 days Hetumomab⁺Cell, parental cell and Hetumomab^-Cell.Obtained cell will be sorted and advance bag is inoculated in identical quantity By in the Transwell cells of Matrigel glue, fixed after 24h, micro- Microscopic observation wears the cell number of film.As a result (table is shown 11), Hetumomab⁺Cell, parental cell and Hetumomab^-The cell number of cell-penetrating for be respectively per the visual field 308 ± 9.5, 210 ± 10.7 and 132 ± 14.7, i.e. Hetumomab⁺The invasion and attack of cell are through the quantity of film apparently higher than other two kinds of cell (p <0.05).Therefore, Hetumomab⁺Cell is than parental cell and Hetumomab^-Cell has stronger invasive ability.

Hetumomab in the liver cancer cells of table 11⁺Cell, parental cell and Hetumomab^-The comparison of cell invasion ability

Using flow sorting techniques Hetumomab is isolated from the human lung cancer SPCA-1 sphere cells of culture⁺Carefully Born of the same parents, parental cell and Hetumomab^-Cell.Obtained cell will be sorted and coating Matrigel in advance is inoculated in identical quantity Fixed after 24h in the Transwell cells of glue, micro- Microscopic observation wears the cell number of film.As a result (table 12) is shown, Hetumomab⁺Cell, parental cell and Hetumomab^-The cell number of cell-penetrating for be respectively per the visual field 222 ± 11.5, 193.7 ± 5.7,154.3 ± 12.1, i.e. Hetumomab⁺The invasion and attack of cell are through the quantity of film apparently higher than other two kinds of cells (p<0.05).Therefore, Hetumomab⁺Cell is than parental cell and Hetumomab^-Cell has stronger invasive ability.

Hetumomab in the lung carcinoma cell of table 12⁺Cell, parental cell and Hetumomab^-The comparison of cell invasion ability

Using flow sorting techniques Hetumomab is isolated from the human gastric cancer SNU-5sphere cells of culture⁺Cell, Parental cell and Hetumomab^-Cell.Obtained cell will be sorted and advance coating Matrigel glue is inoculated in identical quantity Fixed after 24h in Transwell cells, micro- Microscopic observation wears the cell number of film.As a result (table 13) is shown, Hetumomab⁺ Cell, parental cell and Hetumomab^-The cell number of cell-penetrating is respectively per the visual field 247.5 ± 19.1,142.5 ± 9.2 With 145.0 ± 11.3, i.e. Hetumomab⁺The invasion and attack of cell are through the quantity of film apparently higher than other two kinds of cell (p<0.05). Therefore, Hetumomab⁺Cell is than parental cell and Hetumomab^-Cell has stronger invasive ability.

Hetumomab in the stomach cancer cell of table 13⁺Cell, parental cell and Hetumomab^-The comparison of cell invasion ability

The above result illustrates that a variety of cancer cells (liver cancer, lung cancer, stomach cancer) of monoclonal antibody Hetumomab identifications are respectively provided with more Strong invasive ability, i.e., with one of tumor stem cell principal character：High invasion.

5th, the cancer cell of monoclonal antibody Hetumomab identifications has the ability of stronger tolerance chemotherapeutics

In order to detect Hetumomab⁺The resistance ability of liver cancer cells, human liver cancer cell Bel7402-V13sphere is thin The Hetumomab that born of the same parents' airflow classification is obtained⁺Cell, parental cell and Hetumomab^-Cell is inoculated in the quantity in 5000/hole In 96 orifice plates, every group of cell is with containing 0 μ g/mL, 0.0625 μ g/mL, 0.125 μ g/mL, 0.25 μ g/mL, 0.5 μ g/mL, 1 μ g/ The complete medium culture of the cis-platinum of mL, 2 μ g/mL and 48 kinds of various concentrations of μ g/mL, changes 1 subculture, after 7 days after 3 days Detect that OD values determine the IC50 of its resistance ability of reflection with CCK8 methods.Experimental result shows (table 14, Fig. 4), Hetumomab⁺Carefully Born of the same parents, parental cell and Hetumomab^-The IC50 of cell is respectively 0.739 μ g/mL, 0.502 μ g/mL and 0.313 μ g/mL, i.e., Hetumomab⁺The drug resistance of cell is apparently higher than parental cell and Hetumomab^-Cell, the statistically significant (p of difference< 0.05).Therefore, Hetumomab⁺Liver cancer cells are than parental cell and Hetumomab^-Cell has stronger tolerance chemotherapeutics Ability.

Hetumomab in the liver cancer cells of table 14⁺Cell, parental cell and Hetumomab^-Cell tolerance chemotherapy medicine ability Comparison

In order to detect Hetumomab⁺The resistance ability of lung carcinoma cell, will flow in human lung carcinoma cell SPCA-1sphere cells The Hetumomab that formula sorting is obtained⁺Cell, parental cell and Hetumomab^-Cell is inoculated in 96 holes with the quantity in 5000/hole In plate, every group of cell is with containing 0 μ g/mL, 0.2 μ g/mL, 0.4 μ g/mL, 0.6 μ g/mL, 0.8 μ g/mL, 1 μ g/mL and 2 μ g/mL The complete medium culture of the cis-platinum of 7 kinds of various concentrations, is determined with CCK8 methods detection OD values reflect its resistance ability afterwards IC50.Experimental result shows (table 15, Fig. 4), Hetumomab⁺Cell, parental cell and Hetumomab^-The IC50 difference of cell For 0.707 μ g/mL, 0.513 μ g/mL and 0.180 μ g/mL, i.e. Hetumomab⁺The drug resistance of cell is apparently higher than parental cell And Hetumomab^-Cell, the statistically significant (p of difference<0.05).Therefore, Hetumomab⁺Lung carcinoma cell than parental cell and Hetumomab^-Cell has the ability of stronger tolerance chemotherapeutics.

Hetumomab in the lung cancer cancer cell of table 15⁺Cell, parental cell and Hetumomab^-Cell tolerance chemotherapy medicine energy The comparison of power

In order to detect Hetumomab⁺The resistance ability of stomach cancer cell, we are by gastric carcinoma cells SNU-5sphere cells The Hetumomab that middle airflow classification is obtained⁺Cell, parental cell and Hetumomab^-Cell is inoculated in the quantity in 5000/hole In 96 orifice plates, every group of cell is with containing 0 μ g/mL, 0.0125 μ g/mL, 0.025 μ g/mL, 0.05 μ g/mL, 0.1 μ g/mL, 0.2 μ The complete medium culture of the cis-platinum of g/mL, 0.4 μ g/mL and 0.8 8 kinds of various concentrations of μ g/mL, afterwards with CCK8 methods detection OD Value determines the IC50 for reflecting its resistance ability.Experimental result shows (table 16, Fig. 4), Hetumomab⁺Cell, parental cell and Hetumomab^-The IC50 of cell is respectively 0.285 μm of ol/L, 0.155 μm of ol/L and 0.094 μm of ol/L, i.e. Hetumomab⁺Carefully The drug resistance of born of the same parents is apparently higher than parental cell and Hetumomab^-Cell, the statistically significant (p of difference<0.05).Therefore, Hetumomab⁺Stomach cancer cell is than parental cell and Hetumomab^-Cell has the ability of stronger tolerance chemotherapeutics.

Hetumomab in the stomach cancer cell of table 16⁺Cell, parental cell and Hetumomab^-Cell tolerance chemotherapy medicine ability Comparison

The above result illustrates that a variety of cancer cells (liver cancer, lung cancer, stomach cancer) of monoclonal antibody Hetumomab identifications are respectively provided with more Strong tolerance chemotherapeutics ability, i.e., with one of tumor stem cell principal character：Strong drug resistance.

6th, the cancer cell of monoclonal antibody Hetumomab identifications has stronger internal tumorigenesis ability

Whether examine a kind of cell is that " goldstandard " of tumor stem cell is internal strong oncogenicity.Therefore, by human liver cancer The Hetumomab that cell Bel7402-V13sphere cells airflow classification is obtained⁺Cell, parental cell and Hetumomab^-Cell The nude mice by subcutaneous of 4 week old sizes is seeded to respectively, and it is observed for a long time in vivo into the situation of knurl.As a result it is as shown in table 17,1 × 10⁴Individual Hetumomab⁺Cell when being inoculated with 3 weeks can in Mice Body tumorigenesis, and parental cell then needs 1 × 10⁵Individual cell exists It is inoculated with 3 Zhou Houcai and goes out knurl, Hetumomab^-Cell does not go out knurl all the time during observing.The result explanation of this Classic Experiments Hetumomab⁺The internal oncogenicity of cell is compared with parent and Hetumomab^-Cell is significantly strong.Point out Hetumomab⁺Cell has The high oncogenicity feature of tumor stem cell, meets the judgement " goldstandard " of tumor stem cell.Therefore, monoclonal antibody Hetumomab is recognized Cell be hepatic carcinoma stem cell.

Hetumomab in the liver cancer cells Bel7402-V13 of table 17⁺Cell, parental cell and Hetumomab^-Caused in cell body The comparison of knurl ability (into the animal number of elements of knurl)

The Hetumomab that gastric carcinoma cells SNU-5sphere cells airflow classification is obtained⁺Cell, parental cell and Hetumomab^-Cell is seeded to the nude mice by subcutaneous of 4 week old sizes respectively, and it is observed for a long time in vivo into the situation of knurl.As a result such as Shown in table 18,2 × 10³Individual Hetumomab⁺Cell when being inoculated with March can in Mice Body half mouse tumorigenesis, and parent is thin Born of the same parents 2 × 10³Individual cell does not go out knurl, Hetumomab yet after being inoculated with 4 months^-Cell does not go out knurl all the time during observing.This The result of Classic Experiments illustrates Hetumomab⁺The internal oncogenicity of cell is compared with parent and Hetumomab^-Cell is significantly strong.Prompting Hetumomab⁺Cell has the high oncogenicity feature of tumor stem cell, meets the judgement " goldstandard " of tumor stem cell.Therefore, The cell of monoclonal antibody Hetumomab identifications is gastric cancer tumor stem cell.

Hetumomab in the stomach cancer cell SNU-5 of table 18⁺Cell, parental cell and Hetumomab^-Tumorigenesis ability in cell body Comparison (into the animal number of elements of knurl)

It is stronger that the above result illustrates that a variety of cancer cells (liver cancer, stomach cancer) of monoclonal antibody Hetumomab identifications are respectively provided with Internal tumorigenesis ability, i.e., with the high oncogenicity of one of tumor stem cell principal character.

The monoclonal antibody Hetumomab of embodiment 4 can suppress the self-renewing, invasion and attack and resistance function of tumor stem cell

First, monoclonal antibody Hetumomab significantly inhibit the tumor stem cell of kinds of tumors (liver cancer, stomach cancer, lung cancer) self more New ability (one of tumor stem cell principal character).

The form of expression that the self-renewal capacity of tumor stem cell is main is the balling-up ability in serum free medium, its When mode is referred to as the cell division of Asymmetric division, i.e., one into two daughter cells, one of daughter cell remains and parent For the identical feature of cell, and another daughter cell can continue division, form normal daughter cell.In order to prove Whether monoclonal antibody Hetumomab is that can directly suppress the Functional antibody of liver-cancer stem cell, and we will be in serum free medium Bel7402's Bel7402-V13sphere cells of culture 5 days are prepared into after single cell suspension, with the monoclonal antibody of purifying Hetumomab (250 μ g/mL) is experimental group, using PBS as negative control group, and 2h is incubated at 37 DEG C with cell, during which small every half When cell and antibody or negative control are mixed once.It is solid that each group takes 500 cells to be inoculated in half containing 0.8% methylcellulose In state sphere culture mediums (containing EGF, LIF, bFGF etc.), cultivated in 24 orifice plate plates of ultralow adhesion, every other day fluid infusion 1-1.5mL, Two groups of cell balling-up quantity are observed after 14 days.Experimental result (Fig. 5) shows that the balling-up quantity of experimental group is 212 ± 2.8, and cloudy Property control group balling-up quantity be 278.5 ± 0.7, hence it is evident that higher than experimental group.Monoclonal antibody Hetumomab is thin to Bel7402-V13 The balling-up inhibiting rate of born of the same parents is up to 23.9%, p<0.05, there is significant difference.As a result show, monoclonal antibody Hetumomab can be done directly on Liver-cancer stem cell simultaneously suppresses its self-renewal capacity.

In order to prove whether monoclonal antibody Hetumomab is that can directly suppress the Functional antibody of lung cancer stem cell, using same The method of sample have detected inhibitory action of the monoclonal antibody Hetumomab to human lung cancer cell line's SPCA-1 balling-up.As a result (such as table 19, figure 5) show, the balling-up quantity of experimental group highest antibody concentration is 88.3 ± 7.2, and the balling-up quantity of negative control group is 151.3 ± 9.1, hence it is evident that higher than experimental group, monoclonal antibody Hetumomab is to the balling-up inhibiting rates of SPCA-1 cells up to 41.6%, p<0.01, There is significant difference.As a result show, monoclonal antibody Hetumomab can be done directly on lung cancer stem cell and suppress its self-renewing energy Power.

The detection monoclonal antibody of table 19 Hetumomab suppresses the result of human lung cancer cell line's SPCA-1 balling-up

In order to prove whether monoclonal antibody Hetumomab is that can directly suppress the Functional antibody of stomach cancer stem cell, using same The method of sample have detected monoclonal antibody Hetumomab to human gastric cancer cell line SNU-5 CD44 positive cell tumor stem cell subgroups into The inhibitory action of ball.As a result (such as table 20, Fig. 5) is shown, the balling-up quantity of experimental group highest antibody concentration is 18 ± 2.0, and cloudy Property control group balling-up quantity be 128 ± 4.0, hence it is evident that higher than experimental group, monoclonal antibody Hetumomab is to SNU-5 CD44⁺Cell Balling-up inhibiting rate up to 85.9%, p<0.01, there is significant difference.As a result show, monoclonal antibody Hetumomab can be done directly on stomach Cancer stem cell simultaneously suppresses its self-renewal capacity.

The detection monoclonal antibody of table 20 Hetumomab suppresses human gastric cancer cell line SNU-5 CD44⁺The result of cell balling-up

The above result illustrates that monoclonal antibody Hetumomab can directly significantly inhibit a variety of cancer cells (liver cancer, lung cancer, stomach Cancer) self-renewal capacity, it was demonstrated that monoclonal antibody Hetumomab can not only recognize targeting tumor stem cells, and be directly to press down Feature (therapeutic) resisting tumour stem cells monoclonal antibody of tumor stem cell processed.

2nd, monoclonal antibody Hetumomab significantly inhibits the invasion and attack energy of the tumor stem cell of kinds of tumors (liver cancer, stomach cancer, lung cancer) Power (the two of tumor stem cell principal character).

High invasion is another important biomolecule feature of tumor stem cell.In order to whether prove monoclonal antibody Hetumomab It is that can directly suppress the Functional antibody of liver-cancer stem cell, monoclonal antibody Hetumomab is suppressed using Transwell Matrigels The invasive ability of liver cancer cells is analyzed.Experimental result shows (Fig. 6) that the invasion cell number of PBS negative control groups is (301.0 ± 16.3)/visual field, the invasion cell number of monoclonal antibody Hetumomab (0.5mg/ml) groups is (148 ± 16.4)/visual field.It is real Test result to show, the cell invasion ability after monoclonal antibody Hetumomab directly effects substantially weakens, and it is to Bel7402- The inhibiting rate of V13sphere cell invasions is 50.8%, p<0.05, there is significant difference.Should test result indicates that, monoclonal antibody Hetumomab can be done directly on liver-cancer stem cell and suppress its invasive ability.

In order to prove whether monoclonal antibody Hetumomab is that can directly suppress the Functional antibody of lung cancer stem cell, using same The method of sample have detected the inhibitory action that monoclonal antibody Hetumomab is attacked to human lung cancer cell line SPCA-1.Experimental result shows (table 21st, Fig. 6), the invasion cell number of PBS negative control groups is (232.3 ± 3.1)/visual field, and the invasion and attack of monoclonal antibody Hetumomab groups are thin Minimum (153.0 ± the 6.1)/visual field of born of the same parents' number.Test result indicates that, monoclonal antibody Hetumomab directly act on after cell invasion energy Power substantially weakens, and its inhibiting rate to SPCA-1 sphere cell invasions is 34.1%, p<0.05, there is significant difference.The reality Test result to show, monoclonal antibody Hetumomab can be done directly on lung cancer stem cell and suppress its invasive ability.

The detection monoclonal antibody of table 21 Hetumomab suppresses the result of human lung cancer cell line SPCA-1 invasion and attack

In order to prove whether monoclonal antibody Hetumomab is that can directly suppress the Functional antibody of stomach cancer stem cell, using same The method of sample have detected monoclonal antibody Hetumomab and human gastric cancer cell line SNU-5 CD44 positive cell tumor stem cell subgroups invaded The inhibitory action attacked.Experimental result shows (table 22, Fig. 6), and the invasion cell numbers of PBS negative control groups is (231 ± 7.0)/regard Open country, minimum (56 ± the 4.0)/visual field of invasion cell number of monoclonal antibody Hetumomab groups.Test result indicates that, monoclonal antibody Hetumomab Cell invasion ability directly after effect substantially weakens, and it is to SNU-5 CD44⁺The inhibiting rate of cell invasion is 75.8%, p< 0.05, there is significant difference.Should test result indicates that, monoclonal antibody Hetumomab can be done directly on stomach cancer stem cell and suppress it Invasive ability.

The detection monoclonal antibody of table 21 Hetumomab suppresses human gastric cancer cell line SNU-5 CD44⁺The result of cell invasion

The above result illustrates that monoclonal antibody Hetumomab can directly significantly inhibit a variety of cancer cells (liver cancer, lung cancer, stomach Cancer) invasive ability, it was demonstrated that monoclonal antibody Hetumomab can not only recognize targeting tumor stem cells, and be that can directly suppress swollen Feature (therapeutic) resisting tumour stem cells monoclonal antibody of knurl stem cell.

3rd, monoclonal antibody Hetumomab significantly inhibits the tolerance chemotherapeutics of the tumor stem cell of kinds of tumors (liver cancer, lung cancer) Ability (the three of tumor stem cell principal character).

Resistance is one of biological property of tumor stem cell.In order to prove monoclonal antibody Hetumomab whether be can be direct Suppress the Functional antibody of liver-cancer stem cell, the Bel7402-V13sphere cells of 5 days will be cultivated with the quantity in 5000/hole It is inoculated in 96 orifice plates, and adds the monoclonal antibody Hetumomab (0.5mg/mL) and PBS of purifying in each hole, in 37 DEG C, 5%CO₂ Cultivated in incubator after 24h, remove the culture medium containing antibody, every group of cell with containing 0,0.0625,0.125,0.25,0.5,1,2, The medium culture cell of 4 and 8 μ g/mL totally 9 various concentrations cis-platinums, 48h changes 1 complete medium containing cis-platinum, is pressed after 5 days CCK-8 reagents are added according to CCK-8 kit specifications, OD450 absorbances are detected, and calculate each group IC50 values.Experimental result Show (Fig. 7), the resistance ability of the cell after monoclonal antibody Hetumomab directly effects is decreased obviously, and its IC50 value is 0.334 μ g/ ML, and the IC50 values of control group are 0.9 μ g/mL, the cells resistance ability that monoclonal antibody Hetumomab is directly acted on is significantly lower than control Group.Should test result indicates that, monoclonal antibody Hetumomab can be done directly on liver-cancer stem cell and suppress the ability of its resistance.

In order to prove whether monoclonal antibody Hetumomab is that can directly suppress the Functional antibody of lung cancer stem cell, using same The method of sample have detected inhibitory action of the monoclonal antibody Hetumomab to human lung cancer cell line's SPCA-1 resistances.Experimental result shows (figure 7), the resistance ability of the cell after monoclonal antibody Hetumomab directly effects is decreased obviously, the minimum 0.136 μ g/mL of its IC50 value, And the IC50 values of control group are 0.351 μ g/mL, the cells resistance ability that monoclonal antibody Hetumomab is directly acted on is significantly lower than control Group.Should test result indicates that, monoclonal antibody Hetumomab can be done directly on lung cancer stem cell and suppress the ability of its resistance.

The above result illustrates that monoclonal antibody Hetumomab can directly significantly inhibit a variety of cancer cells (liver cancer, lung cancer) It is resistant to the ability of chemotherapeutics, it was demonstrated that monoclonal antibody Hetumomab can not only recognize targeting tumor stem cells, and be directly to press down Feature (therapeutic) resisting tumour stem cells monoclonal antibody of tumor stem cell processed.

The monoclonal antibody Hetumomab of embodiment 5 has in animal body suppresses the growth of tumour transplatation knurl, the pharmacodynamics of collaboration chemotherapy Effect

A series of experiments result above proves that monoclonal antibody Hetumomab is the monoclonal antibody for targetting kinds of tumors stem cell；And Pharmacodynamics in vitro result of study shows that monoclonal antibody Hetumomab can significantly suppress the self-renewing of kinds of tumors stem cell, invade Attack and resistance ability.For the shadows of further clear and definite monoclonal antibody Hetumomab in vivo to kinds of tumors growth, transfer and resistance Ring, using a variety of Gene expressions, have rated monoclonal antibody Hetumomab in vivo to kinds of tumors growth, transfer and resistance Pharmacodynamic action.

First, monoclonal antibody Hetumomab significantly inhibits the growth of human liver cancer transplantable tumor in vivo, and can substantially cooperate with enhancing chemotherapy Curative effect, significantly extend life cycle, with significant antitumor pharmacodynamic action.

Experimental studies of the monoclonal antibody Hetumomab in nude mice interior therapeutic liver cancer has been carried out, has been observed and relatively more alone monoclonal antibody, list The curative effect of liver cancer is treated with chemotherapeutic and monoclonal antibody combined chemotherapy medicine, can com-parison and analysis monoclonal antibody Hetumomab treat liver in vivo The growth of cancer and resistance, and inquire into the optimal case of liver cancer targeting stem-cell therapy liver cancer.

By Bel7402-V13 sphere cells with 30,000/be only seeded to nude mice by subcutaneous, nude mice is randomly divided into 6 group (6 Only/group), be respectively：Independent chemotherapeutic group (cis-platinum 0.3mg/kg, 6)；Antibody high dose group (10mg/kg, 6)；Antibody is high Dosage+chemotherapy group (6)；Antibody low dose group (2.5mg/kg, 6)；Antibody low dosage+chemotherapy group (6)；PBS groups (6 Only).Start within second day after inoculating cell to terminate treatment after treatment, weekly treatment 2 times, 5 weeks.Subcutaneous transplantation knurl is measured 2 times a week Major diameter and minor axis, with formula V=(π/6) × (major diameter × minor axis × minor axis) calculate knurl volume, observe each group gross tumor volume, The change of growth rate.After drug withdrawal, continue to observe the growing state of transplantable tumor, and record tumor size.Tumor growth rate= (V_t-V₀)/number of days, V_tGross tumor volume when being each measurement, V₀It is the gross tumor volume (V before administration₀Refer to tumour when stopping administration Volume).

The growth curve of transplantable tumor is as shown in figure 8, monoclonal antibody Hetumomab can significantly inhibit transplanting in nude mouse in Mice Body The growth of knurl.And also gradually risen with the dosage rise transplantable tumor inhibiting rate of antibody, there is dose-dependence.Experiment As a result such as Fig. 9 is shown, when treating the drug withdrawal of 5 week, and the monoclonal antibody Hetumomab of high and low dose divides the inhibiting rate of transplantable tumor Not Wei 71.5%, 54.4%, the inhibiting rate of chemotherapeutic group is 83.5%, the inhibiting rate of monoclonal antibody high and low dose combined chemotherapy medicine group It is similar, all reach 97% or so.As a result point out, compared with alone monoclonal antibody and alone chemotherapeutic group, monoclonal antibody combined chemotherapy medicine group Preferable therapeutic effect is shown in the treatment of transplantable tumor.

When being discontinued one month, there is dead situation in mouse.At this moment as shown in Figure 10, monoclonal antibody is high and low for the inhibiting rate of each group Inhibiting rate to transplantable tumor is respectively 49.1%, 34.4%, and the inhibiting rate of the high and low combined chemotherapy medicine group of monoclonal antibody is similar, about 84.5% or so, but the inhibiting rate of alone chemotherapeutic group is 48.6%.The result is pointed out, and monoclonal antibody combined chemotherapy medicine group is moved to mouse The inhibiting rate for planting knurl is higher than other several therapeutic scheme groups, P<0.05, difference is statistically significant.Shifting when being discontinued latter month Compared with planting knurl volume when being discontinued, the tumor growth rate of monoclonal antibody combined chemotherapy group is 0.051cm³/ day, than PBS control group Tumor growth rate (0.239cm³/ day) it is low 4.7 times, and than the tumor growth rate of monoclonal antibody high and low dose group and chemotherapeutic group (0.148cm³/ day, 0.185cm³/ day and 0.154cm³/ day) respectively it is low by 2.9,3.6 and 3.1 times, the result show monoclonal antibody combine The method of chemotherapeutic can effectively suppress the growth and recurrence of tumour.

Whole treatment and observation process continue six months.Mouse shows in six middle of the month survivorship curves (Figure 11), this 6 groups small The survivorship curve difference in distribution of mouse is statistically significant, P<0.05.Illustrate that the mouse survival situation of monoclonal antibody combined chemotherapy medicine group is bright It is aobvious to be better than PBS control group, monoclonal antibody group and chemotherapeutic group.Prompting, monoclonal antibody combined chemotherapy medicine treatment can extend the life cycle of mouse.

It these results suggest that, monoclonal antibody Hetumomab, which is used alone, can significantly inhibit the growth of human liver cancer transplantable tumor, have The significant pharmacodynamic action for suppressing liver cancer.Monoclonal antibody Hetumomab combined chemotherapy medicine groups all show the suppression high to transplantable tumor Rate processed, can significantly inhibit the growth of tumour, and therapeutic effect is better than alone antibodyome and alone chemotherapeutic group, show that monoclonal antibody is combined Chemotherapeutic can effectively suppress the growth of tumour, reduce chemotherapy resistance.The survival time of mice of drug combination group is significantly greater than alone anti- Body group and alone chemotherapeutic group, tumour can not only be treated by showing the scheme of monoclonal antibody Hetumomab combined chemotherapies medicine treatment tumour Growth, moreover it is possible to extend the life cycle of mouse, it may be possible to significantly suppress dead mouse caused by the transfer of tumour in vivo.

2nd, monoclonal antibody Hetumomab significantly inhibits the growth of human lung cancer transplantable tumor in vivo, with significant antitumor drug effect Act on.

Experimental studies of the monoclonal antibody Hetumomab in nude mice interior therapeutic lung cancer has been carried out, and has observed alone monoclonal antibody, aloneization Treat the curative effect that medicine treats lung cancer.

Specifically, harvest human lung cancer cell line SPCA-1 sphere cells, with 2.5 × 10⁵Individual cell/be only inoculated with is naked small Mouse.It is divided into 5 groups, every group 5：PBS control group, independent chemotherapy group (cis-platinum 0.3mg/kg), Hetumomab antibody high dose groups (40mg/kg), Hetumomab antibody middle dose group (10mg/kg), Hetumomab low dose groups (2.5mg/kg).It is inoculated with lung cancer Start Antybody therapy within the 2nd day after cell, experimental group and control group are treated by being injected intraperitoneally, treatment altogether is extremely inoculated with 28 days Afterwards, it is discontinued.Chemotherapeutic group is treated 2 times a week.The major diameter and minor axis of subcutaneous transplantation knurl are measured 2 times a week, calculate knurl volume, formula V=(π/6) × (major diameter × minor axis × minor axis).Observe the tumor volume change of each group.

When inoculation stops treatment after 28 days, observe and measure mouse tumor volume growth situation, calculate inhibiting rate.In Mice Body As shown in figure 12, as shown in table 22, monoclonal antibody Hetumomab can significantly inhibit nude mice to knurl volume inhibiting rate to the growth curve of transplantable tumor The growth of internal transplantable tumor, and also accordingly raised with the dosage rise transplantable tumor inhibiting rate of antibody, it is high, medium and low when being discontinued The monoclonal antibody Hetumomab of dosage is respectively 55.97%, 43.56%, 35.58% to the inhibiting rate of transplantable tumor, the suppression of chemotherapeutic group Rate processed is only 24.91%.

It these results suggest that, monoclonal antibody Hetumomab, which is used alone, can significantly inhibit the growth of human lung cancer transplantable tumor, have The significant pharmacodynamic action for suppressing lung cancer.

The monoclonal antibody Hetumomab of table 22 suppresses the result of human lung cancer SPCA-1 growth of transplanted human in animal body

3rd, monoclonal antibody Hetumomab significantly inhibits the growth of Human gastric cancer xenografts in vivo, and can substantially cooperate with enhancing chemotherapy Curative effect, with significant antitumor pharmacodynamic action.

Experimental studies of the monoclonal antibody Hetumomab in nude mice interior therapeutic stomach cancer has been carried out, and has observed more alone monoclonal antibody, list The curative effect of stomach cancer is treated with chemotherapeutic and monoclonal antibody combined chemotherapy medicine, can com-parison and analysis monoclonal antibody Hetumomab treat stomach in vivo The growth of cancer and collaboration enhancing chemotherapeutic efficacy.

Specifically, harvest human gastric cancer cell line SNU-5-V13 sphere cells, with 2.5 × 10⁵Individual cell/be only inoculated with is naked Mouse.It is divided into 7 groups, every group 8：PBS control group, mouse IgG control groups, independent chemotherapy group (cis-platinum 0.3mg/kg), Hetumomab Antibody high dose group (20mg/kg), Hetumomab low dose groups (1.25mg/kg), Hetumomab high doses+chemotherapeutic, Hetumomab low dosages+chemical drug.It is inoculated with the 2nd day after stomach cancer cell and starts Antybody therapy, experimental group and control group passes through abdominal cavity Injection is treated, after treating one month altogether, is discontinued.Chemotherapeutic group is treated 4 weeks, is discontinued afterwards 2 times a week.Skin is measured 2 times a week The major diameter and minor axis of lower transplantable tumor, calculate knurl volume, formula V=(π/6) × (major diameter × minor axis × minor axis).Observe the swollen of each group Knurl Volume Changes.

When inoculation stops treatment after one month, observe and measure mouse tumor volume growth situation, calculate inhibiting rate.Mice Body As shown in figure 13, as shown in table 23, monoclonal antibody Hetumomab can significantly inhibit naked knurl volume inhibiting rate the growth curve of interior transplantable tumor The growth of transplantable tumor in mouse body, and also accordingly raised with the dosage rise transplantable tumor inhibiting rate of antibody, it is high and low when being discontinued The monoclonal antibody Hetumomab of dosage is respectively 57.62%, 30.68% to the inhibiting rate of transplantable tumor, and the inhibiting rate of chemotherapeutic group is only 33.16%, the inhibiting rate of monoclonal antibody high and low dose combined chemotherapy medicine group has respectively reached 70.68% and 47.93%, as a result points out, Monoclonal antibody combined chemotherapy medicine group is to the inhibiting rate of mice-transplanted tumor higher than independent chemotherapy and the therapeutic scheme group of independent Antybody therapy, p< 0.05, monoclonal antibody combined chemotherapy medicine group shows more preferable therapeutic effect in the treatment of transplantable tumor.

It these results suggest that, monoclonal antibody Hetumomab, which is used alone, can significantly inhibit the growth of Human gastric cancer xenografts, have The significant pharmacodynamic action for suppressing stomach cancer.Monoclonal antibody Hetumomab combined chemotherapy medicine groups all show the suppression high to transplantable tumor Rate processed, can significantly inhibit the growth of tumour, and therapeutic effect is better than alone antibodyome and alone chemotherapeutic group, show that monoclonal antibody is combined Chemotherapeutic can effectively suppress the growth of tumour, reduce chemotherapy resistance.

The monoclonal antibody Hetumomab of table 23 suppresses the result of human gastric cancer SNU-5 growth of transplanted human in animal body

Suppress the results show of tumour in this range of animal body above, monoclonal antibody Hetumomab is in vivo to a variety of The growth of human tumour (such as liver cancer, lung cancer, stomach cancer) and resistance have significant inhibitory action (pharmacodynamic action), many for treating Planting tumour growth, transfer and resistance has important application value.

Claims

1. a kind of monoclonal antibody or its antigen-binding fragment, the monoclonal antibody is because on March 16th, 2016 is with preserving number The Mouse Hybridoma Cells that CGMCC No.12251 are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center are thin Born of the same parents produce.

2. a kind of hybridoma, it was preserved in China Microbiological with preserving number CGMCC No.12251 on March 16th, 2016 Culture presevation administration committee common micro-organisms center.

3. a kind of pharmaceutical composition, its monoclonal antibody comprising claim 1 or its antigen-binding fragment and pharmaceutically may be used The carrier of receiving.

4. the pharmaceutical composition of claim 3, wherein the monoclonal antibody or its antigen-binding fragment with selected from cytotoxin, The therapeutic moieties of radio isotope or biological activity protein are conjugated.

5. a kind of malignant tumour for the treatment of in patients, prevention and/or treatment Malignant tumor of bonal metastasis or the method for recurrence, methods described Including the monoclonal antibody or its antigen-binding fragment or claim 3 or 4 of the claim 1 that effective dose is applied to the patient Pharmaceutical composition.

6. the method for claim 5, wherein the malignant tumour be selected from breast cancer, colorectal cancer, cancer of pancreas, prostate cancer, liver cancer, Lung cancer and stomach cancer.

7. the method for claim 5 or 6, in addition to apply other antineoplaston means to the patient, such as using chemotherapy Agent, the antibody of the other tumour specific antigens of targeting or radiotherapy.

8. the pharmaceutical composition of the monoclonal antibody of claim 1 or its antigen-binding fragment or claim 3 or 4 is preparing use Purposes in treatment malignant tumour, prevention and/or treatment Malignant tumor of bonal metastasis or the medicine of recurrence.

9. the purposes of claim 8, wherein the malignant tumour be selected from breast cancer, colorectal cancer, cancer of pancreas, prostate cancer, liver cancer, Lung cancer and stomach cancer.

10. a kind of method for detecting that tumor stem cell is present in biological sample, including：

A) biological sample is made to be contacted with the monoclonal antibody of claim 1 or its antigen-binding fragment；

B) combination of the monoclonal antibody or its antigen-binding fragment and the target antigen in the biological sample is detected,

Wherein detect the combination and represent in the biological sample and there is tumor stem cell.

11. a kind of method for separating tumor stem cell, methods described includes：

(a) the doubtful cell mass for including tumor stem cell is provided；

(b) subgroup of the cell is identified, it combines the monoclonal antibody or its antigen-binding fragment of claim 1；With

(c) subgroup is separated.

12. the method for claim 10 or 11, wherein the tumor stem cell be selected from breast carcinoma stem cell, large intestine cancer stem cell, Pancreas cancer stem cell, prostate cancer stem cells, liver-cancer stem cell, lung cancer stem cell and stomach cancer stem cell.

13. a kind of method for detecting the presence of malignant tumour in patient, including：

A) biological sample for being derived from the patient is made to be connect with the monoclonal antibody of claim 1 or its antigen-binding fragment Touch；

B) combination of the monoclonal antibody or its antigen-binding fragment and the target antigen in the biological sample is detected, wherein Detect the combination and represent in the patient and there is malignant tumour.

14. a kind of be used for the method for Malignant Tumor Recurrence or progress in prognosis patients, methods described includes：

(b) biological sample comprising circulating cells and the monoclonal antibody or its antigen-binding fragment of claim 1 are made Contact；With

(c) presence with reference to the monoclonal antibody or the circulating cells of its antigen-binding fragment is identified, so as to suffer from described in prognosis The recurrence of malignant tumour or progress in person.

15. the method for claim 14, wherein the progress of the malignant tumour includes the transfer malignant tumour in patients.

16. any one of claim 13-15 method, wherein the biological sample include blood sample, lymph sample or Its component.

17. any one of claim 13-15 method, wherein the malignant tumour be selected from breast cancer, colorectal cancer, cancer of pancreas, Prostate cancer, liver cancer, lung cancer and stomach cancer.