CN103732271B - Detection and the method and apparatus of the disease such as treatment tumor and virus infection - Google Patents

Abstract

Present invention discloses the infection that treatment is caused by virus, and antibacterial infects and parasitic infection and the method for the treatment disease such as cancer and autoimmune disease.The invention provides a kind of method, treat pathogenic infection by inactivateing the pathogen in blood circulation in vitro.Over the course for the treatment of, blood flows out and is separated into blood plasma and cell component from patient.Blood plasma fractions is processed by physical method, if ultraviolet radiation is with the pathogen in inactivation, then returnes in the patient.Present invention also provides for a kind of method, is used for treating cancer, including two steps；1) remove tumor or process tumor with Therapeutic Method, such as, by surgical operation, chemotherapy, tumor is treated in radiotherapy or combination；2) remove from blood by extracorporeally circulating blood and/or inactivate circulating tumor cell.

Description

Detection and the method and apparatus of the disease such as treatment tumor and virus infection

Technical field

The present invention relates to by detection and treatment tumor, virus infects, and the method and apparatus of the diseases such as autoimmune disease is special Do not relate to reach detection and the purpose for the treatment of disease by the morbid substance in the blood circulation of periphery is purged.This Invention relates to a kind for the treatment of and the equipment of monitoring tumor and method.Particularly relate to a kind of thin by removing free tumor in blood Born of the same parents prevent tumor recurrence and the equipment of transfer and method, the invention still further relates to a kind of device treating pathogenic infection and side Method.Particularly relate to a kind of treat the apparatus and method of infection by pathogen in inactivation blood.

Background technology

The concrete operations of blood purification can be found in document and research report at many teaching materials.Blood purification includes Hemodialysis, hemofiltration, hemoperfusion, plasmapheresis etc..Hemodialysis, is called for short hemodialysis, and popular saying is also referred to as people Work kidney, wash kidney, be the one of blood purification technology.It utilizes semipermeable membrane principle, by diffusion, in convection cell various harmful and Unnecessary metabolic waste and the removal of too much electrolyte are external, reach to purify the purpose of blood, and suction reaches to correct Water-Electrolyte And the purpose of acid-base balance.Hemofiltration is by machine (pump) or the blood pressure of patient self, makes blood flow through in extracorporeal circuit A filter, under the effect of filtration pressure, leach big quantity of fluid and solute, i.e. ultrafiltrate；Meanwhile, supplement to become with plasma lipid Electrolyte solution as split-phase, i.e. displacement liquid, to reach the purpose of blood purification.Hemoperfusion is that a kind of solid-state of a kind of application is inhaled The perfusion device of attached type, with absorption some exogenous or detoxification device of endogenous toxin in the patient, introduces the blood of patient In perfusion device, enter internal by being back to vein again after dress perfusion device absorbing toxin.Plasmapheresis is also the important hands purifying blood One of section.Its ultimate principle is to utilize blood cell separator, in vitro the blood separation of patient is become blood plasma and blood cell composition (erythrocyte, leukocyte, platelet).Then discard the blood plasma containing harmful morbid substance, replace with the displacement liquid of equivalent, then blood Cell component feeds back to the internal of patient together with plasmapheresis liquid.Plasmapheresis liquid is usually blood plasma or the replacement of normal person Blood plasma, to alleviate the deficiency in blood plasma source.Plasmapheresis can reduce the harmful substance in blood, removes macromolecule in the patient Protein, such as heterologous protein, anaphylactogen, autoantibody, and fat-soluble (or water solublity) medicine, poisonous substance etc..Exempt from Epidemic disease is adsorbed on plasmapheresis development foundation, applies further the antigen of high degree of specificity, antibody or have individually defined thing physics and chemistry Learn the material (aglucon) of affinity, be combined with solid phase material, make adsorbent with corresponding in circulation-type bodies for selective adsorption Virulence factor.The common operation flow process of immunoadsorption therapy is by outside blood samples of patients lead body, sets up extracorporeal circulation, blood Flow through plasma separator and isolate blood plasma, blood plasma is introduced immunoadsorption device and contacts with immunoadsorbent, with selective absorption Mode removes morbid substance, is then fed back in the patient by the blood plasma purified, reaches therapeutic purposes.Some immunoadsorption devices are not Need separated plasma, and can directly carry out hemoperfusion formula immunoadsorption therapy.The key component of immunoadsorption therapy is absorption Post, connects including carrier part and ligand moiety.With the core that absorption object (morbid substance) occurs adsorption reaction Heart part is referred to as carrier, and the material being fixed on carrier, having immunoadsorption activity is referred to as part.The adsorption activity of part is originally Matter is the selectivity between absorption object (morbid substance) or specificity affinity, i.e. intermolecular interaction, including biology Learn affinity (such as antigen-antibody reaction) and physical chemistry affinity (such as hydrophobic reciprocal action).

The various methods carrying out blood purification have respective advantage.Existing plasma separator has a variety of, such as membrane type Plasma separator, centrifugal plasma separator etc..Much these device the most commercializations.Suitable plasma separator is permissible Blood cell separates pathogen with blood plasma then concentrate in blood plasma owing to its volume is significantly smaller than blood cell.

Neoplasm metastasis is the principal element of tumor lethal.1 year relapse rate 60% of China's tumor post-operation, the patient of 90% is five In year, death is in recurrence and transfer.Research shows, Iisolated tumor cells in blood (have another name called circulating tumor cell, Circulating tumor cell, CTC) it is the major reason causing neoplasm metastasis.And its quantity is also predicting tumors transfer The important indicator of probability.After operation at present and chemotherapy, the monitoring of the Iisolated tumor cells quantity in blood is made in developed country For the assessment guiding treatment the most further to antineoplaston result.Such as in metastatic breast cancer patient, periphery after treatment Progression free survival (PFS) and total existence (OS) phase of the higher patient of blood circulation tumor cell are appreciably shorter.Due to excision and Some chemotherapy radiotherapy is to body and the damage of tumor body, and that usually can cause Iisolated tumor cells discharges into blood in a large number.And swell After tumor people have passed through operation, the treatment of long-term radiotherapy chemotherapy, somatic damage is very big, hypoimmunity, and the tumor cell of remaining is special Easily do not revive, cause relapse and metastasis.Multiple studies have shown that tumor resection can cause massive tumor cell to flow into blood Liquid also finally causes recurrence.And a lot of patient is also to there will be the Iisolated tumor cells in blood after chemotherapy and radiation a large amount of Raise.

Summary of the invention

One aspect of the present invention disclose treat by specific antibodies cause the method for autoimmune disease/disease (the "/" labelling in current invention refer to " with " or "or").Numerous disease is all owing to immune system disorder causes, the most It is found that more than 30 kind of disease is closely related with the generation of anti self antibody, the most both included the autoimmune disease such as erythema wolf of classics Skin ulcer, myasthenia gravis, include again multiple common chronic disease such as rheumatoid, some class patients with type Ⅰ DM etc..These diseases do not have more Good Therapeutic Method.

The method of present disclosure includes that two steps, the first first step are that the way applying extracorporeal blood treatment is removed Going internal antibodies harmful, suitable blood purification means to include hemoperfusion, Blood index purifies, and immunoadsorption, blood is saturating Analysis, plasmapheresis etc..Total antibody that nonspecific way is removed in blood can be taked (such as application protein A adsorption column, activity Charcoal adsorption column, the method such as membrane filtration), it would however also be possible to employ specific way is optionally removed in extracorporeally circulating blood The antibody (having the solid-phase adsorbent etc. of specific antigen as used surface to connect) of specific antigen；Use said method by specific harmful (immunocyte can being combined with specific antigen in blood can also be simultaneously by clearly by extracorporeal blood treatment removing for antibody Remove), the blood purification operation used in present invention can be that whole blood (including hemocyte and blood plasma) purifies or blood plasma is clean Change.These technology are well known to the skilled person.Remove the example of specific antibodies in blood to join in many Examine in document and find.Second step is that patient is given the medicine that can inactivate the cell (such as B cell) producing specific antibodies harmful Thus stop the continuation of specific antibodies harmful to produce, reach the therapeutic purposes to disease.Suitable medicine includes that agent-cell goes out Active material junctional complex (is referred to alternatively as again conjugate or conjugate).Cell inactivation material can be cytotoxin, Carbazole alkaloid Agent, the siRNA of the suppression specific immunologic function of cell, radioactive substance, the antisensenucleic acids etc. of suppression cytoactive.Cytotoxin- Antigen junctional complex/conjugate/conjugate can have with surface expression after being imported into human body and can be combined with this antigenic specificity The B cell of albumen (such as specific antibody) and T cell combine, so that cytotoxin plays a role (such as by intracellular Gulp down) reach effect that this specific cells is inactivated, thus inhibit generation and the immunocompetence of the antibody for this specific antigen. Similar antibody-cytotoxin connection/conjugate is successfully applied to the treatment of tumor.It is suitable for the antigen of the present invention Both can be albumen can also be a part such as polypeptide fragment of albumen, and other kinds of antigen such as polysaccharide, nucleic acid or little Molecular substance.

Present disclosure is a kind of brand-new therapy treating the immune dysfunction diseases that multiple autoantibody mediates.It is former Reason is the specific B cell clone's subgroup removed and produce specific anti self antibody, thus eliminates anti self antibody thoroughly Produce and cure relevant disease.Its method is to use autoantigen-drug conjugate (Suicide antigen), thus and expresses corresponding The B cell subgroup of antibody combines so that it inactivates, the antibody-drug conjugates kind anti-cancer drugs of this principle and present clinical practice Species is seemingly.But the free autoantibody in necessary first purged body, otherwise these substantial amounts of autoantibody meeting and medicines before administration Serious side effects is produced in conjunction with producing the most poisonous antigen antibody complex.Remove free autoantibody and can use one The blood purification of the adsorbent containing fixing corresponding antigens, the autoimmune disease that some T cell can also be mediated by this therapy Play a role.

After this kind of Suicide antigen or the like is supplied to patient, blood purification can be carried out again, with further The immune complex of the target antibody removing residual and the antibody-suicide antigen formed or the like.If necessary, may be used To apply the medicine of multidose to patient.Additionally, this kind of suicide antigen or the like can be by using extra blood clean Change to remove from the blood samples of patients after treatment.These suicide antigens or the like can also selective inactivation specific T cells group, Therefore its autoimmune effect caused is reduced.Some T cell can optionally combine target antigen and produce immunoreation, kills Extremely/inactivate these T cell and can reduce bad immunoreation effect, this is the pathogenesis of certain form of diabetes.Should Antigen can be whole antigen (such as albumen) or its part (such as antigenic determinant, such as polypeptide), or can be with anti- The peptidomimetics of body combination or little molecule, it is also possible to be other affinity molecule can being combined with the unique tag of target cell, Such as protein, peptide or little molecule.These affinity ligands (such as antibody) can be connected with toxin/cytostatics/inactivator and these Target immunocyte combines.This method can suppress the immunoreation of some antigen selectively, without causing antibody-anti- The side effect that former immune complex produces, therefore it can also be used to the disease such as such as device that treatment is caused by some antibody Official's transplant rejection, some antibacterial, virus infection etc..Many this kind of suicide antigens or the like are in the news the most, and present invention tries Agent and method can obtain by revising existing method and steps.

Toxin/cytostatics/the inactivating substance of current invention includes but not limited to any to kill cell or press down Normal or the specific function of cell processed (such as, some molecule such as protein (such as antibody) of manufacture, replicate, differentiation, growth, Exhibition is for mature cell or other type of cell) reagent.They can be radiosiotope, albumen, little molecule, SiRNA, antisense molecule, enzyme etc., their example includes NK cytotoxic factor, tumor necrosis factor such as TNF-α and TNF-β (LT), perforin, granzyme, cell death inducer/activator, radical-forming agent, cell membrane disruption agent, lipase, albumen Enzyme, hydrolytic enzyme, toxic agents, chemotherapeutics, for the siRNA of normal function or the antisensenucleic acids of host cell, cytotoxin etc., it Can be active precursor type, only the most active after they with target cell or are absorbed by target cell, be such as similar to Above-mentioned antibody-daunomycin conjugate etc..Similar antibody-cytotoxin connection/conjugate is successfully applied to The treatment of tumor.If cell injury reagent is to come into force intracellular, it typically requires by crossing over cell membrane such as endocytosis work It is used for realizing.

In one example, acetylcholinergic receptor-Ricin conjugate can be used to inactivation generation acetylcholine antibody Immunocyte such that it is able to be used for myasthenia gravis is treated.Before being administered (acetylcholinergic receptor-cytotoxin), Need first to be removed by a large amount of acetylcholine receptor antibodieses in patient body, otherwise can produce high amount of drug-antibody immune complex And have side effects.Suitable method includes patient is carried out blood extracorporeal circulation purification, and blood flows through one containing surface admittedly Determine the adsorption column of the solid phase carrier of acetylcholinergic receptor, thus the acetylcholine receptor antibodies in blood has been removed.At one In example, the blood of patient carries out extracorporeal circulation, and blood passes through a plasma separator, and the blood plasma of separation flows through one and contains 50g secures acetylcholinergic receptor cell membrane outer segment (such as according to Ann N Y Acad Sci. 2008;In 1132:291-9 Prepared by method) the adsorption column of solid phase carrier (such as glucan particles), then blood plasma merges with hemocyte and flows back in patient body, Velocity of blood flow is 150ml/ minute, and extracorporeal circulation immunoadsorption purifies and carries out two hours.So can be effectively in purged body Acetylcholine receptor antibodies.Non-specific method can also be used to remove total antibody, such as application Immunosorba blood Cleaning system.This operation can be repeated to the cleaning antibody level reaching to need.Then patient is injected administration to inactivate product The immunocyte of raw acetylcholine receptor antibodies, such as with acetylcholinergic receptor cell membrane outer segment-ricin A conjugate Or acetylcholinergic receptor cell membrane outer segment-daunomycin conjugate or acetylcholinergic receptor cell membrane outer segment-I125 put together Thing etc..Concrete dosage can be determined by test.In some example, Myasthenia Gravis is given 10 mCi's by single (I125: receptor fragments ratio is 0.9: 1, according to Ann N Y for the acetylcholinergic receptor cell membrane outer segment of I125 labelling Acad Sci. 2008;In 1132:291-9 prepared by method).Patient can also be injected administration acetylcholinergic receptor cell membrane Outer segment-ricin A is (according to J. Immunol. 1984;133;In 2549-2553 prepared by method) or acetylcholinergic receptor Cell membrane outer segment-daunomycin, dosage is 0.1ug/ kg body weight.

In other examples, suffer from the diabetic that autoantibody causes and first pass through the method for blood purification and remove (as GAD 65, IA-2, beta cell surface antigen, insulin, insulin is subject to diabetes autoantibody in blood The antibody of body).First patient's autoantibody can check to determine its corresponding antigens kind, then can be with being connected with phase The adsorbent answering antigen removes specific antibody, it is also possible to remove total antibody with protein A adsorption column.Then it is correlated with to patient injection Antigen-toxin-conjugate produces B cell or the T cell can being combined with related antigen to remove associated antibodies.Use insulin Need the fragment taking non-insulin receptor binding domain as antigen to avoid itself and Insulin receptor INSR during antigen-toxin-conjugate In conjunction with and cause to other cells produce toxic and side effects.

Equally, the treatment of rheumatoid arthritis can be used A47, GPI, HLA-DRB1-Binding peptide, and HA308-317 polypeptide as the corresponding antibodies in antigen purged body and antibody-producing B cell and related T-cell to reach treatment effect Really.Can first check patient's autoantibody to determine its corresponding antigens kind.It is, for example possible to use be fixed with these The adsorption column of antigen optionally removes described antibody when blood flows through.The method that can also use non-selectivity, such as activity Charcoal absorption or protein A post/filter or selective membrane are filtered and are eliminated all of antibody in blood.Then, antigen-toxin is puted together Thing, such as I-125-GPI, GPI-daunorubicin, A47-ricin A chain immunotoxin can be injected to patient, to select Property ground inactivation specific immune cell.The example of antigen-toxin-conjugate can be prepared according to above-cited reference method.Suitable Suitable drug dose can be determined by experiment.Appropriate consumption should have the inactivation capacity of high targeted immune cell and low pair Effect.

Equally, the method can also be used to treat allergy, by should result in allergy antigen solid phase carrier and Free antibodies corresponding in patient body can be removed and make associated immune cells to inactivate by antigen-toxin-conjugate, thus reaches to control Therapeutic effect.

Can also be by corresponding autoantibody or corresponding B cell, T cell is separated by being combined with corresponding antigens, then May determine that itself and antigen-binding proteins sequence and the mRNA nucleotide sequence of corresponding antibodies.By to implanting needle in patient body to phase Answer the antisensenucleic acids of antibody or siRNA can also reach to suppress producing thus reaching the effect for the treatment of of corresponding antibodies.Can hinder Only the affinity ligand (such as antibody, little molecule, nucleic acid ligands) of these antibodies bind antigen also apply be applicable to patient with treatment accordingly Disease.Separate after associated antibodies and determine that mRNA then every patient can provide personalized treatment.Data can also be produced Storehouse, to cover the most common antibody mRNA in the sample of the patient of specified disease, and uses most common sequence data to make For target, all patients are treated.

Many antibacterials and virus can cause human immune system to attack own cells and tissue.The same present invention can To be used for treating corresponding disease.The present invention discloses a kind of to antibacterial, the method that virus and parasitic infection carry out treating.

When virus infected cell, cell surface can be expressed the component (such as virus antigen) of virus thus be disappeared by T cell Go out.Similar strategy can be used to infect virus treat.Virus component will there be is the molecule of affinity (such as antibody, disease Poison entry inhibitors, aptamer) it is connected with toxin, the cell inactivation of virus can be infected thus suppress virus infection.Example As, then can be used to inactivate the T cell of HIV with Ricin after being connected by the antibody of anti-gp120.The method can also expand Open up the treatment such as HBV to other viruses, HCV and antibacterial and the infection of parasite, as long as it understands infection cell and at cell Surface expression identifies thing accordingly.The way that can first pass through blood purification before administration removes pathogen free in blood and disease Substance component (such as virus antigen) and infected cell, thus lower the generation of poisonous immune complex to alleviate medicine Side effect.This policy class is similar to the therapeutic strategy to autoimmune disease described above.Equally, can also carry out after administration Blood purification is to remove the adverse immune complex that Residual Disease substance generates.

In one example, aids patient first passes through blood purification and removes the inhibition of HIV in blood and free gp120 Albumen.Blood first flows through a hollow fiber plasma separators.The aperture of hollow-fibre membrane is 0.5um, can allow inhibition of HIV Pass through.Blood plasma fractions flow through HIV/gp120 remove post (be filled with in such as one post 50ml 90um diameter with The affiliation carrier that the Sepharose 4B microsphere of the CNBr activation of goat-anti gp120 antibody key sum is formed, capacity is 10mg Antibody/ml) right reprocessed blood plasma merges with hemocyte and flows back to patient.Velocity of blood flow is 150ml/ minute, continues 2 little Time.The way that can also use whole blood perfusion (is filled out in such as one post by whole blood flows through a HIV/gp120 removing post Filled 100ml 150um diameter and goat-anti gp120 antibody key and the Sepharose 4B microsphere of CNBr activation formed Affiliation carrier, capacity is 10mg antibody/ml;Or the living with goat-anti gp120 antibody key and CNBr of 100ml 300um diameter The affiliation carrier that the Sephadex G-50 microsphere changed is formed) to remove further in blood infected by HIV and to express gp120's Cell and inhibition of HIV are with free.Velocity of blood flow is 150ml/ minute, continues 2 hours.Then by 3B3-PE in one week Medicine to intravenous injection of patient (20 ug/kg) to inactivate the cell of HIV.C1q blood purification can also be used upon administration The immune complex containing 3B3-PE produced is removed by adsorption column further.Also can also be before administration by Blood of Patients liquid stream Secure the adsorption column of the Sepharose 4B microsphere of gp120 through one containing surface, so can remove in blood anti- The antibody of gp120, reduces its combination to target cell of competitiveness with 3B3-PE.

Toxin/cytostatics/inactivating substance includes but not limited to any can killing carefully at the example of current invention Normal or the specific function of born of the same parents or suppression cell (such as, some molecule such as protein (such as antibody) of manufacture, replicates, differentiation, life Long, develop into mature cell or other type of cell) reagent.They can be radiosiotope, albumen, little molecule, The antisense molecule of siRNA, enzyme etc., their example includes NK cytotoxic factor, tumor necrosis factor such as TNF-α and TNF-β (LT), perforin, granzyme, cell death inducer/activator, radical-forming agent, cell membrane disruption agent, lipase, albumen Enzyme, hydrolytic enzyme, toxic agents, chemotherapeutics, for the siRNA of normal function or the antisensenucleic acids of host cell, cytotoxin etc., it Can be active precursor type, only the most active after they with target cell or are absorbed by target cell, be such as similar to Above-mentioned antibody-daunomycin conjugate etc..

Toxin/its precursor or inactivation/inhibitor can also be targeting substances, and it is for virus/antibacterial or parasite, in order to It connects conjugate and can be used to optionally to kill/inactivation of viruses or antibacterial or parasite rather than host cell.Such as, it Can be antiviral drug poison, antibiotic bacterium, antiparasitic, radiosiotope, radical-forming agent, pathogen film Disrupting agent, pathogen toxic agents, lipase, protease, hydrolytic enzyme, for the siRNA of pathogen and antisensenucleic acids etc., Ta Menke To be pro-drug or non-live ejector half, only when it is combined with target pathogen or is just activated by target pathogen picked-up.Such as, interior The conjugate of lysin or polymyxin and escherichia coli humanized antibody can be used for treating coli-infection, can be injected into In blood, it is used for treating disease.

Additionally, toxin or its precursor or cell (or pathogen) inactivation/inhibitor can also be a kind of drug delivery systems. Affinity ligands, such as antibody or antigen, can be connected with drug delivery system.Described drug delivery system comprise can serve as toxin or Its precursor or the material of inactivation/inhibitor.Such as, drug delivery system can be that polymer (such as connects with multiple daunomycin The polylysine connect), anti-gp120 antibody also couples with this polymer.In another example, drug delivery system is containing ricin The liposome of element, there is anti-gp120 antibody on its surface.These examples can be used to treat HIV.Other medicines delivery system example Such as microsphere, nano-particle is also applied for the present invention.Such conjugate can be used for treating pathogenic infection or autoimmune Sick.

For treatment by the infection that virus in host cell, antibacterial or parasite do not cause, affinity groups needs pin Unique surface marker to this virus, antibacterial or parasite, such as their surface protein, antigen or protein called membrane transporters. Affine material can be can in conjunction with the molecule on its surface or the substrate of transport protein or can by pathogen absorb easily point Son, such as the antibody for surface composition (antigen), the little molecule being combined with surface protein (such as Virus entry inhibitor), for spy Determining the agglutinin of pathogen, some has the antibiotic of pathogen surface affinity.

In one example, can with the little molecule inhibition of HIV entry inhibitors of the combination of gp120 with daunomycin even Connect.Because it is a little molecule, it is possible to the oral host cell being used for killing HIV (human immunodeficiency virus) infection.Little molecule HIV is sick Poison entry inhibitors can also be connected with outer virionic membrane disrupting agent, i.e. can directly kill inhibition of HIV.

Described kill/inactivate/inhibitor can also be the protein of complement system or their fragment or their imitation Thing.Such as, it can be c1q or activation c1q or c3b or c3bbb or c3 invertase or c5 invertase or MAC or Their analogies or there is their molecule of similar functions or molecular combinations.When they are connected with affinity molecule, its for The chemotaxis of pathogen, phagocytosis or dissolution will be enhanced.If affinity molecule is not that (such as, it is antibody The Fc fragment of aptamer aptamer, IgG can be connected with affinity molecule or be connected with described inactivation/inhibitor, right to improve Cracking/the phagocytosis of pathogen.Described kill/inactivate/inhibitor can also be by pathogen associated molecular pattern, or from super Molecule in level antigen.

When it is used to kill infected cell, the labelling molecule of apoptotic cell (such as occurs in the multiple of cell surface Intracellular molecules, such as calprotectin, Phosphatidylserine, annexin α 1 and the LDL of oxidation) can also be used to connect institute Stating affinity molecule and be used as cell inactivation/inhibitor, so these cells infected will be eliminated by macrophage. for disease The specific IgG dimer of substance or oligomer can provide more preferable antipathogen effect, because the dimer of IgG or oligomeric Thing is conducive to the activation of complement system.

The method can also be used for treating HIV or other virus/antibacterial infects, and these infection can produce harmful antibody.It is subject to The cell infected can present some antigen of pathogen on its surface.Such as, either gp120 and its antibody are all HIV diseases Progress is necessary.Either remove GP120 or its antibody all will stop progression of disease, and help immune reconstruction. First patient can carry out blood purification process, to remove gp120 antibody in blood and inhibition of HIV and gp120, connects Getting off, patient will accept Immudel-gp120 medicine or its analog, and to eliminate gp120 antibody produced cell, it is by product The selective destruction of its B cell of life realizes.The Clonal toxin of B cell, may be used for optionally eliminating the anti-of gp120 Answering property B cell.Detailed process can find at relevant list of references.

Many medicines are had all to come into force by being combined with the surface marker of pathogen or human body cell.The medicine of these kinds The example of thing includes but not limited to antibody-drug conjugates, affinity ligand-drug conjugates and Virus entry inhibitor.Therefore, It is similar to the blood purification treatment in above-mentioned method, the circulating antigen/pathogen in blood/can be with these types can be removed The blood that combines with high-affinity of medicine in material.This will reduce side effect, reduce those and cause potentially harmful the exempting from of generation Epidemic disease complex, reduces the medicine of dosage, and adds the curative effect of medicine.A kind of method is to use to be fixed with the one of medicine or medicine Part, or the solid phase carrier of its analogies carries out extracorporeal blood treatment.Other method, such as selectivity plasmapheresis or blood Liquid filters and can also apply, as long as the blood part containing these circulating antigen/pathogen/cells can be removed.Do not remove this During a little circulating antigen/pathogen/cell, medicine by connection to form harmful complex (such as, the exempting from of antibody-antigene Epidemic disease complex, if medicine contains antibody moiety), medicine can be combined with and circulate the molecule of soluble antigen (as suffered from HIV Solubility gp120 in the blood of person), or other has the material having high-affinity to medicine, to medicine and its desired target Mark combines competition and reduces the curative effect of medicine.If they removed, this medicine will more effectively, because can be combined with therapeutic goal Medication amount higher, can be administered less and reduce side effect.Even if desired therapeutic goal (pathogen/cell) is at blood Liquid, the target removing significant quantity from blood is also advantageous, and so can more effectively treat with less medicine and reduce Side effect.Medicine preferably be the circulating antigen/pathogen/cell of significant quantity before blood purification recovers again to disease People.

Antibody-drug conjugates (ADC) is a kind of target therapeutic agent, can be used for the treatment of numerous disease, including cancer Disease.They generally include an antibody (or antibody fragment, such as single chain variable fragment) and are connected to a carrying medicament (generally For cytotoxic molecule).Blood purification can be used before antibody-drug conjugates to remove antigen in blood, separately Outward, blood purification can also be used after patient is used ADC to remove produced immune complex.In one example, Brentuximab vedotin is that approval is for treating primary cutaneous type (ALCL) and the antibody of Hodgkin lymphoma -drug conjugates medicine, is linked to resist for chimeric mAb Brentuximab (its targeting is epicyte protein CD30) Mitogenic agent monomethyl auristatin E forms.Patient first passes through blood purification and processes to remove in CD30 and blood (such as, the blood of patient flows through CD 30 adsorption column with the 150ml/min flow velocity of 2 hours to the cell of expression CD30, inside fills out Fill 100 milliliters of 150um diameters CNBr activation Sepharose 4B pearl combine Brentuximab or 100 milliliters The Brentuximab that 300UM diameter polydextran gel pearl combines), patient can also process with blood cell separator, with Remove most leukocyte (including the cell expressing CD3).Then patient treats with Brentuximab vedotin. In another example, enfuirtide is HIV fusion inhibitor, and it combines GP41 and prevents cell entry cell.HIV (human immunodeficiency virus) The patient infected first processes to remove HIV and gp41 in blood with blood purification.The hollow fiber plasma that the blood of patient passes through Separator separated plasma, the aperture of hollow-fibre membrane is 0.5 micron, and its size allows virion to pass through.Blood plasma fractions passes through It is filled with the Sepharose 4B of 100 milliliters of 100um diameters, connects and have the antibody for gp120 and the antibody of anti-gp41), Then the blood plasma processed and hemocyte are merged to form blood clean.Blood after cleaning is sent back in patient body.Blood Flow velocity 150ml/min treatment continues 2 hours.Next treat to patient's enfuirtide, it is possible to use standard dose or subtract Few dosage.

The solid phase carrier of blood purification can be post, film, fiber, granule or other suitable surface mass any, its Suitable surface characteristic (including the surface within loose structure) should be comprised for the direct-coupling of affinity molecule or repair Couple after decorations or surface derivatization.If solid support is porous, inside it, can also be used for being fixed with the molecule of affinity.

Working as virus infected cell, this cell will present some virus component (such as virus antigen) on cell surface.Institute The solid phase carrier to virus affinity ligand (for the virus antigen on infection cell surface) is had also by virus infected cell to connect In conjunction with.Therefore treatment virus infects and can also realize by taking viruliferous cell in removal blood.

In some embodiments, blood passes through a box including doughnut, it is characterised in that the parent to virus Fix on the perforated membrane of doughnut with molecule.Suitable virus instance includes HIV-1, HBV and HCV.The example of affinity molecule Can be antibody, aptamer, agglutinin or viral entry inhibitor, the affinity molecule of these viruses can also be attached to Solid matrix, is placed in blood purification.The movement (such as pump or agitating device) of liquid inside and outside doughnut can be helped Device can be attached increase diffusion rate.One example of solid phase carrier is agarose.The example of hollow-fibre membrane can be United States Patent (USP) 6528057 and United States Patent (USP) 7226429 find.Apparatus for purifying blood and step can also be easily from these The document of patent and other blood purifications obtains.The molecule of affinity can also be connected on solid phase carrier, and is placed on Blood purification is flowed through by blood and is not used doughnut.Such as ultraviolet, can radiate, heat with the means of inactivation of viruses, micro- Ripple, light can also be applied in depurator or solid phase carrier is to inactivate this virus.Such as, low temperature (such as-10 degree) or high temperature (as 40～60 degree) solid phase carrier (such as post, filter, fiber and film) or filter or the blood plasma fractions of separation can be applied to. Light (ultraviolet light or visible ray), microwave radiation can also be employed.The method of inactivating pathogens is preferably at pathogen and normal blood Slurry composition has certain selectivity.Such as, if ultraviolet is used as means with inactivating pathogens, the most excellent The wavelength of choosing is to have high absorption at nucleic acid, but protein has the wavelength of low absorption, such as wavelength 260nm, because blood Without nucleic acid, pathogen contains nucleic acid to slurry composition.Owing to this virus can stop the longer time on solid phase carrier/filter, They will accept longer time cold heat/light or radiation, and by carefully controlling the intensity for the treatment of, virus will be killed, but healthy Cell/blood plasma fractions will remain in that activity, because they are quickly through solid phase carrier/filter.Blood flowing speed, processes Intensity (such as temperature, light or radiant intensity) can be adjusted, in order to rests on the cause of disease of a very long time on solid phase carrier Cognition is killed.Therefore, even if this virus or pathogen are discharged back into blood and still can not cause new infection.A kind of method is come Keeping virus to rest on the way of inactivating device long period is to have the inactivating device of many microporous particles in application.Granule hole The size of gap/cavity is more than the size of virus, but less than blood cell.Therefore, when whole blood passes through, virus will be trapped in granule Face and it needs to be lot more time to get away, but hemocyte can flow away rapidly.This mechanism is similar to size exclusion chromatography.Therefore, The inactivation time that this virus is longer by accepting treatment.If photon, as infrared, it is seen that light or ultraviolet are used for killing virus, light Sensitizing drug (the pathogen inactivated material of photochemistry as blood products of sterilizing), such as phenothiazine dyes, methylene blue, vitamin B2, psoralen (such as 8 MOP, AMT), photodynamic therapy use photosensitizer, it is also possible to join blood with increase virus/ The inactivating efficacy of pathogen/infection cell.These reagent also can the affinity ligand of coupling pathogen, to increase their selection Property.They can be added to whole blood or blood plasma fractions.They can also be added in the patient or add to be removed body Outer blood/plasma.Additionally, these reagent can be after the pathogen inactivated process in blood/blood constituent, blood/blood becomes Divide to feed back and therefrom remove to before patient, to reduce the potential side effect of these medicines.Such as, by making blood/blood constituent lead to Cross and be filled with adsorbent (such as activated carbon, adsorbent resin etc.), or use hemodialysis apparatus for purifying blood；Can absorb or remove These medicines.The equipment having these types a lot of is available for blood purification/hemoperfusion/hemodialysis and removes the medicine in blood. Such as, the agar embedding attapulgite clay of crosslinking, Paar MB1 filter, equine pharmacy Blueflex filter or LeucoVir MB filter can be used for removing methylene blue at blood or blood constituent.If it is only viral/pathogen inactivated in blood plasma fractions application Means (such as use plasma separator to come the virus contained in washed corpuscles and blood plasma, then treating blood disorders at application inactivating device Slurry part) process, it can not always need to utilize solid phase adsorption or filter to remove pathogen, although combines pathogen and goes out Live and solid phase adsorption or double filtration plasma clearance virus/pathogenic bacteria can increase therapeutic effect.Have many methods can by blood plasma from Whole blood separates, for example with hollow fiber type plasma separator or based on centrifugal blood component separation device.Because it is many Pathogen is in blood plasma, only processes blood plasma and can also reach to reduce pathogen/inactivating efficacy, and reduces the damage to hemocyte Wound.If hollow fiber type plasma separator is used, the hole of doughnut is sufficiently large, to allow pathogen to pass through, but not Most of hemocyte can be allowed to pass through.In certain embodiments, blood plasma flows through a defecator to filter pathogen therein, and And give pathogen inactivated before or after filtering.Filter and pathogen inactivated combination will cause more preferable therapeutic effect.Control Treatment can periodically repeat, until desired effect has reached.For example, it is possible to patient is carried out per week or every three days Treat one time 2 hours.

The method of the present invention may be used for treating other pathogenic infections, such as antibacterial or parasite, as long as they are at blood In liquid.Treatment can be in the way of being continuously flowing or intermittent flow mode in batches.Such as, blood is continuously taken out and continuously Be processed, and return to patient continuously.In another example, blood/blood constituent with a certain amount of taking-up and is treated Regular hour, then returning to patient, then blood/the blood constituent of next batch is drawn out of process.This will allow for Time enough carries out pathogen inactivated or removes.It can also be the combination of flowing/intermittent flow continuously.Such as, blood leads to Cross plasma separator and adsorbent is to be continuously completed, but it is to be conducted batch-wise that pathogen inactivated and blood plasma returns to patient.If The mode that whole blood taking-up and backflow are intermittent flow being carried out, single needle/mono-conduit can carry out blood extraction and return.At some In embodiment, blood or blood constituent flow through adsorbent and are repeated several times.Such as, pass through to be filled with at blood or blood constituent It is reintroduced into again after the device of adsorbent in device to allow it again by adsorbent, is then return in the patient.

Many methods are had to may be used for coupling molecule to solid carrier.These methods can carry from ready-made Scientific Periodicals For the supplier of coupling agent, or related web site obtains.Such as, the molecule containing primary amine can be formed by amido link and be connected to The solid support of carboxylic group;Being generally formed with EDC [1-(3-dimethyl of amido link between amine and carboxylic group Aminopropyl)-3-ethyl-carbodiimide hydrochloride] or other carbodiimides complete.The chemical substance being combined with virus, may Needing suitably to modify or derive to be beneficial to coupling, it is modified and derivative should not significantly reducing combines activity with viral.Also can lead to Overcoupling is coupled to solid carrier again to another part.In some embodiments, viral conjugates matter itself can be used for being formed admittedly Phase carrier.

One aspect of present invention is the group utilizing and having the strongest negative charge on solid phase carrier or has the strongest bearing The group (such as sulfonic acid, benzenesulfonic acid or their salt) of electric charge removes virus.The polyanion combined as virus includes, but not Being limited to, maleic acid and styrene sulfonic acid, the polymer of polyvinyl phthalic acid ester sulfate, sulfated polysaccharides is (such as gel Polysaccharide sulfate, dextrin, sulphuric acid fucoidan, and PPS, the copolymer of dextran sulfate, heparin, sulphuric acid Heparin, carrageenin), polyvinyl (PVS) and polyanethole sulphonic acid ester, their copolymer and acrylic acid and they Salt.Most preferably, these polymer have high density sulfonic group or sulfuric ester functional group or phosphate group or hydroxy-acid group (example As, polyacrylic acid, poly etc.).One example of polymer includes the copolymer of maleic acid and styrene sulfonic acid.Polymer Another example include polyvinyl alcohol phthalate sulfate polymer, they can be to include polyethylene backbone ester and sulfur The mixed ester of acid esters, can be obtained through the esterification of phthalic anhydride and sulphuric acid acyl chlorides by polyvinyl alcohol.The compound of these types There is high density acidic functionality.One example is maleic acid and Styrene Sulfonic Acid Copolymer, maleic acid p styrene sulfonic acid Molecular weight ratio can be that (such as, molecular weight ratio can be 9:1 to 1:9,7:3～3:7 to any amount；And about 1:1).At one In example, maleic acid, it is about 1: 3 with the ratio of the molecular weight of styrene sulfonic acid.Maleic acid and the copolymerization of styrene sulfonic acid (PSMA) Thing can be prepared by the method using maleic acid copolymerized sulfonated phenylethylene, or being total to by maleic anhydride and thereof The hydrolysis of polymers.United States Patent (USP) 2835655 seen from the synthesis of the copolymer of maleic anhydride and styrene sulfonic acid).They are the most commercially available From Sigma-Aldrich, Inc.Other viral conjugates matter includes lectin, antibody and aptamer.

The bound substances of these viruses is fixed on solid support to remove virus from blood.An embodiment In, PSMA is coupled to microsphere and can be carried out as follows: 20 milligrams of aminated silicon dioxide or agarose particle (diameter 200 microns) are used 0.1M MES, pH are 5.0 washing three times, and wash with deionized water three times again.Granule wet cake is suspended in 0.5mL 20 milligrams/in the least The PSMA deionized water solution (Sigma-Aldrich company) risen, the carbonization two being subsequently added into 0.5 milliliter of 20 mg/ml is sub- Amine [1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, EDC] deionized water solution, the most immediately Preparation.Then with 0.1M NaHCO 3 solution by pH regulator to 7.5.Microsphere is at room temperature mixed 2 hours.10mg EDC and 10mg NHS(N-N-Hydroxysuccinimide) join in mixture, the most overnight mix.By microsphere pH 7.5 10mM HEPES buffer solution 3 times, is washed with deionized water 5 times, is then suspended in 1.0 ml deionized water.This reagent is existing Can be in being encapsulated in viruses adsorption device.

Solid support can also be derivatized/modify the group with strong negative charge its surface and internal (if It is porous).Such as polystyrene microsphere can be sulfonated, thus obtained microsphere will contain highdensity styrene sulfonic acid with and sick Poison combines.In one embodiment, the sodium salt of Amberlite IR120 resin can be used.

Because virus is with antibodies in blood sometimes, it is possible to use the affinity molecule of immobilized antibody is (such as, Protein A or virus antigen, because each antibody has two basic change site) capture virus-antibody complex.Described affinity molecule Preferably antigen antibody complex there is high-affinity, such as complement molecule (such as, C1Q .).In one example, it is fixed with benefit The immunoabsorbent column of body C1q and the combination of virus sweep post can be used on blood purification treatment virus and infect, because it can be simultaneously Remove freely virus and the virion of binding antibody.Or the adsorbent of adsorption column is fixed with C1q and viral surface antigen Affinity ligand (such as antibody), or adsorbent is to secure the adsorbent of C1q and secure the adsorbent of virus affinity ligand Mixture.Other materials arrange such as TR350 or PH350 it can also be used to remove antigen antibody complex, although they specificitys The highest.Many molecules, can be combined (as described by U.S. Patent application 10/803246), such as C1q with antigen-antibody complex Derived molecules, gC1q, gaC1q, gbC1q or gcC1q, structurally or the most similar C1Q., A, B or C chain divide Son has the polypeptide of a glutamic acid-X-lysine-X-lysine basic sequence antibody ball heads, and wherein X is a kind of aminoacid, mends Body C1q fragment/analog etc., they can serve as affinity molecule and use in the present invention.In one embodiment, solid on microsphere Surely there is the mixture of the affinity ligand of C1q and virus.

Because virus (such as hepatitis B, hepatitis C) proteolipid protein sometimes, the blood removed for lipoprotein is clean Change device and method can also be used for virus sweep.It is, for example possible to use heparin-induced external lipoprotein pellet, remove disease further Poison protein-lipid complex.Lipoprotein removes post, as dextran sulfate cellulose column and LIPOSORBER system can be linked external Virus removed by blood circulation path.It is affine that carrier in the removing use of lipid filtration/lipoprotein can also be filled in described virus Material is removed post and is removed for virus to form mixed adsorbent.Can also use and there is foregoing strong negative charge group Solid support is to remove virus protein-lipid complex.

It is an object of the present invention to provide a kind of device removing pathogen in blood and inactivated by this device The method of the pathogen in blood.The method uses physical means, it is possible to reduce side effect.This apparatus structure is simple, Ke Yiyu Other apparatus for purifying blood connects use.

The technical scheme is that for achieving the above object

A kind of remove the device of pathogen in blood, depend on including by blood effuser, plasma separator and bleed back tube The extracorporeal circulation path that secondary connection is formed, connects in the blood plasma effuser and blood plasma return duct of plasma separator and has pathogen Inactivating device.

Some examples of described pathogen inactivated device are to be a transparent vessel being provided with ultraviolet radiation facility or The individual container being provided with microwave radiation device.Described blood effuser or bleed back tube are provided with blood pump.

A kind of remove the method for pathogen in blood, utilize said apparatus, blood to flow out from the patient, followed by external Hemocyte is separated by the plasma separator in ring path with blood plasma, blood plasma fractions by pathogen inactivated device to the disease in blood plasma Substance carries out inactivation treatment, then flows back to extracorporeal circulation path and merge with hemocyte, then flows back in patient body.

Described inactivation treatment uses physical deactivation method.Described physical deactivation method includes: ultraviolet radiation, radioactive radiation Process, microwave radiation, radio frequency processing, heating or freezing.Described pathogen is antibacterial present in blood, virus or parasite.

When described pathogen is hepatitis B virus or hepatitis C virus or HIV (human immunodeficiency virus), and the most described pathogen is gone out Removable mounting is set to a transparent vessel being provided with ultraviolet radiation facility, and described inactivation treatment is to irradiate under the uviol lamp of 253nm 30 seconds, radiant intensity was 60 μ W/cm².Other exposure rate, wavelength and time can also use.Such as 220～280nm Ultraviolet wavelength, 30～2000 μ W/cm²Exposure rate, 20 seconds～the irradiation time etc. of 2 minutes.Irradiation time and container body Long-pending, shape is relevant with plasma flow rate.Above-mentioned suitable inactivation parameter value should reach higher pathogen inactivated rate and relatively low blood Slurry protein inactivation rate, can determine according to test value according to different pathogens.When described pathogen is hepatitis B virus or hepatitis C disease Poison or HIV (human immunodeficiency virus), described pathogen inactivated device is a container being provided with microwave radiation device, and described inactivation treatment is also Can be pathogen inactivated device is positioned over microwave generator in make blood plasma be warming up to 50 DEG C-70 DEG C.

Can also add photosensitizer raising in blood plasma when described inactivation treatment and kill efficiency, described photosensitizer includes phenol Thiazine dye, serge blue, psoralen, photosensitizer S59, riboflavin or anticancer photosensitizer, addition is to reach in above-mentioned radiation Be enough to kill pathogen in time and intensity be as the criterion.

In the present invention, the blood of patient carries out extracorporeal circulation, by its blood plasma fractions by a pathogen inactivated device Reason, blood cell fraction is not processed, then by the most defeated time patient body of blood plasma fractions after blood cell fraction and process.Sick Pathogen inactivation device uses physical means that the pathogen in blood plasma carries out inactivation treatment, and suitable means include illumination, especially Being ultraviolet radiation, radioactive radiation processes, microwave radiation, radio frequency processing, heating or freezing etc..Suitable pathogen includes various Antibacterial present in blood, virus (such as hepatitis virus, HIV (human immunodeficiency virus) etc.) and parasite etc..

Plasma separator of the present invention can be existing apparatus, and in some instances, container is contained within many and is used as The doughnut that blood plasma separates, whole blood flows through inside hollow fibre, and the doughnut space outerpace in container can also be filled with Solid phase carrier pathogen adsorbent.Solid phase carrier pathogen adsorbent is that insoluble solid phase carrier is fixed on can be with cause of disease Affinity molecule such as the antibody that body combines, nucleic acid ligands (aptamer), phytohemagglutinin etc. is with by pathogen absorption.Hollow is fine The film hole size of dimension does not allow blood cell to pass through but allows blood plasma and pathogen to pass through.When blood flows through inside hollow fibre Hemocyte will not flow out fiber but can diffuse to containing the blood plasma of pathogen that fiber is outer to be contacted with solid phase carrier adsorbent so that disease Substance (such as virus) is removed.Blood plasma fractions accepts light and irradiates or radiate or heat to inactivate wherein pathogen.At an example In, container itself is contained within the doughnut made by many polysulfone membrane.The film gross area of doughnut is 0.5 square meter, fenestra Footpath 0.2～0.6 micron.Container two ends have the connection of artery and vein pipeline to make blood flow through doughnut, can also fill out outside doughnut There is solid phase carrier pathogen adsorbent (its size is more than the diameter of doughnut fenestra).Due to blood cell not with solid phase carrier Pathogen adsorbent directly contacts, so biological incompatible appearance will not be caused.

Assembly of the invention can be used in conjunction with other apparatus for purifying blood, in blood purification various concrete hemoperfusions and Blood purification technology such as microgranule detoxification system, plug-type adsorption system may pass through suitably modified and is applied in the present invention.

Can also by blood plasma by a defecator to filter pathogen therein, such as carry out double filtration blood plasma clear Division (Double-filtration plasmapheresis) filters pathogen, and then blood plasma carry out above-mentioned physical deactivation hands Section processes pathogen in blood plasma to be killed, and so will filter and combine with inactivation, and effect is more preferable.

The invention also discloses a kind of method and apparatus eliminating inactivation circulating cells/pathogen, this is to special in the U.S. The improvement of profit application 12/227843.Its improvement there are three aspects.First it is in some applications primary bright energy to be accepted Object is changed into blood constituent by whole blood, such as blood plasma.This blood constituent can be that the blood plasma comprising corresponding pathogen is (as divided with blood plasma Obtain from device) or containing the component (as obtained with blood cell separator) of target cell.The energy absorbing substance that second is additional also may be used To be applied only in associated blood composition, it is possible to be removed the most defeated the Huis' body after additional energy process, to reduce additional substance Side effect.3rd be this operation can by be with interruption in the way of blood constituent is conducted batch-wise process, to ensure that it obtains foot Enough circulating cells/pathogen eliminates inactivation time.

Before remove the cell of inactivating pathogens/infection with above-mentioned extracorporeally circulating blood, can be from extracting in the patient A small amount of blood (such as, 10～50ml), the pathogen/sense then removed on a small scale with the method test of same principle/inactivate The vitro efficacy of dye cell.Only when blood sample is removed in the cell of the pathogen/infection of significant quantity, just suggestion the method With extracorporeal blood treatment to patient treatment.Can also test by multiple method with a small amount of blood, find out removal/inactivation The best approach of the pathogen/infection cell of patient.A small amount of blood is drawn out of and merotomizes, and various different pathogen are gone Except method and the result of/external inactivation compare, if they have similar safety, the method demonstrating optimal usefulness And device, the patient treatment for the treatment of will be used for.If they have different safeties, there is the low side effect of method of high effect Method will be used.Because only that a small amount of blood (such as 1～200ml) is tested rather than external circulating therapy in vitro The blood of Shi Yisheng opinion, it is possible to test with a small-scale device/reagent and shorter time.To be used for patient's The part of method or whole program can be tested in vitro and be predicted its external circulating therapy curative effect.If making to survey in this way Try not have during these a small amount of blood samples significant pathogen/infection cell (as < and 15 %) reduce/inactivation, this method will not Can be used.Only when in blood sample test in a small amount in the blood sample of the pathogen of significant quantity/cell that infects can be removed Or inactivation the method to be just used for patient's (such as, in some cases, > 25% be required, in another case, > 50% be Required).For example, it is possible to patient, 20 milliliters of blood of taking-up are in vitro tests, with the adsorbent containing a small amount of pathogen Less size device tests this blood sample, predicts whether to be used for extracorporeal circulation systemic blood stock size device External circulating therapy.The quantity of sorbent of device size and pathogen can be accordingly based on the volume of described a small amount of blood sample With the difference between the blood flow volume of patient reduces.Such as, if the device treating patient contains 100 grams of pathogen Adsorbent, it is possible to use a little post is filled with 1～2 gram of pathogen adsorbent to 20 milliliters of blood testing in vitro.At one In example, in vitro in test, 30 milliliters of blood extract from patient, and 15 milliliters of blood samples are filled with 1G pathogen by one The little adsorption column of adsorbent, another 15 milliliters of blood do not process.Then the cause of disease scale of construction in two samples is checked.If with little More than 50% of pathogen in the blood sample that post processes is removed, and carries out corresponding Full Scale Unit and can be used for this patient.? In in vitro tests, the structure of related device, parameter and program need not be the parameter being accurately same as treating patient, example Such as its size, time, flow can regulate, as long as the testing in vitro program being suitable for as in vitro tests can obtain extracorporeal circulation The prediction of the curative effect of patient during treatment.Similarly, such as use medicine or foreign material or physical method as previously described Pathogen inactivated method, it is possible to test with a small amount of blood sample from patient in vitro.The group of several method/equipment Close, it is also possible to use on a small quantity from the blood sample test of patient in vitro, if the cell of its pathogen/infection is removed/gone out Active fruit is gratifying, and this combination will can be used for treating patient.

Further object is that and provide a kind of by the Iisolated tumor cells in specific removing blood Method thus after operation and chemotherapy radiotherapy, reduce tumor recurrence and the probability of transfer, and then improve the survival rate of cancer patient.

The method that the present invention provides is after removing tumor or treating tumor, such as, after ocal resection, change Treat, radiotherapy, photodynamic therapy, photon radiotherapy, laser therapy, microwave therapy, cryotherapy, heating therapy or it Combination after, by circulating tumor cell (the circulating tumor removed and in/inactivation (such as kill) blood Cells, is called for short CTC) treatment cancer, prevent transfer and the tumor recurrence of tumor.In some embodiments, therapeutic hands Section is for primary tumo(u)r.In order to prevent neoplasm metastasis and tumor recurrence, the method at present invention includes that two step 1) remove Tumor or it is treated, such as operative treatment tumor, chemotherapy, radiotherapy, photodynamic therapy, photon radiotherapy, Laser therapy, microwave therapy, cryotherapy, heating therapy or combinations thereof；Then 2) from blood, circulating tumor cell is removed And/or inactivate circulating tumor cell by extracorporeal circulation of blood.It also provides a side measuring circulating tumor cell quantity Method.

The present invention is by the Iisolated tumor cells in specific removing blood thus is implementing operation and chemotherapy radiotherapy etc. Reduce tumor after removing the treatment of tumor focus and turn recurrence and the probability of transfer, and then improve the survival rate of cancer patient.This A kind of tumor cell scavenging system of bright offer, the principle of this system is similar to nephropathy hemodialysis and blood purification.The blood of patient Liquid is exported, and flows through a container, and the solid phase carrier in container is fixed with the antibody that can combine with tumor cell specific Or ligand substance, or carrier itself i.e. has adsorption to tumor cell, and therein swollen when patient blood flows through this container Oncocyte is just lived without flowing back to patient blood again by specific adsorption, thus is disposed from blood.And normal blood Cell and blood material then will not be removed thus flow back to again in patient body.This device is mainly used in particularly non-blood system After the tumor post-operation of cancer and radiotherapy chemotherapy, extend its life cycle to reduce the recurrence of tumour patient.This device can be with existing Having dialysis machine, hsempdiakysing systems, perfusion machine is compatible.

In general, these circulating tumor cells are by blood purification means (extracorporeally circulating blood passes through blood purification) Being eliminated or inactivate, it is swollen that this blood purification can remove when the blood that circulating tumor cell circulates in vitro/kill circulation Oncocyte and/or inactivate it.By blood purification or can be the blood of whole blood or CTC through the means of CTC inactivation treatment Composition.Blood purification and haemodialysis equipment are widely used in numerous disease such as renal failure.Have tumor cell The solid-phase adsorbent having affinity can be placed on blood purification for blood purification.For example, it is possible to optionally with The affinity molecule that tumor cell combines can be fixed on solid-phase adsorbent (such as post, filter, fiber, film, granule) and be positioned over To remove these cells in apparatus for purifying blood.These affinity molecules preferably to great majority other normal hemocytees have without or Low affinity.

Combine on the apparatus owing to the tumor cell of patient is adsorbed, can be by this dress after completing tumor cell removing Put and take off the tumor cell number checked above, thus provide the accurate prison of blood circulation tumor cell after patient Control, contributes to therapeutic outcome assessment and treatment further.So this device existing treatment function has again diagnostic function.Such as Complete tumor cell remove after can by surfactant adding apparatus with cell lysis, effluent is carried out ELISA or PCR includes tumor cell quantity to the detection of cell marker to determine, it is also possible to tumor cell eluent is (as sweet in low pH Propylhomoserin solution or 0.25% tryptic 0.15M PBS solution) after solid phase carrier elutes, it is counted.

The present invention includes a kind of method differentiating treatment of cancer effect simultaneously.Concrete grammar is that patient is carried out chemotherapy or hands After art removes tumor focus, to the circulating tumor cell in patient body, with the inventive method or other similar approach are carried out clearly Remove.Then after a period of time (such as after the week or one month), its internal circulating tumor cell quantity is again measured, If there being rise that the cancer focus that internal existence is not eliminated i.e. is described, need treatment further.

Cancer therapy drug-cancerous cell affinity molecule conjugate is used to be widely used in the treatment of tumor.People can be easy to Ground uses the method and their principle to be applied to the CTC adsorbent of the present invention.Fixing on solid phase carrier can be with The affine materials such as the antibody that tumor cell specific combines can be to tie with kinds of tumor cells or surface epithelial cell marker The material such as antibody or part closed, as anti-cell Keratin (Cytoketatin) and/or epithelial specific antigen ( EPCAM, Epithelial specific antigen) antibody or nucleic acid ligands (aptamer)；Can also be with specific The antibody of tumor cell combination or part, as used for prostatic specific membrane antigen (prostate-in urological cancer Specific membrane antigen for prostate cancer) antibody or part.These antibody and part can To be macromole biological substance such as albumen and nucleic acid, it is also possible to be synthesis macromolecule such as molecularly imprinted polymer, it is also possible to be The little molecule such as folic acid and the like that can combine with tumor cell surface.They can be single molecule can also be multiple The mixing of molecule.This kind of affine material should not have affinity or the lowest affinity with normal blood cell.So due to solid phase It is combined with the molecule that tumor cell is had affinity on carrier, or carrier itself i.e. has adsorption, blood to tumor cell Cause tumor cell to be combined with solid phase carrier when flowing through carrier during circulation in vitro and be captured thus decrease in blood Iisolated tumor cells (has another name called circulating tumor cell, circulating tumor cell, CTC).If tumor cell affinant Confrontation leukocyte there is no affinity but erythrocyte or platelet are still had affinity then can first by erythrocyte in patient blood and Then platelet separates removes tumor cell again by defeated for blood ex vivo from the mixture of tumor cell and leukocyte.If it is swollen Oncocyte affinant confrontation leukocyte has affinity but does not has erythrocyte or platelet the affinity then can be first by patient blood Then middle leukocyte separates removes tumor cell again by defeated for blood ex vivo from tumor cell with erythrocyte/hematoblastic mixture In.Glycosyl on tumor cell film often can produce specific change, available agglutinin and its specific bond.Such as gastric cancer is thin It is positive that born of the same parents mostly are phaseolus vulgaris agglutinin PHA, and BSA is then positive to malignant breast tumor.So agglutinin can also be as affinity molecule Remove tumor cell.Affinity ligand can also be for other tumor cell marker, such as HER-2(HER-2/neu albumen), EGFR, mammanaglobin albumen, PMSA, GA733-2 and MUC1.Many can be found easily from document to be suitable for Tumor cell surface mark.

Special marker on tumor cell can also be artificially introduced.Its ultimate principle is as follows.For tumor cell surface Self material-specific A, can add to connect has its affinity molecule B of special marker C, and after such B with A is combined, tumor is thin Cellular surface just has special marker C.Will be by swollen by being solidified with the solid phase carrier of the affinity molecule for special marker C Oncocyte adsorption removal.For example, it is possible to by the EPCAM antibody (affinity molecule B) of biotin (special marker C) labelling and tumor Cell binding, then with being combined with the solid phase carrier of affinity element or streptavidin by clear for tumor cell with biotin in blood Remove.It is removed principle and is similar to CellPro Ceprate SC Stem Cell system.Biotin labeled EPCAM antibody is permissible To patient injection, it is also possible to add so that it is combined with tumor cell before flowing through solid phase carrier after blood is flowed out patient.Affine Molecule B can be a kind of molecule can also be the mixture of different kinds of molecules.In some implementations, C and B can be a molecule. Such as it can be Yang Yuan anti-EPCAM antibody, and is combined with rabbit anti-goat-anti body on solid phase carrier to remove You Yang source, surface antibody Tumor cell.It is to say, the most special marker of affinity molecule B.

Can also adsorb simultaneously or connect on solid phase carrier and have anticoagulative substance such as heparin etc., to prevent container intravascular coagulation Occur.Blocking factor (blocking factor) is ever-present a kind of material in Serum of Cancer Patients.It can be closed or hinder Disconnected lymphocyte is to the identification of tumor cell and attack, thus promotes the growth of tumor cell, therefore claims blocking factor.Blocking factor It is soluble tumour antigen or antigen antibody complex or blocking antibody (blocking antibody).Soluble antigen and effect The antigen receptor answering cell surface combines, and antigen antibody complex can blocking effect cell antigen receptor, it is possible to close tumor thin Cellular surface antigen.The identification of both the above situation all interference effect cells against tumor and killing.Tumor antigen also can be non-by some Specific component (such as sialomucin etc.) covers, thus disturbs immunocyte to the identification of tumor antigen and killing.Tumor is thin Born of the same parents also secrete the immunosuppressive factors such as IL-10, TGF-β, VEGF and PGE2 and DC precursor can be suppressed to grow, stop its to Ripe DC differentiation；Suppression DC(the most tumor-infiltrated position DC) express MHC-class Ⅱmolecule and B7.Under above-mentioned factor effect, DC The TIL tolerance to tumor antigen can be induced.In addition these factors are also lowered other immune cell functions, beneficially tumor cell and are escaped Ease immune attack.Such factor also includes Fas ligand, MHC I, MHC II, CD44, placental alkaline Phosphatase, TSG-101, MHC I-peptide complexes, MHC II-peptide complexes etc., also Will be reduced self to the suppression of tumor cell and killing.Immune described in U.S. Patent application 20090304677 Suppressive microvesicular particles also can reduce self to the suppression of tumor cell and killing.Above-mentioned Immunosuppressive factors, blocking factor, the affinant of microvesicular particles such as corresponding antibodies, affinity coagulates Collection element etc. can also be simultaneously attached and be applied to the present invention on solid phase carrier, to strengthen immunocompetence, improves antitumor effect Really.

Owing to some tumor cell surface is covered so being unfavorable for that the solid phase carrier by securing antibody captures by antibody.Can This kind of cell has been removed to use antagonism original antibody complex to have specific molecule to be fixed on solid phase carrier.Such as, may be used To be fixed on solid phase carrier with complement molecule, such as C1Q.. additionally, due to antibody, there are two active binding site, So can also be combined with tumor cell-antibody complex so tumor-cell antigen can also be fixed on solid phase carrier and incite somebody to action It removes.Further whole blood can also be divided into cell component and plasma fraction two parts, only wherein cell component is carried out solid Phase carrier adsorption, thus can be secured in solid phase to remove with the molecule of binding antibody such as protein A with utilizing Remove tumor cell-antibody complex, and the adsorption column such as TR350, PH350 is removed.

Suitable solid phase carrier can be column or dress up column in granular form, it is also possible to be film, filter membrane, fiber, in Hollow fiber, pipe, granule, microsphere, magnetic bead or other forms, allow connection affinity to divide as long as it has suitable surface texture Son.Two kinds of preferred types of solid-phase adsorbent are granule or the fibers connecting and having affinity ligand.Fiber can be made into netted or weaving Become there is suitable pore size (such as, aperture 10～150um).It is, for example possible to use at Toraymyxin PMX- Like fibrous in 20R device.Toraymyxin PMX-20R is fixed on styroflex by polymyxin B covalency Extracorporeal blood perfusion device.This device context amasss and includes 56 grams of fibers for 225mm X 63 mm.CTC removes device then at polyphenyl Connect CTC affinity ligand on vinyl fiber, rather than use polymyxin.Owing to still there being some free aminos on polymyxin B, Can also be the most covalently bound with CTC affinity ligand with the fiber that polymyxin B is fixing.Other kinds of fiber can also make With, such as cellulose fibre, polysulfone fibre, polyether sulfone fiber, vinal, cellulose acetate fibre, polyethylene fibre, Polypropylene fibre, polymethylmethacrylate fibers, polyacrylonitrile fibre, tri acetic acid fiber cellulose fiber or combinations thereof.This A little fibers can easily derivatization with affinity ligand coupling.Fiber can also make netted or weaving there is suitable aperture Size (such as 10～250 microns), therefore can play filtration selectively or relative obstacle, to help the parent of CTC With and capture.Bigger microsphere (the most a diameter of 50 microns) is much larger so can be by properly due to its volume ratio blood cell In the defecator (a diameter of 30 microns of such as its filter opening) in aperture blocks and do not flows into patient body, normal blood is thin at the same time Born of the same parents then can be flow through defecator thus flow back in patient body.Less microsphere particle has bigger specific surface area, is beneficial to inhale If but attached diameter too small (such as diameter is less than 10 microns), it is unfavorable for owing to its size is similar to blood cell according to big Little selective filter separates and easily flows back to internal together with blood cell.Use magnetic microsphere then can rely on externally-applied magnetic field Microsphere is made to be confined to specific region and to suppress microsphere to flow into internal thus allow to use relatively minimicrosphere.When magnetic-particle is used for removing When removing CTC, the blood constituent containing CTC can mix in blood purification with magnetic particle.Use magnetic particle separates The method of cell is well-known.Similarly, the non magnetic microparticle with affinity ligand may also be used for and containing CTC's Blood constituent mixes, and then separates with remaining cellular portions with the filter unit of suitable pore size, to prevent microparticle from entering Enter human body (such as, using microgranule and the 30 micron pore size filters of 50 microns of sizes).

Application tumor affinity molecule is connected formation targeted drug and has been widely used for oncotherapy with anticarcinogen.Equally, These affinity molecules can be connected to solid phase carrier for the present invention.These affinity molecules can be to have kinds of tumor cells Specificity (such as EPCAM antibody), it is also possible to be only certain tumor cell to be had specificity, such as special to certain lung carcinoma cell Antibody then can be used to such pulmonary carcinoma is treated.Kinds of tumor cells affinity molecule can also can be mixed by one Conjunction is connected on carrier.There are many methods affinity molecule can be connected to solid phase carrier.This quadrat method is it is known that can be Document, books obtain.Such as, the affinity molecule containing amino can be by forming amido link and the solid phase carrier containing carboxyl Key and.This key and can being completed by the application polypeptide condensing agent such as EDC.

In some implements, blood flows through hollow-fibre membrane, and the surface of film connects tumor cell affinity molecule Such as tumor cell surface molecular antibody or part.Can be lived to provide by chemical modification such as graft polymerization in hollow-fibre membrane surface Sexual function group (such as amino or carboxyl) be beneficial to affinity molecule key and.Hollow-fibre membrane has been widely used in blood Dialysis and blood filtration.

In some implements, blood is extracted out from the patient, flows through the such as film table of the solid phase carrier containing affinity molecule Face.In other implement, blood is extracted out from the patient, is then split into blood plasma and cell two parts.A lot of methods Separate blood components can be used for, as used various plasma separator.Then cellular portions flows through the solid phase containing affinity molecule Carrier flows back in patient body to remove in then tumor cell flows back to patient body or to merge with blood plasma.This operation can repeat many Secondary to arrive expected effect.For example, it is possible in art, postoperative or chemotherapy one day after, three days, one week, one month, Yi Jisan Respectively carrying out once after individual month or determine according to tumor cell quantity in blood, each 2 hours, hemoperfusion flow was 100-200 Milliliter is per minute.The concrete operations carrying out hemoperfusion and blood purification can be looked in document and research report at many teaching materials Arrive.Various concrete hemoperfusions and blood purification technology such as microgranule detoxification system, plug-type adsorption system may pass through suitable It is applied in the present invention when amendment.Before blood purification and purify after can take blood measure wherein tumor cell quantity with weigh control Therapeutic effect and guides whether carry out more blood purification operation.If in blood, free tumor is thin before and after operation, radiotherapy chemotherapy etc. Born of the same parents' quantity is the lowest and does not increase, and can not carry out blood purification operation, if increasing more or the highest, needs Carry out blood purification operation until its number reduces the state reaching satisfied.Although by blood Iisolated tumor cells remove for The patient not carrying out other antineoplaston any is the most meaningful, but owing to Iisolated tumor cells can constantly produce, so Good certain antineoplaston such as Radiotherapy chemotherapy that first carries out, to remove the focus producing Iisolated tumor cells, then carries out blood Liquid purifies and removes remaining Iisolated tumor cells to prevent recurrence and transfer.

The whole blood extracted out can be divided into several cellular component with blood cell separation device.By containing a large amount of CTC's in treatment Component is through CTC removal/inactivation treatment.Such as, separating from whole blood to contain CTC blood constituent, many methods can With application, such as Leukapheresis, filtration based on cell size, it is centrifuged, elutriation etc..Many blood cell separation devices can be used In this purpose, such as cs3000plus blood cell separator, COBEVR spectroscopic system and Elutra system (Caridian BCT).Such as, people can also use the device of similar hollow fiber plasma separators type, separates CTC from other hemocytees, this The film that hollow-fibre membrane separates application at blood plasma has bigger aperture.Aperture is large enough to make erythrocyte and platelet pass through, But less than CTC (such as aperture can be 8 microns, 10 microns, 12 microns or 15 microns).After whole blood passes through, doughnut, especially It is inside it, to will be enriched in CTC and liquid outside doughnut will be containing erythrocyte, some leukocyte, blood plasma and platelet, Outside component can be the most defeated back to patient.Described coupling has the solid-phase adsorbent (its particle diameter > membrane aperture) of CTC affinity ligand also Doughnut can be filled in.The film of doughnut also can be coated with affinity ligand.If such structure is used, at certain In a little application, membrane aperture can also be greater than CTC(such as 20-50 micron).CTC absorption can also be filled with outside doughnut Agent.

A kind of CTC method of removing is to use blood cell separator.When blood processes through blood cell separator, mostly During in the case of number, CTC will be left in white cell component.In some cases, CTC will be at monocyte fraction；Certain given patient The accurate distribution of concrete CTC can be determined by the experiment of a small amount of blood of test given patient.People can use hemocyte Seperator separates these components.Containing CTC part continuously or to be given CTC removal/inactivation treatment with batch (-type).Other blood Liquid composition can directly send back to internal or with.Other blood constituents can also inactivate hands by different blood purifications or CTC Section returns internal.White cell component containing CTC can also be by centrifugal equipment reprocessing.The invention provides multiple CTC clear Except/ablation method.These methods can be applied in combination to reach more preferable effect.

The method removing the tumor cell in blood after another kind of postoperative or chemotherapy or radiotherapy is to filter.Due to tumor cell Volume is more than most of blood cells, allows patient blood flow through and has the filter in suitable aperture and just can remove tumor cell and allow strong In health blood flowing patient body.Such as 10 microns or 15 microns of aperture of application or the filter membrane of 20 microns.Although leukocyte volume is also Relatively big still morphotropism is higher such that it is able to be more easy to pass through filter membrane than tumor cell.Tumor cell due to easy bonding agglomerating so It is more easy to blocked.Application has the polycarbonate membrane of 8-15 micron pore size and can effectively remove most tumors cell and allow mostly Number leukocyte passes through.Single-stage can be used to filter and can also use multistage filtering.The series connection of multiple filter membrane such as can be used to carry out Filter.Use multistage filtering can use the filter of larger aperture.The microsphere that addition can be combined with tumor cell specific is then Owing to itself and tumor cell are combined into the bigger complex of volume thus it are more favorable for being blocked by filter selectivity and allow blood Cell passes through.Can also will by the less filter membrane in aperture or other ways (such as leukocyte separator, blood cell separator, centrifugal) Leukocyte all removes with tumor cell, then by other hemocytees and blood plasma first defeated ex vivo, if it is desired to by the most defeated for leukocyte time Internal, then can remove in the most defeated ex vivo of tumor cell in the leukocyte collected, removing tumor cell in leukocyte can use Tumor cell adsorbent equipment in the ways such as widely used immunomagnetic beads or the present invention.First leukocyte selectivity can also be removed Go, then tumor cell and other blood cells are passed through the filter membrane (the such as filter membrane of 5 or 10 or 15 microns of filter openings) that aperture is less And remove tumor cell and allow erythrocyte and platelet by and in defeated time patient body.The method that leukocyte selectivity is removed The most a lot, the nylon fiber that can optionally adsorb leukocyte according to nylon fiber in the presence of calcium ion is such as applied Filter, granulocyte based on cellulose acetate pearl adsorbs the various leukocyte adsorbers such as filter.These leukocyte separated Can by again from leukocyte adsorber eluting and in defeated time patient body.In one example, operative treatment is first carried out to breast Adenocarcinoma patients's tumor resection, then carries out hemocyte separation Leukapheresis to patient.Collected white cell component (200- 400ml) (1 gram of surface is fixed with the polystyrene magnetic particle of 1 micron of size of EpCAM antibody, or 5 milliliters with CTC adsorbent The surface of 100um diameter is fixed with the Sepharose 4B granule of EpCAM antibody) mix 15 minutes, then CTC adsorbent is removed Go (such as, use a Magnet remove magnetic particle or remove sepharose 4B granule with the filter in 60um aperture), the most clearly Clean leucocyte fraction is then returned to patient.

The carbohydrate of tumor cell surface is the most different compared with normal cell, it is possible to use particularly for it Coagulation usually combine CTC.Such as, PHA (phaseolus vulgaris agglutinin) agglutinin can be combined with stomach cancer cell, and BSA agglutinin can To be combined with breast cancer cell.Fixing lectin on solid phase carrier can be used for removing CTC.Tumor cell surface due to The reason such as sialylated often has more negative charge than normal cell.So the material the most poly-amino Portugal containing positive charge Grape sugar, chitosan, the synthetic polymer etc. containing polyamino group can be combined by electrostatic interaction therewith.By such material with solid Its combination of the filters such as phase carrier or filter membrane, then flows through blood and can also be blocked by wherein tumor cell.In order to avoid red Cell is also adsorbed, and wherein erythrocyte first can separate (as centrifugal, filtration etc.), then by thin to remaining leukocyte and tumor Born of the same parents flow through the solid phase carrier with positive charge or filter to remove wherein Iisolated tumor cells；Then by defeated to erythrocyte and leukocyte Return.

Owing to different cells has different density, it is also possible to by centrifugal way, tumor cell is separated, such as whole blood After centrifugal, erythrocyte is in lower floor, and leukocyte and tumor cell are on upper strata.Can be by microsphere particle bigger for density (such as glass, silicon Or magnetic microsphere) upper connection tumor cell specific binding molecule mixes centrifugal such tumor cell with blood will be in combination and close Degree become drop to greatly bottom and with other cell separation then by defeated for healthy blood time patient.Multiple hemocyte has been had to separate at present Different cell masses in blood can be separated by machine according to the size of cell/density difference.Different tumor cells and tumor Cell mass has different distributions on different blood cell separators, and concrete separation parameter can be determined by experiment.From blood Other hemocytees a large amount of are contained in the groups of cells branch rolled into a ball containing tumor cell and tumor cell that separator cell obtains, can be by This cellular component with removing tumor cell therein and sends normal plasma cell back to body through above-mentioned tumor cell adsorbent equipment In.

Because the treatment in current invention may cause loss or the inactivation of some blood constituent, so after the treatment may be used To give the blood constituent that patient supplements loss.Such as, if erythrocyte or platelet are lost or the patient that is killed can be to Giving the erythrocyte come from healthy donors or the platelet of appropriate amount, if needing to supplement specific leukocyte, can be used to from strong The leukocyte (such as cultivating from bone marrow or the stem cell of patient) of health donor or the resource from patient self.Can improve The medicine that hemocyte produces can also be applied.Liquid/buffer (such as artificial plasm), it is also possible to add in the treatment to help CTC removes/inactivates and separates with blood constituent.

The means that can inactivate CTC are equally applicable to extracorporeally circulating blood or the blood constituent containing CTC, due to only at body The blood constituent of outer circulation carries out processing and therefore reduces its side effect to whole body other system.Specific means can be heating (such as extracorporeally circulating blood or the blood constitutent containing CTC being heated to 42-48 degree, concrete grammar can be heat exchange, micro- Ripple or infrared), return internal blood and can be cooled to normally to return internal.In one example, obtain through blood separator The white cell component containing CTC arrived heats 30 minutes at 42 degree, is then returned to internal.

In certain embodiments, UV is used for inactivateing CTC, to extracorporeal circulation whole blood or the blood constituent (example containing a large amount of CTC As being centrifuged the leucocyte fraction obtained from blood cell separator) in vitro it is irradiated.A kind of preferably wavelength is those Nucleic acid has the wavelength of stronger absorption, such as 250-260nm wavelength.Radiant intensity (such as 10J/ml) and time should be enough to kill Dead CTC and less to normal injury of blood cell.Extracorporeal circulation and/or UV process can by be with flowing continuously in the way of or interval The mode of flowing is carried out, to guarantee enough irradiation times.Ultraviolet therapy condition can pass through a small amount of the containing of experiment test The blood of CTC determines.CTC removes and flows through the blood/blood after affinity adsorbent can also be applied to pre-irradiation or irradiate Composition, again that it is the most defeated back to patient.

In some embodiments, chemical agent (such as anticarcinogen) is used for inactivateing CTC.CTC inactivator can add It is in external extracorporeal circulation whole blood or the blood constitutent (such as the centrifugal leucocyte fraction obtained) containing CTC.Can apply anti-swollen Especially those directly kill/inactivate the medicine of cancerous cell to tumor agent/medicine.Suitably the example of reagent and/medicine include but not It is limited to: alkylating agent (such as, phosphamide chlormethine, sulfur generation-TEPA；Nitrosourea, such as carmustine, lomustine, department Mo Siting and streptozotocin), bleomycin, amycin, mitomycin, cisplatin and paclitaxel.If external blood or blood Composition is the most infrared by photon radiation, it is seen that light or ultraviolet process, and optical active matter is (such as treating the examination of blood product sterilization Agent), such as phenothiazine dyes, methylene blue, vitamin B2, psoralen (such as 8-MOP, AMT), the photosensitizer of photodynamic therapy (as Photofrin or Levulan or nano-size titania) can also be used for inactivateing CTC.These optical active matter/photosensitizer can also Coupling affinity ligand is to provide more preferable selectivity to CTC.Preferably they blood in vitro time be added to the blood containing CTC Composition or whole blood are to avoid these medicines to cause internal side effect.The amount of the reagent used should sufficiently achieve therapeutic effect, The IC 50 of such as 10 times, or determine inactivation CTC required dosage by document or experiment.Except those half-life are short before feedback With regard to deactivated medicine, preferably before blood/blood components reinfusion is to patient, these reagent are removed or are inactivated (as neutralized) To reduce the impact on patient of potential these medicines of side effect.For example, it is possible to make the blood/blood constituent of pastille by filling There is the apparatus for purifying blood (such as a hemoperfusion post) of adsorbent (such as 100g activated carbon, adsorbent resin etc.), thus absorption removes Remove these medicines；Or from blood, eliminate medicine with hemodialysis.The equipment having these types a lot of is available for blood purification/blood Perfusion/hemodialysis.Such as, the agar of the crosslinking of embedding attapulgite clay, Paar MB1 wave filter, equine pharmacy Blueflex filter or LeucoVir MB filter can be used for removing methylene blue at blood or blood constituent.Because they The difference of molecular size range hemodialyzer based on semipermeable membrane or filter membrane can optionally remove antitumor agent.Absorption The apparatus for purifying blood that agent is filled, it is also possible to be used for removing these antitumor agents through this device at blood or blood constituent.Inhale Receipts can be non-selective or selective.Such as, Linesless charcoal and adsorbent resin are the poor adsorbents of selectivity.It is fixed with specific The solid phase carrier of the affinity molecule of antitumor agent is used as adsorbent and optionally removes antitumor agent.These medicines can also By adding suitable nertralizer to be inactivated.Such as, spermine or protamine can be used for neutralizing the cellulotoxic effect of alkylating agent. Treatment can be being in the way of continuously flowing or the mode of intermittent flow.Such as, blood is continuously withdrawn, and is then continuously added to CTC deactivator also returns patient continuously.In another example, blood/blood constituent is with a certain amount of extraction in batches, through certain Time and drug treating, then return to patient, carries out extraction and the medicine of the blood/blood constituent of next batch the most again Process.This will allow for the enough CTC inaxtivation of drug time.It can also be the combination of flowing/intermittent flow continuously.Such as, Blood is separated by hemocyte and adsorbent is continuously completed, but CTC inactivation and antitumor agent (adding medicament and temperature bath), go Except medicament and defeated time patient of blood constituent are to carry out in batch.If whole blood is extracted out and fed back is to enter in the way of intermittent flow OK, single needle/conduit can be used for performing extract out in different time intervals and return blood.Intermittent flow may apply to whole mistake Journey or a part for overall process.Antitumor cell Drug therapy blood or blood constituent can be intermittent flow (batches), make him Obtain required drug treating time (if cancer therapy drug action times of 5～30 minutes are to come into force, 5-10 minute light power Treatment).Another kind of form is that this medicine is added into blood, but blood the most directly removes the medicine of interpolation before returning to patient Or medicine is injected directly into the blood vessel of patient and does not carry out extracorporeal circulation, only (such as, make medicine in blood after completing treatment The a period of time stopped), carry out extra hemodialysis or blood purification to remove the medicine of addition from blood.

CTC removal/deactivation treatment the process of present invention is repeatable several times to reach required effect.Such as, it is permissible Operation or chemotherapy one day after, three days, one week, carry out when one month and three months;Or according to tumor cell amount in blood Carry out.In many application examples, the volume of the blood extracorporeal circulation for the treatment of should be more than the total blood volume of patient every time.Preferably This volume is more than the twice of the total blood volume of patient.During operation in certain embodiments, blood flow is that 100-200ml/ divides Clock, continues 2 hours.Many blood purification methods and program can obtain from reference material.Before and after CTC blood purification The change of amount can be used to evaluate therapeutic effect, it is possible to be used for determining the need for further blood purification.If hands The amount of the CTC before and after art and chemotherapy is the lowest, does not increases, blood purification then perhaps without.If CTC amount increase or The highest, then need to carry out blood purification and measure desired level to reduce CTC.Although it is single when there is no other oncotherapy It is also useful for solely carrying out blood purification, first carries out removing treatment (the such as hands of the root of the CTC of generation Art, chemotherapy, radiotherapy etc.), then carry out blood purification (process of CTC removal/inactivation) and remain CTC in blood with removing, with Prevent recurrence and the transfer of tumor.First preferably carried out within the ablation of tumors operation later moon in many application implementations Secondary CTC removal/inactivation treatment.CTC monitoring and test can be carried out, if CTC quantity the highest (such as > 5/milliliter), May be repeated blood purification treatment (as every 3 days or weekly), until CTC quantity is satisfactory.In one example, Carry out CTC removal/inactivation treatment for the first time after surgery in the week, then carried out once, then at ensuing one week Ensuing fortnight, next month and afterwards two months, carried out once after following 6 months and every 6 months.CTC monitors Test may be used to determine removes/inactivation treatment the need of more CTC.In another example, after surgery in one day The first time CTC removal/inactivation that carries out processes, and then next week is once, ensuing fortnight, next month and afterwards Within two months, be repeated once, further CTC monitoring test may be used to determine the need of more CTC removal/inactivation process. In the 3rd example, it is immediately performed for the first time CTC removal/inactivation treatment after surgery, the most every 3 days or carry out once weekly CTC removal/inactivation treatment, until CTC satisfactory number.For patients undergoing chemotherapy, CTC removal/inactivation processes for the first time Can give in end of chemotherapy one week after, decide whether to carry out at more CTC removal/inactivation by the detection of CTC quantity Reason, as the highest in CTC counting, again carry out blood purification.CTC removal/inactivation treatment can at chemotherapeutics first for the first time Carry out in being administered a week, the most repeatable removal of CTC more times/deactivation treatment.Such as, chemotherapy one day, three days, one Week, one month and carry out after every 3 months.If patient accepts radiotherapy, photodynamic therapy, photon radiotherapy, laser is treated Method, microwave therapy, cryotherapy or thermotherapy (such as hyperthermia), described first time CTC removal/inactivation processes can be first Carry out in secondary treatment 1 week or after being terminated one week by whole treatment.More CTC remove/inactivate and may be repeated.Such as, one My god, three days, a week, within one month, carry out after treating three months.Whether CTC quantity can often be monitored carrying out to instruct More CTC removal/inactivation treatment, if CTC counting Gao Zeke is carried out.

Nonspecific method can also be used to remove tumor cell from blood/blood constituent and (such as, to use activated carbon mistake Filter, distinction membrane filtration, cryofiltration, there is the filter such as 8um-12um bore filter in suitable aperture Device).Leucocyte filter can be when blood/blood constituent passes through for removing tumor cell.Tumor cell is usually concentrated in Shift together.Filtration based on size can be used for removing the cancerous cell of caking.These cell masses are more than the chi of hemocyte Very little, therefore, use filter can remove the tumor cell of caking, but (filter need to have suitable aperture, greatly not to remove hemocyte In size of blood cells, less than the aperture of tumor cell mass, such as 20um), purify for post-operative blood and can reduce transfer Risk.Similar blood purification method, can find in foregoing many lists of references.

Tumor cell absorption and removing device in the present invention can be added a tumor cell simultaneously and be killed device.Due to swollen Oncocyte be attracted on the solid phase carrier in container or blocked thus they will in container stay longer or always Stop other blood cells the most quickly to flow away.In container, so apply tumor cell when cell kills means be then applied relatively Long-time thus be prone to be killed.It can be heating (such as 50-60 degree) or freeze (such as-10 degree) or ultraviolet that cell kills means Or light radiation or microwave, radio frequency or radiation or various means combine.These means are applied only on the solid phase carrier in container. By Accommodation intensity and blood flow flow velocity, tumor cell will be killed by selectivity and blood the most not to be adsorbed is thin Born of the same parents are the most unaffected.Accordingly even when tumor cell again by refunds body owing to being inactivated so also will not cause a disease again.If Inactivate CTC with photon, optical active matter can be added in blood, such as phenothiazine dyes, methylene blue, vitamin B2, Psoralen Fat, photodynamic therapy photosensitizer agent etc., to increase CTC inactivating efficacy.These optical active matter/photosensitizer can also be joined plus affine Body provides more preferable selectivity to CTC.Can also flow back to internal before by optical active matter remove to reduce side effect.Can also lead to Crossing blood and pass through a container, optionally slow down the motion of CTC, therefore CTC will accept longer one section of inactivation time.Such as, The hole pattern (such as film or fiber alignment or fabric) of multilamellar can be comprised in box, mesh size bigger than erythrocyte but unlike CTC size much larger (such as 20 microns, 30 microns, 50 microns or 100 micron mesh sizes).Net can also scribble affinity ligand To catch CTC.Container can also be containing having relative blocking structure to block CTC, such as distance 80 microns each other A lot of micro-column structure.

The carrying out Therapeutic Method for tumor and cancer and comprise the following steps of present disclosure: 1) tumor patient is performed the operation Excision or chemotherapy or radiotherapy or other tumor body is had lethal effect such as phototherapy, microwave, radio frequency, freezing etc. treat to remove oncosis Stove.2) then patient carrying out hemoperfusion purification run to remove the Iisolated tumor cells in blood, hemoperfusion purifies can With by blood flowing through filter or being fixed with the solid phase carrier of the affine material of tumor cell and complete, its specific procedure is permissible For first patient blood being imported a container, the solid phase carrier that container is contained within to combine with tumor cell maybe can be by swollen The filter of oncocyte retardance, thus Iisolated tumor cells is removed from blood, the defeated time patient of blood after then purifying.

In an example, tumor patient removes circulating tumor cell through whole blood purified treatment after tumor removes operation. The risk of neoplasm metastasis is increased owing to operation can cause tumor cell to be released into blood.Some chemotherapy or radiotherapy also can cause tumor thin Born of the same parents are released in blood.After operation/chemotherapy/Patients During Radiotherapy neutralizes, remove them through blood purification can reduce tumor recurrence With shift risk.Blood purification technology is optional for such as the above method.Such as, a kind of method is to use to be fixed with leaf The adsorption column post of acid is optionally through the tumor cell removed in the blood flowing through post.Another example is to use non-selectivity Method such as activated carbon adsorption or leucocyte filter or selective membrane be filtered to remove tumor cell in blood.

The solid phase carrier that whole blood or blood constituent carry out blood purification can be post, film, fiber, granule or any its Its suitable surface mass, it should comprise suitable surface characteristic (including the surface within loose structure) for affinity molecule Direct-coupling or carry out is modified or is coupled after surface derivatization.If solid support is porous, can also be used for solid inside it Surely there is the molecule of affinity.

In some embodiments, blood flows through hollow-fibre membrane, and the affinity molecule of tumor cell is fixed on perforated membrane Outside.The example of affinity molecule is anti-cell Keratin (cytoketatins) antibody and epithelial cell adhesion molecule (EpCAM) (such as to tumor of prostate, antibody is for prostate specific membrane antigen to any other antibody of antibody or antitumor cell Antibody).Affinity molecule can be subsequently placed in the periphery of film prior to being fixedly attached to solid phase carrier substrate.In solid matrix one Individual example is agarose or glucosan.The example of hollow-fibre membrane can be in United States Patent (USP) 6528057 and United States Patent (USP) 7226429 Find.Blood purification method and program can also obtain easily from these patents and other hemodialysis reference materials.

In an embodiment of the method for the present invention, blood is extracted out from patient and is contacted and is fixed with affine point of tumor cell The filter membrane of son.In another embodiment, blood or blood constituent contact have the absorption of tumor cell specific affinity molecule Agent granule is to remove them and to be back to patient.Treatment can periodically repeat, until desired effect has reached.Example As, this process can carry out 2 hours per week.Therefore, the illustrative steps of the present invention is: (a) by body fluid under certain condition With and tumor cell have the immobilized molecule contacts of affinity, this condition can make the formation complex of this molecule and target cell B () collects unconjugated material;And (c) feeds back unconjugated part in the patient.

Many methods are had to may be used for coupling molecule to solid carrier.These methods can carry from ready-made Scientific Periodicals For the supplier of coupling agent, or related web site obtains.Such as, the molecule containing primary amine can be formed by amido link and be connected to The solid support of carboxylic group;Being generally formed with EDC [1-(3-dimethyl of amido link between amine and carboxylic group Aminopropyl)-3-ethyl-carbodiimide hydrochloride] or other carbodiimides complete.The molecule being combined with tumor cell, can Can need suitably to modify or derive to be beneficial to coupling, it is modified and derivative should not significantly reducing is combined activity with tumor cell. Also solid carrier can be coupled to again by being coupled to another part.In some embodiments, tumor cell bound substances itself can For forming solid phase carrier.

Accompanying drawing explanation

Fig. 1 is that the blood in the present invention, patient being contained morbid substance such as pathogen flows through after plasma separating unit with disease The structural representation of the example that pathogen inactivation device processes.

Fig. 2 is an example of the plasma separator device containing pathogen adsorbent removed for pathogen in the present invention The schematic diagram of son.

Fig. 3 is remove circulating tumor cell (circulating tumor cells is called for short CTC) in the present invention one Plant the structural representation of device.

Fig. 4 is the structural representation of a kind of device removing circulating tumor cell including CTC adsorbent in the present invention.

Fig. 5 is the structural representation of a kind of device removing circulating tumor cell not having CTC cell outlet in the present invention.

Fig. 6 is the dress removing circulating tumor cell that in the present invention, a kind of CTC of including adsorbent does not has CTC cell outlet The structural representation put.

Fig. 7 is the structure of a kind of device removing circulating tumor cell without doughnut based on filter membrane in the present invention Schematic diagram.

Fig. 8 is a kind of structural representation by the device removing circulating tumor cell of three filters in series in the present invention.

Fig. 9 is the signal of a kind of apparatus and method that blood carries out extracorporeal circulation removing circulating tumor cell in the present invention Figure.

Detailed description of the invention

In order to be better understood from the present invention, below in conjunction with embodiment the present invention done and describe in detail further, but this The scope that invention claims is not limited to the scope that embodiment represents.

Example 1: a kind of remove the device of pathogen in blood, as shown in Figure 1: include by blood effuser 1, plasma separator 3 and bleed back tube 7 be sequentially connected with formed extracorporeal circulation path, blood plasma effuser 4 and blood plasma at plasma separator 3 return Flow tube 6 connects and has pathogen inactivated device 5.

Flow through in connection has the blood plasma of pathogen inactivated device 5 can also directly flow back to patient body or flow into and connect Blood of Patients Pipe and the pipeline of plasma separator blood outflow end.Now plasma separator can not have blood plasma return duct.

Described pathogen inactivated device 5 can be an outside or inside be provided with ultraviolet radiation facility transparent vessel or One outside or inside is provided with the container of microwave radiation device.Described blood effuser 1 is provided with blood pump 2.This device also may be used With additional HIV (human immunodeficiency virus) adsorbent equipment or other virus filtration devices, blood plasma flows out or blood plasma flows back on pipeline the most in FIG Connect again a HIV (human immunodeficiency virus) adsorbent equipment (solid support particle of the particle diameter 20um such as having included HIV antibody covalently bound Adsorption column) or to use membrane aperture be the virus filtration device (HIV (human immunodeficiency virus) size is 100nm) of 60 nm filter membranes.

Example 2: described in include pathogen adsorbent for HBV/HCV treatment of infection plasma separator one as figure Shown in 2, blood is separated into the blood plasma fractions containing virus and blood cell fraction after flowing through this plasma separator, in plasma separator Containing the doughnut 8 made by many polysulfone membrane, the film gross area of doughnut is 1 square meter, membrane aperture 0.5 micron.Container Two ends have the connection of artery and vein pipeline to make blood flow through doughnut 8, are filled with solid phase carrier pathogen adsorbent outside doughnut 8 9.Have pathogen affinant matter on solid phase carrier pathogen adsorbent 9, the most covalently bound anti-hepatitis B surface antigen antibody or The glucosan Sepharose 4B granule of the crosslinking of the particle diameter 90um of PSMA.The blood plasma flowed out from blood plasma effuser can lead to further Cross an inactivation of virus equipment (such as ultraviolet irradiates or isotope irradiates), then return to plasma separator.Extra light Activating agent (such as those are at the medicine of the pathogen inactivated middle use of photochemistry) or photosensitizer can be at blood plasma through inactivation treatment Before be added in blood plasma, and remove with activated carbon after inactivation.The blood plasma entering and leaving inactivating device can shape in batches Formula carries out (such as, by increase valve open/closed valve after a certain time), to guarantee that blood plasma enough time of staying is to reach The time is processed required for inactivating device.

Example 3: the blood samples of patients first arteriovenous passage suffering from hepatitis C carries out extracorporeal circulation, and its blood is in one Hollow fiber membrane plasma separator, blood flow is 200ml/ minute, the blood plasma separated flow through one flat saturating to ultraviolet Bright container (the such as quartz container of an internal capacity 10x10x1cm), container is shone by the uviol lamp that wavelength is 253nm Penetrating, on container, irradiation intensity is 60 μ W/cm², it is 30 seconds that blood plasma flows through the container time, then merges with blood cell and flows back to In patient body.Whole operation continues 2 hours.After the ultra-vioket radiation of above intensity, test through Virus culture, in plasma sample Hepatitis C virus more than 95% can be inactivated.The mode (such as 50～70 degree) that can also take heating kills virus, such as Container is placed in a microwave generator, and regulation microwave intensity makes blood plasma therein be warming up to 56 DEG C.After said temperature processes, Testing through Virus culture, the hepatitis C virus more than 95% in plasma sample is all inactivated.Other radiant intensity, wavelength, flow velocity Can also apply with the time, such as 220～the ultraviolet of 280nm, 30uW～3000uW/cm², during the radiation of 20 seconds to 120 seconds Between (time of staying of the blood plasma in radiation path is determined by the size of flow velocity, shape and radiation path), these parameters Selection should make it have high inactivation of virus efficiency and low plasma protein inactivation efficiency.When application ultraviolet or other wavelength light According to during to kill pathogen, it is also possible to adding photosensitizer such as phenothiazinium dye in blood plasma, serge blue, Psoralens resistance is (such as 8- MOP, AMT), photosensitizer S59, riboflavin, anticancer photosensitizer etc. kills efficiency to improve.Can divide with hemocyte at blood plasma Can also add in whole blood in vitro from rear addition, addition is to reach to be enough to kill disease in above-mentioned radiated time and intensity Substance is as the criterion.Can also be directly to patient injection or oral addition.Can also be flowed through after illumination photosensitizer adsorbent (as Activated carbon granule or be connected to the solid support particle of the affine material of photosensitizer) with photosensitizer is removed in the most defeated time patient body with Reduce the photosensitizer side effects to patient.Photosensitizer can also with affinity molecule (such as the antibody) covalency for pathogen even Connect and re-use, thus improve the specificity that pathogen is killed.Such as, vitamin B2 can be added to (concentration in blood plasma 100uM), with radiant intensity 1mW/cm²Wavelength at 260nm-370nm or 450nm carries out inactivation treatment.Vitamin B2 absorbs Device (being such as filled with the container of the activated carbon granule of 100 grams of agarose parcels) can be placed on the downstream of radiation path, with The vitamin B2 preventing excess enters patient.In addition to the container of box-shape, and other kinds of radiation path is such as Helical tubular structure around UV lamp can also be used for inactivating device.Additionally, be filled with container or the filter of HCV adsorbent (pore size of 60nm) may also placed in the downstream of radiation path, to clean blood plasma further.The absorption of hepatitis C virus The example of agent includes affinant (the such as antibody or coagulation being associated with HCV affinant and its immune complex on solid support Element: the mol ratio of C1Q. is the mixture of 1:1), solid support can be the Sepharose 4B of 50 milliliters of 90um diameters Pearl.Similar, during treatment HBV infection, B-type hepatitis blood samples of patients flows into the hollow fibre being contained within made by many polysulfone membrane Plasma separator.The film gross area is 0.5 square meter, membrane aperture 0.2～0.6 micron.Container two ends have the connection of artery and vein pipeline to make blood Solution travels through hollow fiber, is filled with solid phase carrier pathogen adsorbent outside hollow fibre.Blood flow is 100ml/ minute, and solid phase carries An affine material of hepatitis B virus is had, the most covalently bound particle diameter 0.5 of anti-hepatitis B surface antigen antibody on body pathogen adsorbent The glucan particles of the crosslinking of millimeter.Blood plasma containing pathogen diffuses to outside hollow fibre by micropore on film and solid phase carrier Pathogen adsorbent contacts, and then flows out through upper outlet, and the blood plasma of outflow processes (253nm through a ultra-vioket radiation inactivating device Ultra violet lamp, irradiation intensity is 200 μ W/cm²) flow back to plasma separator by end opening, spread back again in doughnut with Then blood cell component can and flow back in patient body.Whole operation continues 2.5 hours.Ultra-vioket radiation through above intensity After, to test through Virus culture, the hepatitis B virus more than 95% in plasma sample can be inactivated.

Example 4: as it is shown in figure 1, the patient blood suffering from acquired immune deficiency syndrome (AIDS) flows out from radial artery, menses pumping action is passed through Fig. 2 Plasma separator, blood flow is 100ml/ minute, and the blood plasma separated flows through a flat appearance to UV clear Device (the such as quartz container of an internal capacity 10x10x1cm), container is the ultra violet lamp of 253nm by wavelength, holds On device, irradiation intensity is 60 μ W/cm², (or on 260nm container, irradiation intensity is 200 μ W/cm²) blood plasma flows in series through container, its Time is 30 seconds, then merges with blood cell and flows back in patient body again, and whole operation continues 3 hours.This operation can repeat Repeatedly, continued for several weeks the most weekly.To contain inhibition of HIV blood through above operation process after, through external Virus culture Test, the HIV (human immunodeficiency virus) more than 95% in blood sample can be inactivated. and plasma separator can be filled with HIV adsorbent.Chinese mugwort Grow virus the Sepharose 4B granule that adsorbent is 30 milliliters of 90um diameters being connected with AntiHIV1 RT activity gp120 antibody and 30 milliliters It is associated with the mixture of the Sepharose 4B granule of the 90um diameter of C1q.

Example 5: in certain embodiments, first blood samples of patients extracorporeal circulation is established, and blood is by as described in Figure 3 Case.Container comprises many Polysulfone hollow fibers 10.The suitable interior diameter of fiber can be from 100um～1000um. The gross area of hollow-fibre membrane is 2 square metres, and the aperture of this film is 12 microns.One end of container has blood entry port and tremulous pulse stream Going out blood to connect, container also has blood output makes blood return to vein.Inside such as Fig. 4 doughnut 10 can also be filled Have solid phase CTC adsorption particle or fiber 11(diameter > pore size of film 12 and the aperture of hollow-fibre membrane, such as granular size It is 100 microns and the aperture of filtration membrane 12 is 30 microns).The left part of Fig. 3 and Fig. 4 demonstrates this device longitudinal section, Fig. 3 and The right part of Fig. 4 shows the schematic diagram of the level cross-sectionn of device.At Fig. 3 or Fig. 4, there is the outlet containing CTC cell, It can have a valve, to control the ON/OFF/speed of liquid stream.What blood entry port path can also have that a valve adjusts open/ Close and the blood flow rate of flow, and other liquid such as cleanout fluid is provided.When blood is by container, erythrocyte, platelet, blood Slurry and some leukocyte can pass through hollow-fibre membrane, return in patient body from from blood outlet.CTC can be thin with some white blood Born of the same parents/blood plasma stays doughnut, contains CTC cellular component when the valve is open from outlet flow container.When doughnut aperture Time less (such as 10 microns), most of CTC will be retained, but more leukocyte also will be retained.When bigger hole (such as 20 Micron) time, less leukocyte will be retained, but some CTC are likely to effusion.By from the blood sample analysis of patient On the basis of CTC size, optimal hole dimension can be chosen.Doughnut also can be with other type of synthetic polymer or inorganic Material is made.Pore-forming/fibre wall passage can have identical diameter and in the inside/outside side of doughnut 10 or have different big Little.Such as, the inwall aperture of doughnut can be more than outer wall aperture.So the diameter of whole hollow fiber walls passage from inboard to Outside reduces.Internal void size (such as 30～50 microns) can size than CTC bigger.Valve can be normally opened or periodically open Or when being only opened in EP (end of program) with discharge containing CTC cellular component.Other liquid can be added on entrance, to help to discharge carefully Born of the same parents.This valve can also remain turned-off all of time.Effluent containing CTC can enter by the means that other CTC removes/inactivates Row processes.Such as, it can be then return in patient body by the device containing CTC affinity adsorbent.It can also be by another The box of one same type isolates hemocyte (can be defeated back to patient) to contain from CTC further component.

Example 6: first blood samples of patients extracorporeal circulation is established, blood passes through case as described in Figure 5.Container bag Containing many Polysulfone hollow fibers 14.The suitable interior diameter of fiber can be from 100um～1000um.In one embodiment, A diameter of 300um.The gross area of hollow-fibre membrane is 5 square metres, and the aperture of this film is 15 microns.One end of container has blood Liquid entrance 13 flows out blood with tremulous pulse and is connected, and container also has blood output 15 makes blood return to vein.Also blood can be led to Cross case as described in Figure 6.The inside 14 of Fig. 6 doughnut is filled with CTC adsorbent, such as granule or fiber (its Diameter > aperture of hollow-fibre membrane and the aperture of filter membrane 16, such as, granular size is 200 microns, and filter film 16 aperture is 50um).The left part of Fig. 5 and Fig. 6 shows the longitudinal cross-section of equipment, and the right part of Fig. 5 and Fig. 6 is of equipment The schematic diagram of cross section.Equipment in different Fig. 3 and Fig. 4, this device does not contains the outlet of CTC cell.At doughnut The other end is sealed.When blood is by container, erythrocyte, platelet, blood plasma and some leukocyte can pass through doughnut Film, returns in patient body from blood outlet.CTC can stay doughnut with some leukocyte/blood plasma.When doughnut aperture Time less (such as 10 microns), most of CTC will be retained, but more leukocyte also will be retained.When bigger hole (such as 20 Micron) time, less leukocyte will be retained, but some CTC are likely to effusion.By from the blood sample analysis of patient On the basis of CTC size, optimal hole dimension can be chosen.Doughnut also can be with other type of synthetic polymer or inorganic Material is made.Pore-forming/fibre wall passage can have identical diameter and in the inside/outside side of doughnut 10 or have different big Little.Such as, the inwall aperture of doughnut can be more than outer wall aperture.So the diameter of whole hollow fiber walls passage from inboard to Outside reduces.Internal void size (such as 30～50 microns) can size than CTC bigger.At doughnut containing CTC Cellular component C can be eluted out from blood entry port.At Fig. 4, the device described in 6 is similar to Fig. 3, the device of 5, but in There is extra CTC adsorbent the inside of hollow fiber.Filter membrane prevents CTC adsorbent from entering in the patient in being placed on device.Cross The aperture of filter membrane is more than cell size, but less than the particle diameter of CTC adsorbing material.

Example 7: Fig. 7 demonstrates the embodiment of another type of CTC removal device.This equipment has multiple different pore size Filter membrane or filter plate 19 are in container.Aperture close to the filter of the entrance 18 in blood exports near blood more than filter The pore size of 20.Such as, as shown in Figure 7 A, the first filter (top 1) has for 35um aperture, described second filter (middle one) has the aperture of 20um and the 3rd (bottom 1) has 12um aperture.In another example, pore size Change is from 30 microns to 15 microns to 8 microns.Outlet 21 can be had, such as containing of discharging in Fig. 7 b between top and filter CTC cellular component can further process (means such as, removed through other CTC/inactivate).CTC adsorbing material 22, Described box can also be filled in, as seen in figure 7 c.In one example, filter 19 is polysulfone membrane filter, each surface layer 0.1 Square metre, aperture is respectively 45 microns, 30 microns and 20 microns.In some cases, CTC remover only comprises one layer of filtration Device is to remove CTC.Groove outlet above filter can periodically be opened with discharge accumulation containing CTC cellular component, and this may heap Amass and block the hole of filter, therefore affect blood flowing and pass through.

In Fig. 8, there is in box the device of different hole dimension filters and sequentially placed and each box only has One filter.3 filter box with different pore size are placed on extracorporeally circulating blood path.Each filter has 0.05 The filter membrane of per square meter of surface area.Filter 24 has the aperture of 30 microns, and filter 25 has size and the mistake in the hole of 20um Filter 26 has 12 micron pore size.Blood entry port 23 and filter 24；Blood outlet 27 is connected with filter 26.Can there is cell Flow export is further processed with derivation cell before filter membrane.Similarly, the various purifiers described in that patent are also Blood access can be placed them in be applied in combination.

Example 8: the blood of extracorporeal circulation is flowed into container, the most proper amount of antitumor agent (such as, adds dose to reach In container, drug level is 20 times of its IC50) it is added in a certain amount of blood (such as 200ml) or the blood Han CTC Composition (such as 50ml).Now liquid in container flowing stops, through the time of one (such as 20 minutes) this batch of drug treating Terminating, blood/blood constituent discharges in this container, and then next batch (can only have one batch for blood constituent In secondary) enter container and medicament addition.The addition of CTC inactivator and mix with blood or blood constituent can be in the way of intermittent flow Carry out, so will have time enough by medicine and CTC effect.Other process, such as blood are discharged, and blood backflow becomes with blood Point separate can by be with continuously flowing in the way of can also be in the way of intermittent flow.Can also give at blood/blood components reinfusion Additional process is applied, as removed CTC with affinity adsorbent and/or removing medicine before patient.After whole treatment completes, Ke Yijin The extra dialysis of row or blood purification are to remove the medicine in blood.

Example 9: blood circulation or blood constituent add the microsphere can being combined with tumor cell in vitro will provide a big chi Very little CTC combines complex and therefore will assist in and remove tumor cell with filter.First surface is fixed with anti-CTC surface mark The Sephadex pearl of 300 micron diameters of the antibody knowing thing joins extracorporeally circulating blood to remove CTC, is then flow through by blood There is the filter in aperture of 200um to remove microballon and the CTC being combined with pearl.In one embodiment, the method be with Batch processing form completes.Extract blood 300 milliliters out from patient, in being then used in a chamber and secure EpCAM containing 3 milliliters The cross-linking dextran Sephadex pearl mixing of 300 micron diameters of antibody.Blood and Sephadex pearl mix 5 points in the chamber Clock, then by the filter membrane in the 200um aperture, exit of described chamber to remove microballon and the CTC of combination.Ensuing filter Liquid (blood) is returned to patient, 300 milliliters of blood of another batch be then sent to described chamber with newly added 3 milliliters micro- Pearl mixes, and repeats said process.Cross-linking dextran pearl after filtration can be from this chamber quilt after each batch or after several Remove.The mode that this operation can also be flowed continuously is carried out.Blood is extracted out, mixes with microballon, filters and to return blood the most continuous Ground is carried out.Can also use other particle, the most inorganic beadlet (such as silica bead), biodegradable pearl, magnetic bead replaces handing over Connection glucosan or the like.Magnetic particle is used to allow by filtering and the combination described pearl of removing of Magneto separate.It is different from micro- The pearl used in granule detoxification system, it is adaptable to the non-magnetic particle of this method should be greater than CTC, such as: > 50um, > 100um's or > 200um with promote filter.This filter should allow major part hemocyte pass through, but retains the particle added. Additionally, other shape of granule can also be not limited to globule, can be such as fiber, bar-shaped, cube etc., as long as they be permissible The filter used removes.

Example 10: folic acid and solid support particle are combined and can take following steps: the 20 amidized solid phase carriers of mg Granule (such as the glycosaminoglycan granule of crosslinking of particle diameter 0.2-0.5 millimeter) cleans with 10mL 0.1 M MES, pH 5.0 solution After again with 10mL deionized water clean three times.It is subsequently adding the deionized water solution and 0.5 of 0.5 mL 20 mg/mL folic acid The deionized water solution of the EDC of the fresh preparation of mL 20 mg/mL. with 0.1 M NaHCO₃Aqueous solution is adjusted to pH 7.5.? Stirring reaction two hours under room temperature.Add 10mg EDC and 10mg NHS, stirred overnight at room temperature.Solid support particle is right After with 10mL pH 7.5 10 mM HEPES solution clean three times, then with 10mL deionized water cleaning three times, be dispersed in 1mL and go In ionized water.This granule just can load in adsorption column as adsorbent to remove tumor cell.

Example 11: the circulating tumor cell detection method of FDA approval at present uses one group of antibody for all of tumor.They The antibody of the common label being aimed in epithelial cell.Owing to most tumors is epithelial cell, therefore one group of antibody Universality is had for tumor.Such as, antibody and the anti-cytokerantins antibody of EpCAM can be incorporated into and fills at blood purification Solid support in putting, removes the tumor cell in the peripheral blood circulation of patient after the tumor of excision.In this example EPCAM antibody is connected to agarose particle as tumor cell absorption carrier.The agarose of cyanogen bromide-activated be used to EPCAM antibody key and.The agarose of cyanogen bromide-activated is commercially available can also be made by oneself.In brief (see Cuatracasas, Wilchek and Anfinsen. Proc Natl Acad Sci USA 61 (2): 636-643,1968), by 1ml's EpCAM antibody, with the NaHCO of the 0.1M of 10 mg/ml concentration₃PH is that 9.5 solution join the cyanogen bromide-activated of 1ml Agarose (the Sepharose 4B of the diameter of about 100um, such as cyanogen bromide-activated), and make it in reaction overnight.When having reacted Cheng Hou, is sucked out unreacted material, is fully washed by the agarose connecting described antibody with aseptic cold PBS.One specifically grasps Under such as: take the 20ml Sepharose 4B(cut-off footpath granule more than 100 micron diameters) it is placed in buchner funnel and drains, add A small amount of 0.1M pH 9.0 NaHCO₃Liquid washs, and proceeds to immediately in 100ml beaker, and ice bath is placed on magnetic stirring apparatus.2g bromine Changing cyanogen, the 20ml that adds water dissolves, and is subsequently poured in agarose, careful dropping 2M NaOH, makes pH be maintained at about 11, reacts 10 points Clock.Adjusting rapidly pH in 1 ~ 2 minute is 8.0～11.0 maintenance 10 minutes.Pour rapidly the agarose of activation into buchner funnel In, take out with frozen water and wash into neutrality, rapider taking out with 0.1M pH 9.0 NaHCO3 cold for 250ml is washed.The anti-of coupling will be needed in advance Body protein 200mg is placed in 0.1M pH 9.0 NaHCO3 liquid dialysis a few hours.The agarose of activation is poured into rapidly containing 200mg EPCAM antibody-solutions in, 4 DEG C are slowly stirred overnight, make albumen be combined with the agar of activation.Then at sintered glass funnel Middle 200ml coupling buffer (0.1mol/L sodium bicarbonate, 0.5mol/L sodium chloride, pH8.3) washes coupling medium once.Turn Moving in the flask that coupling medium extremely blocks buffer (1mol/L ethanolamine, hydrochloric acid adjusts pH to 8.0) containing 100ml, incubated at room 2 is little Time or 4 DEG C overnight.100ml coupling buffer, 100 ml acetate buffer (0.1mol/L second are used successively in sinter funnel Acid sodium, 0.5mol/L sodium chloride, pH4.0) wash coupling medium once, repeat this step 4 time.Clean with 100mL deionized water again Five times, this granule just can load in adsorption column as adsorbent to remove tumor cell.

Example 12: in this example, Cytoketatin antibody is connected to glass microsphere granule as tumor cell absorption carrier. The 0.1M sodium borate (pH value is 9.5 concentration) of 10 mg/ml anti-cytokerantins antibody joins aldehyde derivatization titanium dioxide Silica glass pearl (diameter 200 microns).This reaction is most effective at alkaline pH, but pH be 7-9 can also, generally excessive with 2-4 times Protein completes.Then in this mixture, add the NaCNBH 3 of 10 microlitre 5M, and make it at room temperature by mixture reaction 2 Hour.Reaction at the end of, the unreacted aldehyde remained on the glass surface add 20 microlitre 3M ethanolamine with every milliliter of reaction (pH is 9.5) neutralizes.After at room temperature 15 minutes, reaction solution is poured out, fully wash in PBS.Product is stored in refrigerator In, until preparing to use.Can also 200 micron grain sizes glass microsphere particle surface introduce carboxyl, then with EDC and NHS Reaction forms NHS Acibenzolar, is filtered to remove unreacted EDC and NHS.10 mg/ of 5 times of excess (relative to NHS Acibenzolar) Ml Cytoketatin antibody is dissolved in the solution of 0.1M sodium bicarbonate pH 8 and is added in glass microsphere granule, room temperature reaction 3 hours, add ethanolamine and close, then clean five times with pH 7.5 10 mM HEPES solution, more clear with deionized water Washing five times, this granule just can load in adsorption column as adsorbent to remove tumor cell.

Example 13: surface is fixed the tumor adsorption particle each 30ml dress containing Cytoketatin antibody and EPCAM antibody The column shape container of outlet is had under entering.Through the blood that anticoagulant processes, 500ml is added 1,000,000 breast cancer cells make containing cancer Cell blood sample, then flows through the adsorbent equipment containing above-mentioned tumor adsorption particle by this blood sample, and the cancer that detection is flowed out in blood is thin Born of the same parents' content.The cancerous cell of more than 90% can be removed by this device.Adsorbent equipment can also be taken off with 200ml 0.15M The tumor cell adsorbed the inside after PBS is with low pH glycine solution or containing 0.1%EPCAM antibody-solutions or containing 0.25% Adsorbent equipment (is mixed 10 minutes with eluent 50ml and then allows eluting liquid stream by tryptic 150 mM PBS solution eluting Go out), the eluent containing CTC concentrates with 1500 revs/min for centrifugal 10 minutes, gained cell is placed in blood cell counting plate micro- Count under mirror, the CTC quantity in blood can be learnt.Conventional CTC fluorescent staining method can also be used with glimmering by gained cell With glimmering after the CK8 antibody of photoinitiator dye labelling and leukocyte common antigen CD45 antibody and nucleic acid dye DAPI dyeing Light microscope count, the cell selecting CK8+ and CD45 mono-is positive cell, after according to DAPI stained cells nuclear morphology, size Confirm tumor cell more accurately.

Example 14: the granule (granule in the most above-mentioned example 9-13) containing tumor adsorption function is filled internal diameter 50mm, Column shape container composition blood purification perfusion device (as shown in Fig. 9 29) of interior high 200mm, there is blood entry port at container two ends and goes out Mouth connects arteriovenous pipeline, and entrance and exit all has its filter opening of filter membrane (such as 80um) less than particle diameter to prevent granule from flowing into In patient body, but more than hemocyte diameter to allow blood and cell inflow and outflow.Patient's ocal resection (such as mammary gland Cancer, skin carcinoma or lung cancer resection) carry out blood purification two days later, first set up arteriovenous passage, by the artery and vein of patient respectively It is connected with the artery and vein pipeline of hemoperfusion apparatus, utilizes blood pump to maintain velocity of blood flow 100ml/ to divide, then will purify Blood is fed back internal again by vein end.Each 2 hours.Adsorbent equipment can be taken off with 500ml after terminating by blood purification CTC eluting is counted by the method described in it with trypsin eluent in 200ml example 13 after 0.15M PBS, Learn the CTC content in patient blood.

Example 15: will act as the hollow fiber hemodialyzer of hemodialysis in this example as 200 micron diameter solid phase carriers The container of granule, hollow fibre internal diameter is 300 microns.The carrier (can chosen from example 9-13) in above-mentioned for 30ml example is poured into In doughnut, use PBS.The blood entry port at dialyser two ends and outlet all have the filter membrane in 100 microns of aperture.Patient's radiotherapy One day laggard row blood purification, first sets up arteriovenous passage, dynamic and static with hemoperfusion apparatus respectively by the artery and vein of patient Vascular road is connected, and utilizes blood pump to maintain velocity of blood flow 100-200ml/ to divide left and right, then by the blood that purified again by vein End feeds back internal.Each 2 hours.Blood purification can also carry out the toxin dialysing to remove in blood simultaneously simultaneously.Can also Need not dialyse and the most only carry out blood purification operation.Can also be with blood purification to remove immunosuppressive factor, by a reservoir (can be outside inside hollow fibre or doughnut) be filled suitable immunosuppressive factor solid-phase adsorbent and is entered in the same time OK.Can also first pass around leukocyte separation equipment by whole blood, the leucocyte fraction comprising only CTC is led to by such as blood cell separator Cross blood purification, be then returned in the patient.The blood constituent (such as blood plasma, erythrocyte and platelet) of another part is straight Connect conveying and do not pass through blood purification back to patient.

Example 16: using hollow fibre or filter membrane itself as solid phase carrier, tumor binding molecule can also be connected directly between On its surface.In one embodiment, the hollow polysulfone fiber separator separated as blood plasma is used as solid phase carrier.This separation Device is first by 4 degree of soaked overnight of 4% human serum albumin, the albumin glutaraldehyde cross-linking then adsorbed on it.By excess Glutaraldehyde washes away.It is subsequently adding the EPCAM antibody of sodium cyanoborohydride solution and 2-3mg/ml to react 4 and spend night.So Close and clean with PBS with ethanolamine afterwards.So surface of doughnut just have cured EPCAM antibody, and blood is from hollow The most adsorbable tumor cell when flowing through in fiber.This device filtrated air disappears with gamma ray (25-40 kGy) after drying After poison, low temperature is stored in dry darkroom with standby.

Example 17: will when implementing blood purification removing tumor cell by the various blood purification perfusion containers in examples detailed above It is placed in a microwave generating apparatus, and regulation microwave power makes liquid in container temperature remain 46-50 degree.The most just may be used To kill the tumor cell of its absorption.

Example 18: a small amount of blood (such as 1-100 milliliter) can be extracted from a patient out, and small-scale external at one CTC removal/inactivating device/method is tested, and uses this method, device to follow patient at whole body blood extracorporeal blood with prediction CTC removal/inactivating efficacy during ring treatment.If the efficacy and saferry of small-scale test is gratifying, then the method/device Can be used for whole body blood extracorporeal circulation of blood treatment patient.

The test of the most external little blood sample amount should simulate the treatment of whole body blood extracorporeal circulation of blood closely.Because only that it is a small amount of Blood is tested, and compared with the treatment for the outer blood circulation of volume of whole body blood, the size of this device can be small-sized Changing, the amount of the reagent used can reduce, and can be shortened in the time.Operation sequence can also be adapted to external Test frame.The blood (such as 1L-4L) of the effect tested in vitro from small size blood and large volume tests effect in vitro Fruit and the relation of the effect to entire patient's blood extracorporeal circulation of blood treatment (real treatment), can first pass through experiment and determine. If the effect in small size blood in vitro tests and large volume testing in vitro or the effect of cardiopulmonary bypass in patients treatment is had good Good dependency, then it is preferably used to effect of the outer blood circle treatment of predictor.In one embodiment, 2 millis it are filled with As in vitro tests, the 0.5 centimetre of pillar of diameter rising the CTC adsorbent from embodiment 11 predicts that in embodiment 14, use contains There is the CTC removal effect of the apparatus and method of the CTC adsorbent of 100 milliliters of examples 11.In vitro in test, first by 30 milliliters Blood extracts from patient.15 milliliters of blood samples use pillar process and another 15 milliliters of blood not to be used for surveying (as comparison) Examination.15 milliliters of blood samples of test cycled through post with the flow velocity of 1ml/min in 20 minutes in vitro.Then to the two sample In CTC quantity count, the in vitro tests clearance rate of such CTC can be easily calculated.Extract 30 milliliters of blood out After liquid, the systemic blood of patient is carried out by the extracorporeal circulation described in example 14 with the CTC adsorbent containing 100 milliliters of examples 11 Blood purification, after treatment, CTC clearance rate is also computed (comparing CTC amount in the blood recorded of blood purification front and rear). Then the relation (such as, a mathematical model or formula) between effect that the effect of in vitro tests and the people of reality treat is permissible Determine from two CTC clearance rate rates.Multiple patients can be carried out by aforesaid operations, and obtained data can be used for providing more preferably Use in vitro tests be the relation that untreated patient predicts actual therapeutic result.Such as, if obtained relation is for using The CTC clearance rate of specific method in vitro tests 60% is relevant to 40% clearance rate in actual therapeutic in this way, then patient The CTC clearance rate showing 60% in his blood in vitro tests is used measurable to make this patient's actual therapeutic CTC in this way Clearance rate will be 40%.Doctor can use this predicted treatment effect, sick to this with determine whether to make in this way People treats.It is known that different real Therapeutic Method needs different in vitro testses, obtained relation is often Non-interchangeable.The parameter tested in vitro can also be revised, as long as it remains able to (provide two CTC clear into actual therapeutic Except the dependency that rate is good) good prediction is provided.It is, for example possible to use different CTC adsorbance (such as 1ml), different big Little post (such as 0.2 cm diameter), in different blood flow volumes (such as 10ml), makes blood disposable with the flow velocity of 2mL/min Flow through this post rather than be circulated through, as long as good dependency yet suffers between two CTC clearance rate.Optimal parameter Value can change the parameter tested in vitro by experiment provides optimum prediction ability.

Example 19: patient accepts whole body blood extracorporeal blood, uses the CTC adsorbent units containing 200 grams of embodiments 12 by real The method described in example 14 of executing is carried out, and device is the adsorption column of internal diameter 5cm, highly 20cm.0.5 cm diameter is (for actual therapeutic Device flows through the 1/100 of area) 2 grams of CTC adsorbents being filled with from embodiment 12 of pillar (use good 1/ in actual therapeutic 100) CTC removal effect during in vitro tests treatment real with prediction it is used as.In vitro in test, patient before the actual treatment Extract 40 milliliters of blood.20 milliliters of blood samples use the most not test (as comparison) of pillar test and another 20 milliliters of blood.

In vitro tests is by 20 milliliters of blood are passed through pillar with the flow velocity of 1ml/min, and collects filtrate.Then to filter In liquid, the CTC number with control sample counts, and such CTC in vitro tests clearance rate just can be calculated.Patient enters simultaneously Row whole body blood extracorporeal circulation of blood is treated.After treatment, CTC clearance rate is also computed.Then the effect of in vitro tests and reality Relation (such as, a mathematical model or formula) between effect of the treatment on border can determine from two CTC clearance rate rates. Multiple patients can be carried out by aforesaid operations, and obtained data can be used for providing preferably use in vitro tests for not treat trouble Person predicts the relation of actual therapeutic result.For example, it is possible to obtain a curve according to data point, the most each point represents a trouble Person, wherein x is the CTC clearance rate of in vitro tests, and y is that (other parameters also can be included for the CTC clearance rate of this patient's actual therapeutic In curve, such as the type of cancer and original CT C quantity etc., it can be used as the value of Z axis or go out as cluster Now on figure).In order to predict the actual therapeutic effect of a new patient, his blood sample in vitro tests proceeded as above.By institute CTC clearance rate is used for as x, obtain the y value using this curve corresponding.Consequent y value is the treatment CTC that prediction is real Clearance rate.

Another way is to carry out in vitro tests to replace the outer blood circulation of whole body blood to control with substantial amounts of blood (such as 1L-4L) Treat the CTC clearance rate projected relationship setting up said method.It can also be used to optimize small size blood in vitro tests parameter To improve the dependency with actual therapeutic.Human body contains the blood of 34 liters.Use jumbo blood sample rather than external Blood circulation can be simplified the procedures, it is provided that an approximation imitates the model of real human body.Containing blood outlet and blood entry port The storage blood container of large volume, anthropometric dummy can be treated as extracorporeal circulation of blood.

Example 0.5 centimetre of pillar of 20: one diameters is filled with 2 milliliters of CTC adsorbents from embodiment 11 and is used as external examination Test the device of the CTC adsorbent that can be used for prediction use containing 100 milliliters of examples 11 by the CTC removal effect of the method in example 14. In vitro in test, before actual therapeutic, patient extracts 30 milliliters of blood.15 milliliters of blood samples use pillar test with another One 15 milliliters of blood the most not tests (as comparison).In vitro test be by by 15 milliliters of blood with the flow velocity of 1ml/min 20 Cycle through pillar in minute, and collect filtrate.Then count with the CTC number of control sample in filtrate, such CTC body Outer test come clear rate can be calculated.In the 4L human blood that anticoagulant processes, add 1,000,000 lung carcinoma cells simultaneously, place In a vessel, this container has a blood entry port and blood outlet.Blood outlet is connected with blood pump, drives blood The CTC such as example 14 flowing through 100 milliliters of CTC adsorbents containing embodiment 11 removes device.Blood is then by it Blood entry port returns to container.Flow velocity is 100ml/min, processes 2 hours.Then clear to calculate it to the counting of CTC in container Except rate.So relation of the effect of the CTC elimination effect in the in vitro tests of corpusculum hematocele and big blood volume in vitro tests can be by Determine (such as, obtaining a mathematical model or formula).The blood sample of multiple large volumes can be carried out by aforesaid operations, and The dissimilar cancerous cell with quantity.Produced data can be used to be more suitable for using small size blood in vitro tests Patient is predicted the relation of actual therapeutic result.Such as, the patients with lung cancer that CTC is counted as when using certain method 50/ml is carried out Test, this patient's small size blood testing obtains the CTC clearance rate of 60%, if the relation table obtained bright in vitro tests small size The CTC clearance rate of 60% is relevant to the clearance rate of the sample 40% that large volume blood testing contains 50/mlCTC, then prediction is to this disease People uses the CTC clearance rate of this therapy for treating to be 40%.Doctor can use this predicted treatment effect, to determine whether to use This patient of this therapy for treating.

The parameter tested in vitro can also be revised, as long as it remains able to (provide two CTC to remove into large volume blood sample The dependency that rate is good) good prediction is provided.It is, for example possible to use different CTC adsorbance (such as 1ml), different size Post (such as 0.2 cm diameter), in different blood flow volumes (such as 10ml), make blood disposably flow with the flow velocity of 2mL/min Through this post rather than circulation etc., as long as good dependency yet suffers between two CTC clearance rate.Optimal parameter value is permissible By experiment, provide optimum prediction ability by changing the parameter tested in vitro.

Example 21: use a small amount of blood to test the CTC being not limited to the described above in vitro and remove application.Same strategy is also Current invention or other extracorporeal circulation of blood method for virus/pathogen removal/inactivation can be used in.A reality Executing in example, 30 milliliters of blood are extracted out from the patient with infection with hepatitis C virus.Blood is through low-speed centrifugal, by blood plasma fractions It is divided into two equal parts.One part compares, and another part is through the ultraviolet light 30x6=180 second 60uW/cm2 of 253 nm Exposure rate irradiates.This condition is to imitate the UV treatment in example 3.In example 3, flow velocity 200ml/min.Due to the mankind Average blood volume be 4000 milliliters, within 2 hours, by blood circulation 120x200ml, this is total blood volume of 6 times in extracorporeal circulation.In other words Say, blood plasma treatment with irradiation (each 30 seconds) 6 times.Therefore, it is 180 seconds in the time of 60uW/cm2,253 nanometers in test in vitro Ultraviolet light will ensure small size blood sample obtain ultraviolet irradiation amount be equal to real patient treatment in embodiment 3 Irradiation dose.It follows that hepatitis C virus inactivation ratio is detected in two blood plasma fractions by (such as using Virus culture method) The HVC virus quantity of survival completes.Inactivation ratio at this testing in vitro HCV is reported and is decided whether that patient should be with external blood to doctor Method described in liquid circulation embodiment 3 is treated.Such as, if the HCV of significant quantity is inactivated (such as > 60%), then Patient is to this treatment sensitivity, and this Therapeutic Method is recommended.In example 3, additional device is filled with hepatitis C virus and inhales The attached dose of hepatitis C virus that may be used in further blood plasma.Being similar to described by embodiment 18～20, pillar is filled with reality Execute hepatitis C virus adsorbent used in example 3 to can also be used to as testing in vitro to predict used in embodiment 3 The device removal effect to the HCV of patients blood.Such as, blood 30 milliliters is taken out from the patient with infection with hepatitis C virus Take.Blood low-speed centrifugal separated plasma, is divided into two equal parts by blood plasma fractions.One part does not process as right According to, and another part is with the diameter 0.5 centimetre of HCV adsorbent by being filled with in 1 milliliter of embodiment 3 of the flow velocity of 1ml/min Pillar.Two blood plasma fractions all carry out the counting (using PCR or ELISA such as) of HCV to determine HCV clearance rate.If significant quantity HCV be removed (as > 60%), then patient is sensitive to the device in embodiment 3, and it can process combination or individually with UV Process for patient without UV.If the most a small amount of HCV is removed (such as < 30%), extra device such as can be removed Device or the double filtration method of lipoprotein-HCV complex can be used for this patient.Additionally, be similar in embodiment 18～ Described by 20, multiple patients can be used a small amount of blood testing in vitro tests and by extracorporeal circulation of blood HCV in embodiment 3 Removal processes, and records test in vitro and the hepatitis C virus clearance rate of actual therapeutic, then may determine that between them Relation (such as, a curve), the result of this relation and new patient's experiment in vitro just can be used to predict the HCV that new patient is actual Remove therapeutic effect.

Use a small amount of blood to test the CTC being not limited to the described above in vitro and remove application.Same strategy can also It is used in current invention or other extracorporeal circulation of blood blood purification technologies.The blood purification technology be suitable to, including but do not limit In hemodialysis, hemofiltration, plasmapheresis, hemoperfusion, immunoadsorption etc..It is similar to CTC/ pathogen is removed/inactivation Application, in test, patient's small size blood imitates specific blood purification technology and can be used to predict and make in patients in vitro By effect of this blood purification technology.Additionally, safety (side effect) can also by the blood in vitro tests of small size be correlated with because of The safety problem that son (level of such as bradykinin, haemolysis reduce beneficiating ingredient such as HDL and the such as change waited) causes Predict the safety of the actual therapeutic to patient.Such as, LIPOSORBER system uses dextran sulfate cellulose as suction Attached dose with remove patient blood plasma in low-density lipoprotein cholesterol (LDLC), pillar is filled with dextran sulfate cellulose can It is predicted patient is used effect of LIPOSORBER system being used to test the plasma sample of a small amount of patient.Such as, The 10 milliliters of blood plasma having the patient of high plasma low density lipoprotein cholesterol flow through with the flow velocity of 1ml/min and are filled with 1ml 0.5 centimetre of pillar of the diameter of the dextran sulfate cellulose used by LIPOSORBER, in the plasma sample before post and after post The level of LDLC and HDL-C (HDLC) is measured.LIPOSORBER system likely removes some patients HDLC thus cause potential safety problem.This patient is then front by LIPOSORBER system and also mensuration treatment and treats After LDLC and HDLC content.By repeating this process, in a small amount blood in vitro tests and LIPOSORBER to multiple patients The model of the relation between therapeutic outcome can be determined, and it can be used to predict curative effect (LDLC clearance rate) and safety (HDLC clearance rate), by patient carrying out above-mentioned a small amount of blood sample in vitro tests and applying correlation model, doctor can predict it Effectiveness and safety, to decide whether LIPOSORBER treatment should be used for this patient.In another example, in vitro Test a small amount of blood heparin-induced external post lipoprotein (HELP) with prediction to the curative effect of patient and impact.In vitro tests Carry out as follows: by there are 10 milliliters of blood plasma of patient of high low density lipoprotein cholesterol by the ratio identical with HELP treatment Mix with blood plasma with the heparin buffer of formula, and use the method identical with HELP to carry out precipitation removal.Test it in vitro Front measured with LDLC and HDLC level in blood plasma afterwards.If the reduction rate of the level in the plasma sample of LDLC and HDLC Being gratifying, patient can be carried out by HELP.Additionally, multiple patients can be carried out by above-mentioned test in vitro.In vitro The relation of the change of LDLC and the HDLC level of test and HELP treatment can be used for producing in vitro tests result and HELP curative effect Between the forecast model of relation.The plasma sample that new patient can use him carries out in vitro tests, and result is input to Forecast model predicts the efficacy and saferry of HELP.Much different LDLC method/equipment is had to list, such as: DALI, HELP, LIPOSORBER, immunoadsorption antilipoprotein post, membrane filtration MDF, dextran sulfate cellulose adsorption (DSCA) etc..A small amount of blood can also be used to set up each method corresponding in vitro tests result and this method curative effect mould Type.When patient comes when, his blood sample can be tested, give best result (such as: best low density lipoprotein Protein cholesterol removal effect or best curative effect/safety) Therapeutic Method, patient can be recommended.In some cases, If these in vitro testses use identical condition (such as, being similar to blood flow volume, flow velocity etc.) then without forecast model.At another In individual example, use a small amount of blood in vitro tests for predicting that Immunosorba removes autoantibody (as anti-ds-DNA resists Body) carry out the effect of the patient of systemic lupus erythematosus (SLE).A small amount of blood can be taken out from patient and test (example in vitro As, flow through a little post of the solid phase being filled with identical protein A coupling, measure himself cleaning antibody rate), its result It is for determining whether to be applied to Immunosorba this patient.

Before removing circulating tumor cell and/or extracorporeally circulating blood inactivation circulating tumor cell from blood, can be from trouble The blood (such as 10-50 milliliter) that in person's body, extraction is a small amount of, removes/inactivates CTC with in vitro tests and test its effect in vitro Simulate the treatment of whole body blood.Only when in blood sample the CTC of significant quantity be eliminated or inactivate, just with the method extracorporeal circulation whole body blood Treatment patient.Separately having a kind of different method is by with a small amount of blood, finds out and patient removes/inactivates the optimal method of CTC Test.The most a small amount of blood is drawn out of and is divided into several part, each external with a kind of CTC removal/method for deactivating Experiment processes and result is compared, if they have similar safety, demonstrates that the method for optimal effect will be used as To patient treatment.If they have different safeties, the method with efficient low side effect will be used.Because only that it is few The blood (such as 1-200ml) of amount is tested rather than in vitro with the blood of liter opinion, it is possible to use the equipment/examination of small-scale Agent and shorter time are carried out.A part or the whole program test blood sample that can carry out the method for patient are predicted Curative effect to patient treatment.If do not have when making to test in this way these a small amount of blood samples significant CTC(as < 15 %) Reducing/inactivation, this method will not be used.When a small amount of blood sample is tested, only when the significant quantity in blood sample CTC is removed or inactivates (the most in some cases, > 50% is required, in other cases, > 25% be required ) the method is just used for patient.For example, it is possible to take out 20 milliliters of blood with patient and in vitro with containing a small amount of The small device sizes of CTC adsorbing material is tested this blood sample and is predicted whether to carry out systemic blood application stock size device CTC adsorbs external circulating therapy.The size of experiment in vitro device and CTC quantity of sorbent and amount can be accordingly based on described blood Difference between volume and the blood flow volume of patient of sample reduces.Such as, if therapy equipment accommodates 100 grams of CTC adsorbents Patient carries out treatment can use a pillar to fill 1-2 gram of CTC adsorbent to 20 milliliters of blood sample in vitro testses.Show at one In example, in vitro in test, first 30 milliliters of blood are extracted from patient.15 milliliters of blood samples use containing 1 gram of CTC absorption The pillar of agent processes and another 15 milliliters of blood are not used for testing (as comparison).Then, two samples carry out CTC counting Check.If more than 50% CTC in the blood sample processed is removed, corresponding treatment can be used for this patient.Test dress in vitro The structure put, parameter and program need not with for treating the most identical of patient, and such as device size, the time, flow is all Can regulate, the prediction of curative effect of patient as long as it can obtain medical treatment as in vitro tests.Due to the size of different CTC, close Degree, surface marker can change, and the CTC minimizing technology being the most sometimes suitable for a patient may be not suitable for another patient. Effect of external circulating therapy is will ensure that a successful result of small-scale in vitro tests.In another example, from 20 milliliters of blood samples of patient flow through reduced size of filter (surface area less but same aperture), if it exceeds 70 % CTC be removed, the filter of normal area will be used for patient.Blood a small amount of in another example is in vitro by using Blood cell separator based on centrifugation or similar whizzer, with check whether CTC can successfully by CTC from greatly Other hemocytees most separate, to decide whether in this approach for patient treatment.Equally, CTC ablation method, such as front The described use medicine of literary composition or foreign material or physical method, it is possible to survey with blood sample from patient on a small quantity in vitro Examination, to decide whether to can be used for this patient.The combination of several method/equipment, it is also possible to use a small amount of from patient's in vitro Blood sample is tested, if its CTC removal/inactivating efficacy is gratifying, this combination will can be used for treating patient.

Additionally, the result of CTC removal/inactivation treatment can be also used for instructing further chemotherapy or other type for the treatment of (such as radiotherapy).If after CTC removal/inactivation processes, the CTC number in blood increases again, in patients may residual Tumor focus, or do not removed by former thorough treatment or have tumor undiscovered.Then may need to carry out again Secondary chemotherapy or other type for the treatment of (such as radiotherapy, excision adjacent tissue, lymph node).Collect from blood CTC can carry out cultivation to select effective drugs on patients to treat with cancer therapy drug, and this can be by checking that described medicine is swollen The killing power of oncocyte incubation is carried out.

Therefore the carrying out treating for tumor and cancer and prevent the method for neoplasm metastasis and tumor recurrence from including of present disclosure Following steps: 1) tumor patient carried out excision or chemotherapy or radiotherapy or tumor body is had the therapy of lethal effect such as to put by other Penetrate treatment, photodynamic therapy, photon radiotherapy, laser therapy, micro-wave therapeutic oncotherapy, cryotherapy, heating therapy or Combinations thereof 2) detection predicted the circulating tumor cell removal/ablation method of one or more originally from a small amount of blood sample of patient Curative effect 3 to patient) select suitable method, with it, patient is carried out blood purification operation to remove or in inactivation blood Iisolated tumor cells, the defeated time patient of blood after then purifying.

Mention all patents in this description and publication is suitable to those skilled in the art's general level.Foregoing invention relates to And many well-known chemistry, instrument, method and skill.Relevant technical staff in the field can be from document, such as chemistry textbook, The paper of Scientific Magazine and other well-known reference sources obtain.

Claims (4)

1. remove a device for circulating tumor cell in blood, reflux including by blood effuser, plasma separator and blood Pipe is sequentially connected with formed extracorporeal circulation path, it is characterised in that also include the removing circulation being connected with extracorporeal circulation path The container of tumor cell, one end of described container has blood entry port, and this blood entry port flows out blood with tremulous pulse and is connected, container Also having blood outlet makes blood return to vein, and described container contains the outlet of CTC cell, during described container comprises Hollow fiber film, is filled with solid phase carrier CTC adsorbing fiber inside described hollow-fibre membrane, the blood entry port of described container with Hollow-fibre membrane entrance communicates, and described bleed back tube communicates with the space outerpace of hollow-fibre membrane；Described solid phase carrier CTC adsorbing fiber makes the netted of 10～250 micron pore size sizes, and on described adsorbing fiber, covalently bound anti-epithelium is special Property the antibody of antigen or the antibody of anti-cell Keratin, solid phase carrier CTC adsorbing fiber also adsorbs or connects heparin simultaneously, and also Connect the affinity agglutinin having blocking factor；And the aperture of described hollow-fibre membrane is large enough to make erythrocyte and platelet Pass through, but less than CTC cell, and this device also includes that a tumor cell kills device simultaneously, and this kills device in container Solid phase carrier CTC adsorbing fiber applies cell and kills means, and described cell kills means selected from heating, ultraviolet or microwave.

Device the most according to claim 1, it is characterised in that described adsorbing fiber is selected from cellulose fibre, polysulfones is fine Dimension, polyether sulfone fiber, vinal, cellulose acetate fibre, polyethylene fibre, polypropylene fibre, polymethylacrylic acid Methyl ester fiber, polyacrylonitrile fibre, tri acetic acid fiber cellulose fiber or combinations thereof.

Device the most according to claim 1, it is characterised in that be also filled with CTC adsorbent outside described hollow-fibre membrane.

Device the most according to claim 1, it is characterised in that described solid phase carrier CTC adsorbing fiber is also connected with complement C1q。