JP2006346096A - Base material for blood processing - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a base material for blood processing, a device for blood processing and a blood processing method, for capturing cells of a target substance from the whole blood. <P>SOLUTION: The base material is for the blood processing to absorb or to remove the target substance in the blood and characterized by followings: (a) the surface of the base material is fixed with recognition molecules of molecular weight ≤130 kDa, having binding affinity to target protein molecules; (b) the dissociation constant of the recognition molecules and the target protein molecules is ≤1 μM; and (c) in the case of supplying the target protein molecule solution of a concentration C(M) which is 50 times to 5,000 times of a dissociation constant concentration to the recognition molecules at a flow velocity v (L/second) for the time t (seconds), if the supply fullfills the relation of 100<Cvt/Sd<2x10<SP>5</SP>with the mol surface density d (mol/mm<SP>2</SP>) of the recognition molecules fixed on a base material surface area S (mm<SP>2</SP>) to which the target protein molecules are supplied, 50% or more of the number of target protein molecule junctions that the recognition molecules have are bounded with the target protein molecules. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a blood processing substrate for specifically adsorbing or removing an object to be separated from a biological fluid such as blood, and use thereof.

  The purpose of blood purification therapy is to maintain homeostasis in the body by removing substances from the blood that are thought to be related to the onset, induction, or exacerbation of the disease. Treatment, also called apheresis treatment. The blood purification therapy currently performed can be roughly classified into four according to the components to be removed and the removal mechanism.

(1) Hemodialysis, hemofiltration, hemofiltration dialysis therapy: Acute renal failure, chronic renal failure, etc. are indicated. This is a therapy that purifies the components (low molecular weight components such as urea and nitrogen) to be excreted as urine directly from the blood, and mainly targets low molecular components in the blood.
(2) Plasma exchange therapy: indications include fulminant hepatitis, acute liver failure, obstructive arteriosclerosis, myasthenia gravis, Guillain-Barre syndrome, multiple sclerosis, myeloma, drug addiction, pemphigus. It is a therapy that separates and removes components accumulated or produced in the body from the blood, and is intended mainly for slightly larger components in the blood such as cholesterol and immunoglobulins.

  It can be understood that the difference between hemodialysis, hemofiltration, hemofiltration dialysis and plasma exchange is the size of the substance removed from the blood. In other words, hemodialysis removes small molecular weight substances with molecular weights of up to several thousand Da, and hemofiltration removes medium molecular weights of 20,000 to 30,000 Da, but plasma exchange is a protein with a higher molecular weight than that. And harmful substances (antibodies, inflammatory cytokines, humoral factors such as immune complexes and addictive substances) present in the blood by binding to proteins and proteins are removed.

(3) Adsorption blood purification therapy: Septic shock due to endotoxin or gram-negative bacterial infection, familial hyperlipidemia, obstructive arteriosclerosis, focal glomerulosclerosis, dialysis amyloidosis, etc. are indicated. This is a therapy in which blood and plasma are brought into contact with an adsorbent and the pathogenic (related) substances are removed using physicochemical phenomena. The removal target is plasma components such as endotoxin, LDL, and β2 microglobulin. A typical example of the adsorbent used here is activated carbon. In addition, a substance that binds to lipid A which is an active center of endotoxin and immobilizes polymyxin B having an action of neutralizing the activity and adsorbs endotoxin is known.

(4) Blood cell component (white blood cell) removal therapy: Although it is a kind of adsorption blood purification therapy, it is classified separately because the substance to be removed is a blood cell component. Indications such as ulcerative colitis, rheumatoid arthritis, multiple sclerosis, skin diseases, and immune diseases are indicated. This is a therapy in which blood is contacted and cells, especially leukocytes, that are pathogenic (related) substances are removed using physicochemical phenomena or immune reactions. What is currently used is the selective adsorption of granulocytes and monocytes by cellulose acetate beads, etc., or the surface state of the substrate that selectively adsorbs activated leukocytes and platelets. Use it to achieve selectivity.

  In recent years, in the above-mentioned blood cell component removal therapy, side effects have been reduced by specifically capturing cells, which are target substances that are theoretically estimated to be pathogenic, using biological interactions such as immune reactions. Studies are underway to increase sex and therapeutic effectiveness. Furthermore, since such blood cell processing technology can be applied to the stem cell collection / infusion field in regenerative medicine, it is considered to extend to general medical technology for separating and processing blood cell components.

  For the purpose of capturing the target substance cells more selectively and specifically, Patent Document 1 reports a separation material on which a peptide or lipid having affinity for T lymphocytes is immobilized, but at least exemplified. There is a practical problem because the binding affinity for T lymphocytes when thymic hormone is immobilized is insufficient. Patent Documents 2 to 5 each disclose a CD4 positive cell trapping material in which a peptide having affinity for CD4 positive cells is immobilized on a non-woven fabric. Alternatively, immobilization of the complementary determinant region partial peptide of the light chain variable region is practically insufficient in binding affinity for CD4 positive cells and cannot be used. Furthermore, Patent Document 6 discloses a CD4 positive cell separation device using a chimeric antibody, a single chain antibody, or an antibody combining them, which binds to a CD4 molecule, but there is still a problem in improving the productivity of the recombinant antibody. There is a problem that the manufacturing cost is expensive and that a method for removing CD4 positive cells from whole blood, not mononuclear cell suspensions, is not described and is not practical. There is. Patent Document 7 discloses an adsorbent in which a ligand having affinity for a substance expressed on the surface of a tumor cell is immobilized on a polymer compound by a chemical bond. The requirement for capturing target cells selectively and specifically from whole blood has not been disclosed or implemented. Patent Document 8 discloses an immune system cell-adsorbing material in which a substance having affinity for the surface antigen of T helper type 2 cells is immobilized on a water-insoluble carrier, but the adsorption rate is insufficient, Also, there is no disclosure or implementation of requirements for capturing target cells selectively and specifically from whole blood.

Known recognition molecules having specific binding affinity for the target substance include antibodies, chimeric antibodies, F (ab ′) ₂ , Fab, Fab ′, peptides, nucleic acids, sugar chains, low molecular weight compounds, etc. However, in general, antibodies have high binding affinity and can easily retain binding activity even when immobilized on a substrate, but are expensive to produce and have practical problems. A recognition molecule of about 130 kDa or less has the merit that the production cost is lower than that of an antibody, but it is difficult to maintain a practically sufficient binding activity when immobilized on a substrate.

  As described above, it is expected that cells that are target substances in blood are specifically captured directly from whole blood, but it has not been established as a practically usable technique.

JP-A-3-32680 JP-A-6-1554316 Japanese Unexamined Patent Publication No. 6-15317 Japanese Patent Laid-Open No. 6-218051 JP-A-6-269663 Japanese Patent Laid-Open No. 11-332594 JP 2000-217909 JP JP 2003-52817 A

  In general medical technology that separates and processes blood cell components as well as blood component removal therapy, cells that are target substances that are theoretically estimated to be etiologically can be obtained using biological interactions such as immune reactions. Despite the expectation of more selective and specific capture to reduce side effects and increase safety and therapeutic effects, cells that are target substances in the blood, specifically directly from whole blood However, there is a problem that the technology to be captured is not established as a practically usable technology. The problem to be solved by the present invention is to provide a practically usable blood processing substrate, blood processing device, and blood processing for specifically and specifically capturing cells as target substances from whole blood. It is to provide a method. Furthermore, another problem to be solved by the present invention is to select a recognition molecule immobilized on the surface of a blood processing substrate under a predetermined condition, a method of selecting a recognition molecule for surface immobilization of a blood processing substrate, and It is providing the manufacturing method of the base material for blood processing.

This inventor repeated earnest research in order to solve the said subject. First, consider the region researchers usually critical, dissociation constant as an index indicating quantitatively the binding affinity of the recognition molecule and the target substance: K _D (the lower the value binding affinity high) measured Furthermore, a substrate on which the recognition molecule was immobilized was prepared, and it was tested whether or not the target substance cell could be directly captured from whole blood. Correlation was found between the dissociation constant and the specific capture ability of the cell. I found that there is a case that can not be seen. Subsequently, the present inventors examined these data in detail, and even a recognition molecule having a similar dissociation constant is immobilized on the surface of the substrate when measured under certain conditions described later. Surprising findings that the target substance cell can be directly and specifically captured from whole blood when 50% or more of the target protein sites in the recognition molecule are bound to the target protein molecule. I found. That is, the target protein molecule solution is supplied to the immobilized recognition molecule at a concentration C (M) that is 50 to 5000 times the dissociation constant concentration and at a flow rate v (L / sec) for a time t (second). And the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the target protein molecule is supplied,
100 <Cvt / Sd <2 × 10 ⁵
Is set so that 50% or more of the number of binding sites of the target protein molecule possessed by the recognition molecule is bound to the target protein molecule, and the binding affinity for the target protein molecule is set. A substrate for blood treatment that can be practically used for directly capturing a target substance cell from whole blood by immobilizing at least one type of recognition molecule having a molecular weight of 130 kDa or less on the surface of the substrate. And a further essential condition for providing a blood processing device and a blood processing method. In other words, the above essential condition is an indicator that can directly evaluate how much activity the recognition molecule used retains in an immobilized state. Since the number of recognition molecules that can be immobilized on the surface is limited by the surface area, even if the amount of recognition molecules immobilized is increased, if this value is 50% or less, target protein molecules on the cell surface are effectively captured. It becomes difficult to do so, making it practically unusable. Through the experiment and detailed analysis as described above, the present invention has been completed.
That is, according to the present invention, the following inventions are provided.

(1) A blood treatment substrate that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in blood,
(A) an artificially prepared at least one recognition molecule having a molecular weight of 130 kDa or less having binding affinity for a target protein molecule present on the surface of the target substance is immobilized on the substrate surface;
(B) the dissociation constant between the immobilized recognition molecule and the target protein molecule is 1 μM or less,
(C) Supplying a target protein molecule solution having a concentration C (M) of 50 to 5000 times the dissociation constant concentration to the immobilized recognition molecule at a flow rate v (L / second) for a time t (second). In this case, the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the target protein molecule is supplied,
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of target protein molecule binding sites of the recognition molecule is in a state of being bound to the target protein molecule.
A blood processing substrate characterized by the above.

(2) The blood processing substrate according to (1), wherein the recognition molecule is immobilized on the surface of the substrate via a covalent bond.
(3) A blood processing device in which the blood processing substrate according to (1) or (2) is filled in a container having an inlet and an outlet.
(4) The blood containing a target substance is passed through a blood processing device in which the blood processing substrate according to (1) or (2) is filled in a container having an inlet and an outlet. Blood processing method.

(5) A recognition molecule immobilized on the surface of a blood treatment substrate that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in blood,
(A) at least one molecule having a molecular weight of 130 kDa or less, which is artificially prepared and has a binding affinity for a target protein molecule present on the surface of the target substance,
(B) the dissociation constant with the target protein molecule is 1 μM or less,
(C) When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied for a time t (second) at a flow rate v (L / second), the target protein molecule is supplied. The molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² )
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of target protein molecule binding sites of the recognition molecule is in a state of being bound to the target protein molecule.
A recognition molecule characterized by that.

(6) (a) an artificially produced molecule having binding affinity for a target protein molecule present on the surface of a target substance, and (b) a molecular weight of 130 kDa or less, and (c) a target protein From a candidate recognition molecule having a dissociation constant of 1 μM or less from the molecule,
When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / second) for a time t (second), the substrate to which the target protein molecule is supplied Between the molar surface density d (mol / mm ² ) of the candidate recognition molecule immobilized on the surface area S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Including selecting a target protein molecule in which 50% or more of the target protein molecule binding sites of the candidate recognition molecule are bound to the target protein molecule.
A method for selecting a recognition molecule for immobilizing a surface of a blood treatment substrate that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in blood.

(7) A method for producing a substrate for blood treatment that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in blood, On the other hand, when the target protein molecule solution is supplied at a concentration C (M) that is 50 to 5000 times the dissociation constant concentration and at a flow rate v (L / sec) for a time t (second), the target protein molecule is supplied. Between the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Is set so that 50% or more of the number of binding sites of the target protein molecule possessed by the recognition molecule is bound to the target protein molecule, and the binding affinity for the target protein molecule is set. A method for producing a blood processing substrate, comprising immobilizing at least one recognition molecule having a molecular weight of 130 kDa or less on the surface of the substrate.

(8) A blood processing substrate for removing 90% or more of CD4-positive cells in blood,
(A) An artificially prepared at least one recognition molecule having a molecular weight of 130 kDa or less having binding affinity for CD4 molecules on the cell surface is immobilized on the substrate surface,
(B) the dissociation constant between the immobilized recognition molecule and the CD4 molecule is 1 μM or less,
(C) A CD4 molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied to the immobilized recognition molecule at a flow rate v (L / sec) for a time t (second). The molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the CD4 molecule is supplied,
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of CD4 molecule binding sites of the recognition molecule is in a state of being bound to the CD4 molecule.
A blood processing substrate characterized by the above.

(9) The blood processing substrate according to (8), wherein the recognition molecule is immobilized on the substrate surface via a covalent bond.
(10) A blood processing device in which the blood processing substrate according to (8) or (9) is filled in a container having an inlet and an outlet.
(11) The blood containing a target substance is passed through a blood processing device in which the blood processing substrate according to (8) or (9) is filled in a container having an inlet and an outlet. Blood processing method.

(12) A recognition molecule immobilized on the surface of a blood processing substrate for removing 90% or more of CD4-positive cells in blood,
(A) at least one molecule having a molecular weight of 130 kDa or less, which is artificially prepared and has binding affinity for CD4 molecules on the cell surface;
(B) the dissociation constant with the CD4 molecule is 1 μM or less,
(C) When a CD4 molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / sec) for a time t (seconds), the group to which CD4 molecules are supplied Between the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the material surface area S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of CD4 molecule binding sites of the recognition molecule is in a state of being bound to the CD4 molecule.
A recognition molecule characterized by that.

(13) (a) an artificially produced molecule having binding affinity for the CD4 molecule on the cell surface, (b) a molecular weight of 130 kDa or less, and (c) a dissociation constant with the CD4 molecule From candidate recognition molecules with a 1 μM or less,
When a CD4 molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / sec) for a time t (seconds), the substrate surface area S to which the CD4 molecules are supplied Between the molar surface density d (mol / mm ² ) of the candidate recognition molecule immobilized on (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Including selecting a substance in which 50% or more of the number of CD4 molecule binding sites of the candidate recognition molecule is bound to the CD4 molecule when supplied so that the relationship of
A method for selecting a recognition molecule for surface immobilization of a blood processing substrate that removes 90% or more of CD4-positive cells in blood.

(14) A method for producing a substrate for blood treatment that removes 90% or more of CD4 positive cells in a solution, wherein the concentration of a CD4 molecule solution is 50 to 5000 times the dissociation constant concentration with respect to the immobilized recognition molecule. When supplying a time t (second) at a flow rate v (L / second) at a concentration C (M), the mole of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the CD4 molecule is supplied Between the surface density d (mol / mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Is set so that 50% or more of the number of binding sites of CD4 molecules possessed by the recognition molecule is in a state of binding to CD4 molecules, and the molecular weight of 130 kDa having binding affinity for CD4 molecules. The manufacturing method of the base material for blood treatment including fix | immobilizing the following at least 1 type of recognition molecule on the base-material surface.

  In general medical technology that separates and processes blood cell components as well as blood component removal therapy, cells that are target substances that are theoretically estimated to be etiologically can be obtained using biological interactions such as immune reactions. Despite the expectation of more selective and specific capture to reduce side effects and increase safety and therapeutic effects, cells that are target substances in the blood, specifically directly from whole blood The technology to capture automatically has not been established as a practically usable technology. According to the present invention, it is possible to provide a practically usable separation device and separation method for specifically and directly capturing cells as target substances from whole blood, meaning that only target target substances can be processed. This contributes to improving the quality and efficiency of medical care in the EBM (Evidence Based Medicine) era.

Hereinafter, the present invention will be described more specifically.
The “target substance” used in the present invention means a specific biological substance to be removed from biological substances, cells, tissues, etc. contained in a biological fluid represented by blood. And at least one substance selected from harmful substances, pathogenic cells, viruses and virus-infected cells. In addition to the case where the target substance is contained in the blood itself, the target substance may be contained in a liquid containing a large amount of contaminants derived from blood.

  Among the target substances, harmful substances include, for example, LDL, β2 microglobulin, drug, bilirubin, bile acid, amino acid, creatinine, endotoxin, anti-A antibody, anti-B antibody, anti-acetylcholine receptor antibody, IgG, anti-cardiolipin antibody, Plasma components such as anti-DNA antibodies, immune complexes, coma, rheumatoid factors. Abnormal prions are also included. Various mediators including inflammatory cytokines are also harmful substances depending on the pathological condition. IL-1, IL-6, IL-8, IL-18, TNF-α, GM-CSF, RANTES / SIS cytokine family and the like are known as inflammatory cytokines. Mediators involved in inflammation include substances generated from arachidonic acid in vivo such as prostaglandins, thromboxanes and leukotrienes, platelet activating factor, histamine, bradykinin and complement factor C5a.

Pathogenic cells are tumor cells, bacteria, blood cells such as leukocytes (neutrophils, eosinophils, basophils, lymphocytes, or monocytes), platelets, etc. It is. For example, when there is a concern about enhancement of cellular immunity in autoimmune diseases, it is desirable to distinguish pathogenic cells using immunoglobulin superfamily molecules expressed on the surface of white blood cells as an index. Specifically, CD2-positive cells, CD4-positive cells, CD8-positive cells, CD28-positive cells, CTLA-4 (CD152) -positive cells, PECAM-1 (CD31) -positive cells, ICAM-1 (CD54) -positive cells, ICAM- 2 (CD102) positive cells. It is also effective to distinguish by integrin family molecules. For example, VLA-1 (CD49a / CD29) positive cells, VLA-2 (CD49b / CD29) positive cells, VLA-3 (CD49c / CD29) positive cells, VLA-4 (CD49d / CD29) positive cells, VLA-5 ( CD49e / CD29) positive cells, VLA-6 (CD49f / CD29) positive cells, LFA-1 (CD11a / CD18) positive cells, Mac-1 (CD11b / CD18) positive cells, p150, 95 (CD11c / CD18) positive cells , GpII
b / IIIa (CD41 / CD61) positive cells, LPAM-1 positive cells, and the like.

  The “base material” used in the present invention means a material in a solid state in an aqueous solution at room temperature, and the shape is not particularly limited, but the surface area is large in order to increase the frequency of contact with target cells in the liquid phase. It is preferable. Examples of shapes include fiber structures such as spheres, cubes, planes, flat membranes, chips, fibers, cotton, cloths, yarns, bundles, woven fabrics or nonwoven fabrics, sponges and other high structures. A molecular porous body, a bead shape, a gel shape, a hollow fiber, etc. are mentioned. Of these, spherical shape is achieved due to the ease of fine packing, the ability to keep the recognition molecules homogeneously on the surface, the fact that a relatively large effective area can be secured, and the flow of biological fluids typified by blood. Granular and fibrous are preferred. In particular, when separating or adsorbing cells in blood, a woven fabric or a nonwoven fabric is preferable from the viewpoint of adsorption efficiency, and a nonwoven fabric is most preferable from the viewpoint that the structure of the carrier can be easily controlled.

  The material of the base material is not limited to inorganic compounds and organic compounds as long as it can stably fix at least a predetermined amount of recognition molecules, but it can be processed into various shapes, and the recognition molecules are directly or indirectly chemically bonded. Those that can be used safely as medical materials are preferable, such as being able to be used and having a small amount of eluate upon contact with a biological fluid typified by blood. For example, inorganic materials such as glass, kaolinite or bentonite, polysaccharide compounds derived from plants such as cellulose, natural polymers such as dextran, chitin, chitosan, starch, agarose, protein (collagen etc.) or natural rubber, polypropylene, polystyrene , Polyamide, polyethylene, polyurethane, polyvinyl alcohol, ethylene vinyl alcohol copolymer, polymethacrylic acid ester, polyacrylic acid ester, polyacrylic acid, polymer and copolymer of vinyl derivatives such as polyacrylic acid, polyvinyl alcohol and derivatives thereof, nylon Examples thereof include polyamide compounds such as 6 or 66, polyester compounds such as polyethylene terephthalate, synthetic polymers such as polyamino acids, or activated carbon.

  When selecting a nonwoven fabric base material, it is preferable that the fiber which comprises a nonwoven fabric base material is a cellulose, polyester, or a polypropylene from a point of intensity | strength, workability, or safety | security. Moreover, the structure of a nonwoven fabric base material is controllable by selecting the average fiber diameter of a constituent fiber suitably. The average fiber diameter of the substrate constituting fibers is preferably 2.5 μm or more and less than 50 μm. This is because if the average fiber diameter is less than 2.5 μm, there is a tendency for the eyes to become finer when made into a nonwoven fabric, and nonspecific cell adsorption that physically captures cells other than the target cells occurs, which is not preferable. On the other hand, when it is 50 μm or more, it tends to be rough when formed into a non-woven fabric, and the contact frequency with the target cell is remarkably lowered, and therefore the removal efficiency is lowered.

The “recognition molecule” used in the present invention is an artificial molecule prepared using protein engineering or molecular biology and genetic engineering techniques, and has a molecular weight of 130 kDa or less. It is selected from F (ab ') ₂ part, Fab part, Fab' part of antibody, peptide, recombinant protein, nucleic acid, sugar chain, low molecular weight compound, etc., preferably a recombinant protein or nucleic acid . Since it is an artificial molecule, it has the advantage of being able to supply a product with excellent thermal stability at a low price by mass production. These recognition molecules are sometimes called artificial antibodies because they bind specifically to the target. Artificial antibodies are usually prepared by randomizing the loop-forming part using a structural molecule similar to the tertiary structure of the antibody V region or incorporating CDR3 of the antibody V gene. Affibody with protein A antibody (K. Nord, O. Nord, M. Uhlen, et al., Eur. J. Biochem, 268, 4269 (2001)), Fluorobody with GFP antibody (ISKim, JHShim) , YTSuh, et al., Biosci. Biotechnol. Biochem., 66, 1148 (2002)), Minibody (V. Batori, A. Koide, S. Koide, Protein Eng. 1015 (2002)), Pepbody (E.Lunde, V.Lauvrak, IBRasmussen, et al., Biochem.Soc.Trans., 30, 500 (2002)), and the structure of camel antibody. Examples include single-chain antibodies Nanobody (A. Muruganandam, J. Tanha, S. Narang, D. Stanimirovic, The FASEB J. 16, 240 (2002)). Also, Spiegelmer (B. Wlotzka, S. Leva, B. Eschgfaller, et al., Proc. Natl. Acad. Sci. USA, 99, Aptamer with nucleic acid structure and RNA enantiomer enatio-RNA) 8898 (2002)).

As an example of such an artificial antibody, there is a custom monoclonal antibody production service by phage display of MorphoSys, Germany. When the artificial antibody HuCAL F (ab ') ₂ against the soluble human CD4 molecule shown in the comparative example was produced, a natural monoclonal antibody was produced. A specific binding affinity comparable to was obtained. The molecular weight of this artificial antibody HuCAL F (ab ′) ₂ was 130 kDa.

A "dissociation constant" used in the present invention is normally represented by K _D, which represents the degree of dissociation. The smaller the number, the stronger the bond. It can be calculated by quantitatively measuring the amount of the target protein molecule released after sufficiently binding various concentrations of the target protein molecule to the recognition molecule. The quantification method is ELISA (Enzyme-Linked ImmunoSorbent Assay), spectroscopic method, method using crystal oscillator, Frontal Affinity Chromatography (M.Nishikata, K.Kasai, S.Ishii, J. Biochem. 82, 1475 ( 1977)), a method using an RI-labeled target protein molecule, and a method using the surface plasmon resonance phenomenon. Among these, for example, in a biosensor using the surface plasmon resonance phenomenon, a glass surface on which a gold thin film is deposited is processed into a sensor chip, a recognition molecule is immobilized thereon, and then a target protein molecule solution is poured to recognize the recognition molecule. And target protein molecules are brought into contact. Light of a certain wavelength is placed on a gold thin film before and after contact, and the time change of reflected light is observed. The reflected light is affected by the density of the material in the vicinity of the gold thin film due to the surface plasmon resonance phenomenon, so the change in the amount of binding in real time can be seen from the change in the surface density, and the dissociation constant can be easily determined by obtaining the binding rate constant and dissociation rate constant I can know.

  More specifically, a sensorgram is obtained by flowing a target substance or target protein molecule as an analyte with respect to a recognition molecule immobilized on the sensor chip under the same conditions as the substrate immobilization. For example, when the target substance is a protein molecule on the cell surface that is recognized by the recognition molecule in the cell, the solubilized target molecule prepared and purified as a soluble molecule by genetic engineering may be used as the analyte.

  The term “specific” used in the present invention basically binds only to a target substance or a target protein molecule with a high binding affinity by a biological interaction represented by antibody antigen binding. The dissociation constant between the immobilized recognition molecule and the target protein molecule (analyte) should be 1 μM or less, preferably 10 nM or less, more preferably 1 nM or less.

  Since the recognition molecule immobilized on the sensor chip can be detected as an increase in mass density on the sensorgram, the number of recognition molecule moles per unit area on the sensor chip can be calculated. In this way, when the target substance or solubilized target molecule is reacted in an excessive amount for a sufficient amount of time as an analyte to the immobilized recognition molecule on the sensor chip, the mass of the analyte bound per unit area is measured and the bound analyte is measured. You can know the number of moles of light. This is Rmax-real.

The excess amount means that the analyte is reacted at a concentration of 50 to 5000 times the dissociation constant concentration. Sufficient time means reaction time from 1 second to about 12 hours. When reacted for an excessive amount of time, the target protein molecule solution having a concentration C (M) that is 50 to 5000 times the concentration of the dissociation constant with respect to the immobilized recognition molecule is flowed at a flow rate v (L / sec). In the case of supplying time t (second), 100 <Cvt between the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the target protein molecule is supplied. The relationship / Sd <2 × 10 ⁵ is established.

  On the other hand, since it is possible to theoretically know how many moles of analyte are bound to 1 mole of recognition molecule, the number of moles of recognition molecule per unit area on the sensor chip can be theoretically bound. The number of analyte moles can be calculated. This is Rmax-theory.

  Here, assuming that (Rmax-real / Rmax-theory) × 100 (%) is referred to as coupling efficiency, an immobilization condition is set such that the coupling efficiency is 50% or more. That is, in the present invention, “50% or more of the number of target protein molecule binding sites possessed by the recognition molecule is in a state of being bound to the target protein molecule” means that the above-mentioned binding efficiency is 50% or more. This coupling efficiency is preferably set to 65% or more, more preferably 80% or more.

  This binding efficiency is very important information for grasping in what state the recognition molecule is immobilized on the substrate surface. Conventionally, as a technique for immobilizing a recognition molecule on the surface of a base material, a technique in which activity is maintained after immobilizing by reacting as many molecules as possible has been selected. If the binding active site of the recognition molecule is sterically hindered during immobilization, or if the binding active site is destroyed during the immobilization reaction, the necessary binding is possible no matter how high the recognition molecule is immobilized. There is no activity. The number of molecules that can be immobilized on the surface of the substrate is limited because it is determined by the surface area and the number of active groups, and a substrate having the desired activity cannot be produced simply by increasing the amount of immobilization. When an antibody is used as a recognition molecule, there is an Fc region that is relatively hydrophobic and not involved in binding activity, so there is a high probability that the Fc region will be immobilized without any particular control, and the binding efficiency will be high. There are many. However, in the case of an artificially produced molecule, it is an original merit that it can be supplied at a low price by mass production. Therefore, a molecule having a low molecular weight is desired, and there is no portion corresponding to the Fc region. In order to immobilize artificial recognition molecules having a relatively low molecular weight than these antibodies on the surface of the substrate while retaining the binding activity, it is devised not to cause steric hindrance on the recognition molecule side, or to the binding active site. It is important to prevent the immobilization site from entering. By controlling the immobilization state using the binding efficiency as an index, effective recognition molecules can be efficiently immobilized on the limited substrate surface, which is necessary for cell removal that requires extremely high binding activity. It becomes possible to produce a substrate having activity.

  The upper limit of the coupling efficiency is 100% in principle. If it exceeds 100%, the target protein molecule is bound to other than the assumed binding active site, and the target binding specificity is not satisfied, so this should not be adopted.

  In immobilizing the recognition molecule on the substrate surface, the recognition molecule may be directly bonded to the substrate surface or may be bonded via an active group. Any binding state may be used as long as the recognition molecule or the active group is basically not detached from the substrate. Examples of the bonding state include a single bond or a plurality of resultant forces of a covalent bond, an ionic bond, a van der Waals bond, a hydrogen bond, or a hydrophobic bond. From the viewpoint of suppressing the outflow of the recognition molecule, a covalent bond and a hydrophobic bond are more preferable, and a covalent bond is most preferable. As the binding reaction, any known reaction can be used as long as the binding efficiency is 50% or more, preferably 65% or more, and more preferably 80% or more. , Addition reaction, condensation reaction, polymerization reaction or ring-opening reaction.

  When the recognition molecule is covalently bonded to the substrate surface via an active group, the active group may be any known functional group as long as the recognition molecule and the substrate can be covalently bonded. As the active group, for example, α-acetaminohalogen group obtained by activating the substrate surface using N-hydroxymethylhaloacetamide or N-hydroxymethyldihaloacetamide, N-hydroxymethyldihalopropion, etc. Α, α obtained by activating the substrate surface using α, β-propionaminohalogen group, N-hydroxymethyldihaloacetamide, etc. obtained by activating the substrate surface using amide or the like Using a haloacetaminoalkane agent such as α, α, α-acetaminotrihalogen group obtained by activating the surface of the substrate using N-acetaminodihalogen group or N-hydroxymethyltrihaloacetamide Active groups obtained by activating the substrate surface, or epichrome Examples thereof include an epoxy group obtained by activating a functional group such as a hydroxyl group or an amino group on the substrate surface using rhohydrin or the like. Other suitable active groups include, for example, an N-hydroxysuccinimide ester group, an isocyanate group, an isothiocyanate group, an amino group, a carboxyl group, a hydroxyl group, a vinyl group, and a maleimide group obtained by activating a carboxy group with N-hydroxysuccinimide. , A tosyl group, a tresyl group, an active group by bromocyan, and the like, but are not limited thereto. From the point that it can react under mild conditions while maintaining the function of the recognition molecule, an active group or an epoxy group, an N-hydroxysuccinimide ester group obtained by activating the substrate surface using a haloacetaminoalkane agent, Maleimide groups are preferred.

  Furthermore, as a method for introducing an active group into the substrate surface, it is also preferable to introduce a functional group or an active group into the substrate surface by graft polymerization, plasma treatment, corona treatment, chemical treatment or exposure to gas. Illustratively, active groups that can be used for immobilization easily by irradiating polystyrene, polypropylene, polyethylene terephthalate, etc. with γ-rays and immersing them in a vinyl monomer solution such as glycidyl methacrylate in a state of blocking oxygen. A base material into which a functional group has been introduced can be produced. A single monomer may be used for these graft chains, or a copolymer obtained by mixing a plurality of monomers may be used. Of course, a hydroxyl group, a carboxyl group, an amino group, or the like may be introduced by graft polymerization, and further activated by the above-described method to form an active group. It is known that various types of functional groups can be introduced into the substrate surface by plasma or corona treatment. However, if it is intentionally exemplified, hydroxyl groups and carboxyl groups can be introduced into the polyethylene terephthalate substrate surface in the presence of oxygen. These surface modifications can also be applied to the present invention. It is also possible to immobilize the recognition molecule by introducing a photoreactive azo group or diazo group onto the substrate surface and irradiating with light or radiation in the presence of the recognition molecule.

  It is also possible to introduce an active group into the surface of the substrate by polymerizing a polymer containing the active group as described above, coating the substrate surface with a suitable solvent, and drying. .

As described above, the blood processing substrate of the present invention has a target protein molecule solution at a concentration C (M) that is 50 to 5000 times the dissociation constant concentration with respect to the immobilized recognition molecule, and a flow rate v ( L / second) when supplying a time t (second) and the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the target protein molecule is supplied Between,
100 <Cvt / Sd <2 × 10 ⁵
Is set so that 50% or more of the number of binding sites of the target protein molecule possessed by the recognition molecule is bound to the target protein molecule, and the binding affinity for the target protein molecule is set. It can be produced by immobilizing at least one type of recognition molecule having a molecular weight of 130 kDa or less on the substrate surface. Such a method for producing a blood processing substrate of the present invention is also included in the scope of the present invention.

  The blood processing substrate of the present invention in which a predetermined recognition molecule that can be prepared as described above is immobilized on the surface is filled in a container having an inlet and an outlet to construct a blood processing device. can do. The container having an inlet and an outlet used here is not particularly limited as long as it can accommodate a blood processing substrate, and, for example, a column or a tube can be used. By processing blood by passing blood containing a target substance through this blood processing device, at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in the blood is obtained. Can be adsorbed or removed. The above-described blood processing device and blood processing method are also included in the scope of the present invention.

Furthermore, the recognition molecule is immobilized on the surface of the blood processing substrate of the present invention,
(A) at least one molecule having a molecular weight of 130 kDa or less, which is artificially prepared and has a binding affinity for a target protein molecule present on the surface of the target substance,
(B) the dissociation constant with the target protein molecule is 1 μM or less,
(C) When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied for a time t (second) at a flow rate v (L / second), the target protein molecule is supplied. The molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² )
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of target protein molecule binding sites of the recognition molecule is in a state of being bound to the target protein molecule.
Such recognition molecules are also included within the scope of the present invention.

The recognition molecule used in the present invention is, for example, (a) an artificially prepared molecule having binding affinity for a target protein molecule present on the surface of a target substance, and (b) a molecular weight of 130 kDa or less. And (c) a candidate recognition molecule having a dissociation constant of 1 μM or less from the target protein molecule,
When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / second) for a time t (second), the substrate to which the target protein molecule is supplied Between the molar surface density d (mol / mm ² ) of the candidate recognition molecule immobilized on the surface area S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Can be selected by selecting those in which 50% or more of the target protein molecule binding sites of the candidate recognition molecules are in a state of being bound to the target protein molecules. Such a method for selecting a recognition molecule is also included in the scope of the present invention.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, this invention is not limited to these Examples.

As an example, the target substance is CD4 positive cells in human peripheral blood, F (ab ′) ₂ artificially prepared from a monoclonal antibody as a recognition molecule, and Fab ′, HuCAL F (ab ′) prepared by phage display ₂ is used as an example.

Example 1: Preparation of F (ab ') ₂ by enzymatic treatment of mouse anti-human CD4 antibody The original mouse anti-human CD4 antibody was clone: 4H5 (Medical and Biological Laboratories). This mouse antibody was digested with pepsin, a proteolytic enzyme, for a certain period of time and cleaved into fragments derived from the F (ab ′) ₂ site and the Fc site. This was purified by gel filtration chromatography to obtain a 4H5 anti-human CD4 F (ab ′) ₂ antibody fragment (hereinafter simply referred to as F (ab ′) ₂ ). The number of target protein molecule binding sites of F (ab ′) ₂ was divalent, and the molecular weight was about 100 kD.

Comparative Example 1
Using F (ab ′) ₂ prepared in Example 1 with ₂ -mercaptoethanol, only disulfide bonds at the portion called the hinge site of F (ab ′) ₂ are selectively used under mild conditions in the presence of EDTA. Reduced to Next, an alkylthiol molecule having an SH group at the end is reacted to block the SH group in a free state, whereby the target protein binding site is a monovalent 4H5 anti-human CD4 Fab ′ antibody fragment (hereinafter simply referred to as Fab ′). Obtained). The molecular weight of Fab ′ was about 50 kD.

Comparative Example 2: Preparation of human anti-human CD4 F (ab ') ₂ antibody by phage display method Human soluble CD4 was produced and purified in the same manner as in Example 1 of JP-A-11-332594. Using the purified human soluble CD4 as a target protein, human anti-human CD4 F (ab ′) ₂ antibody was prepared by the phage display method using a custom monoclonal antibody production service provided by Morphosys. That is, the above human soluble CD4 is adsorbed as a target protein on a plastic substrate, washed with phosphate buffered saline (hereinafter referred to as PBS (-)), and then at the end of the filamentous gIII protein, A variable antibody sequence of a human anti-human CD4 antibody was obtained by binding a library displaying a human antibody Fab (HuCAL GOLD (registered trademark)) and screening those having strong binding. In order to produce this as a form of F (ab ') 2 in which the target protein molecule binding site is divalent, a helix-turn-helix motif capable of self-association is linked after the CH1 region of the antibody heavy chain, His tag sequence for purification was added and recombined into an expression vector for E. coli. By expressing and purifying them, a human anti-human CD4 F (ab ′) ₂ antibody (hereinafter referred to as HuCAL F (ab ′) ₂ ) was obtained. The number of target protein binding sites of HuCAL F (ab ′) ₂ was divalent, and the molecular weight was about 130 kD.

Example 2: Measurement of dissociation constant The human soluble CD4 protein (manufactured by Genzyme techne, USA) used for the measurement of dissociation constant and binding efficiency incorporated human CD4 cDNA of the full length of the extracellular portion into baculovirus and infected with insect cells Sf21. , Purified from the culture supernatant and having a molecular weight of about 40 kD.

F (ab ′) ₂ of Example 1 was diluted with 20 mM acetate buffer pH = 5.0 buffer to a concentration of 10 μg / ml, and activated with EDC and NHS in advance using BIACORE S51 from Biacore. Of the sensor chip series S CM5 (Biacore) is immobilized at a covalent bond via the amino group in the recognition molecule by the amine coupling method by feeding at 5 μl / min to one spot. F (ab ') _{2 A} 6536RU immobilized sensor chip was obtained. Similarly, Fab ′ of Comparative Example 1 was also immobilized to obtain a Fab ′ 5881RU immobilized sensor chip. Further, HuCAL F (ab ′) _{2 of} Comparative Example 2 was immobilized in the same manner except that it was diluted with 20 mM acetate buffer pH = 4.5 buffer to obtain a HuCAL F (ab ′) ₂ 2502RU immobilized sensor chip.

  The above human soluble CD4 final concentration of 250 nM (10 μg / ml) HBS-EP (Biacore) buffer solution is sent to the sensor chip at a flow rate of 30 μl / min for 200 seconds to bind the human soluble CD4 protein. After that, the binding / dissociation behavior of each recognition molecule and the target protein molecule was observed by switching to only HBS-EP buffer. Each rate constant and dissociation constant were calculated from the results of curve fitting using BIA Evaluation soft (Biacore) based on the obtained sensorgrams. These are shown in Table 1.

Even when comparing F (ab ′) ₂ of Example 1, Fab ′ of Comparative Example 1 and HuCAL F (ab ′) _{2 of} Comparative Example 2, the binding rate constant Ka, the dissociation rate constant Kd, and the dissociation significant difference was not observed in the constant K _D, all three have been shown to bind strongly to human CD4 molecule.

Example 3 Measurement of Binding Efficiency Theoretical maximum binding amount: Rmax− based on the immobilized amount and molecular weight of each recognition molecule in the experiment of Example 2, the number of CD4 binding sites, and the molecular weight of human soluble CD4 used in the experiment The theory (RU) can be calculated. Here, 1RU is known to be 1 pg / mm ² . Furthermore, using a sensorgram measured by feeding a 250 nM human soluble CD4 solution corresponding to a concentration 50 to 5000 times the dissociation constant at a flow rate of 30 μl / min for 200 seconds, the maximum binding amount actually measured: Rmax-real (RU) was calculated.

  Here, binding efficiency = (Rmax-real / Rmax-theory) × 100 (%) was calculated. These are shown in Table 2.

It can be seen that F (ab ′) ₂ of Example 1 has a lower binding efficiency loss than Comparative Example 1 and Comparative Example 2 when immobilized on the substrate surface via an amino group. That is, it was found that only the recognition molecule of Example 1 efficiently maintained the target protein molecule binding activity in the immobilized state by the covalent bond via the amino group.

Here, it is shown that the present example meets the measurement conditions described above. When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied to the immobilized recognition molecule at a flow rate v (L / sec) for a time t (seconds), A relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established between the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the target protein molecule is supplied. . In this example, the dissociation constant between the immobilized recognition molecule and the target protein molecule human soluble CD4 is on the order of several hundred pM from that in Example 2, and the 250 nM target protein molecule solution is 50 to 5000 times the concentration. . Since the liquid was fed at a flow rate of 30 μl / min for 200 seconds, the liquid was fed at 100 μl, and Cvt is calculated to be about 2.5 × 10 ⁻¹¹ mol. Regarding the substrate surface area S (mm ² ) to which target protein molecules are supplied, according to BIACORE, the series S CM5 chip has a Y-shaped flow cell with a width of 0.9 mm and a length (between the inlet and outlet) of 2.9 mm. It has three recognition substance immobilization sites including the reference part. Here, for approximation, the flow cell is rectangular and one third of the recognition substance immobilization substrate surface area Then, the area is calculated as 0.87 mm ^{2 at the} maximum.

Here, since F (ab ′) ₂ of Example 1 is immobilized at 6536 RU at 100 kD, 1 RU has a molar surface density d (mol / mm ² ) of 6.54 × 10 ⁻¹⁴ mol / mm from 1 pg / mm ^{2. 2} and Sd is calculated to be about 5.7 × 10 ⁻¹⁴ mol. Therefore, Cvt / Sd = 2.5 × 10 ⁻¹¹ mol / 5.7 × 10 ⁻¹⁴ mol = 439, and the relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established.

Similarly, since Fab ′ of Comparative Example 1 is immobilized at 5881RU at 50 kD, 1RU has a molar surface density d (mol / mm ² ) of 1.18 × 10 ⁻¹³ mol / mm ² from 1 pg / mm ² . , Sd is calculated to be about 1.0 × 10 ⁻¹³ mol. Therefore, Cvt / Sd = 2.5 × 10 ⁻¹¹ mol / 1.0 × 10 ⁻¹³ mol = 250, and the relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established.

Further, since HuCAL F (ab ′) ₂ of Comparative Example 2 is immobilized at 250 kRU at 130 kD, 1RU has a molar surface density d (mol / mm ² ) of 1.92 × 10 ⁻¹⁴ mol / mm from 1 pg / mm ^{2. 2} and Sd is calculated to be about 1.7 × 10 ⁻¹⁴ mol. Therefore, Cvt / Sd = 2.5 × 10 ⁻¹¹ mol / 1.7 × 10 ⁻¹⁴ mol = 1471, and the relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established.

On the other hand, if the area detected for SPR signal measurement of the series S CM5 chip is the minimum area of the recognition substance-immobilized substrate surface area S, according to BIACORE, the length is 0.065mm and the width is 0.125mm Therefore, it is calculated as 8.13 × 10 ⁻³ mm ² .

Using this minimum value of the recognition substance-immobilized substrate surface area S, F (ab ′) ₂ Cvt / Sd of Example 1 is calculated. Since 6536RU is immobilized at 100 kD, 1RU is 1 pg / mm ² The molar surface density d (mol / mm ² ) is 6.54 × 10 ⁻¹⁴ mol / mm ² , and Sd is calculated to be about 5.3 × 10 ⁻¹⁶ mol. Therefore, Cvt / Sd = 2.5 × 10 ⁻¹¹ mol / 5.3 × 10 ⁻¹⁶ mol = 4.7 × 10 ⁴ , and the relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established.

Similarly, since Fab ′ of Comparative Example 1 is immobilized at 5881RU at 50 kD, 1RU has a molar surface density d (mol / mm ² ) of 1.18 × 10 ⁻¹³ mol / mm ² from 1 pg / mm ² . , Sd is calculated to be about 9.6 × 10 ⁻¹⁶ mol. Therefore, Cvt / Sd = 2.5 × 10 ⁻¹¹ mol / 9.6 × 10 ⁻¹⁶ mol = 2.6 × 10 ⁴ , and the relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established.

Further, since HuCAL F (ab ′) ₂ of Comparative Example 2 is immobilized at 250 kRU at 130 kD, 1RU has a molar surface density d (mol / mm ² ) of 1.92 × 10 ⁻¹⁴ mol / mm from 1 pg / mm ^{2. 2} and Sd is calculated to be about 1.6 × 10 ⁻¹⁶ mol. Therefore, Cvt / Sd = 2.5 × 10 ⁻¹¹ mol / 1.6 × 10 ⁻¹⁶ mol = 1.6 × 10 ⁵ , and the relationship of 100 <Cvt / Sd <2 × 10 ⁵ is established.

Example 4 Production of Activated Nonwoven Fabric N-hydroxymethyliodoacetamide, which is an active group of a nonwoven fabric, was produced in the same manner as in Example 10 of JP-A-11-332594. To a glass flask, 0.6 g of N-hydroxymethyl iodoacetamide, 5.7 ml of concentrated sulfuric acid and 7.2 ml of nitrobenzene were added and dissolved at room temperature with stirring, and 0.036 g of paraformaldehyde was further added and stirred. To this, 0.3 g of a nonwoven fabric made of polypropylene P080HW-00F (manufactured by Tonen TAYPRUS, average fiber diameter 3.5 μm) was added, followed by light-shielding reaction at room temperature for 24 hours. After reacting for 24 hours, the nonwoven fabric was taken out, washed with ethanol and pure water, and vacuum-dried to obtain an activated nonwoven fabric.

  Four pieces of this activated non-woven fabric cut into a circle of 0.68 cm in diameter are added to 400 μL (16 μg / 400 μL) of each recognition molecule solution dissolved in a phosphate buffer solution (hereinafter referred to as PBS (−)) not containing calcium or magnesium. It was soaked at room temperature overnight, fixed, and then washed with 2 ml of PBS (−). Next, 0.2% polyoxyethylene sorbitan monolaurate / PBS (-) solution (hereinafter referred to as Tween 20 solution) was added and rinsed, and then washed with 2 ml of PBS (-) to obtain the target cell adsorbent. By the above operation, each recognition molecule of Example 1, Comparative Example 1 and Comparative Example 2 is covalently bonded to the activated nonwoven fabric surface via an amino group in each recognition molecule.

  A cell adsorber was prepared by filling a container of 1 ml capacity having an inlet and an outlet (Mobicol column: manufactured by Funakoshi Co., Ltd.) with each of the four cell adsorbents and a PBS (−) solution.

Example 5: Evaluation of cell adsorbability Immediately before the start of the experiment, the PBS (-) solution was cut off by passing the air through the above column, and ACD-A (Kawasumi Chemical Co., Ltd.) added human healthy human fresh blood 5.0 ml (Blood: ACD-A = 6.7: 1) was sent from the inlet of the cell adsorber with a syringe pump at a flow rate of 1.0 ml / min, the first 5 drops were discarded, and blood after treatment from the outlet of the column Was recovered.

The removal rate of CD4 positive cells at this time was measured by flow cytometry using a change in white blood cell count using a Coulter counter and a flow cytometer (FACS Calibur: Beckton Dickinson). It was calculated from the change in the ratio of CD4 positive cells. For staining of cells, 10 μl of anti-CD4-PE solution (clone SK3, Beckton Dickinson) and 10 μl of anti-CD45-PerCP solution (Beckton Dickinson) are added to 100 μl of collected blood, After staining for 30 min at room temperature, 1 mL of FACS Lysing solution (Beckton Dickinson) was added and hemolyzed. CD45-positive cells were gated as leukocytes, and the proportion of CD4-positive cells among them was analyzed.
These results are shown in Table 3.

  Comparing this result with Tables 1 and 2, it can be seen that only the dissociation constant, which is an index of binding affinity, does not have a sufficient correlation with the cell separation behavior, and the binding efficiency defined in the present invention is important. That is, when the recognition molecule immobilized on the substrate surface is reacted with a large excess of target protein molecules, it can be substantially used when the number of target protein molecule binding sites of the recognition molecule is 50% or more. It turns out that it becomes a cell separation base material.

By using the present invention, not only blood cell component removal therapy but also general medical techniques for separating and processing blood cell components, the biological pathological interaction such as immune reaction is used to theoretically estimate the etiology. By capturing cells that are targeted substances more specifically, side effects can be reduced, and safety and therapeutic effects can be enhanced. In particular, it can be used for a blood processing apparatus and a separation method useful as a practically usable technique for directly and selectively capturing from whole blood.

Claims (14)

A substrate for blood treatment that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, virus-infected cells in blood,
(A) an artificially prepared at least one recognition molecule having a molecular weight of 130 kDa or less having binding affinity for a target protein molecule present on the surface of the target substance is immobilized on the substrate surface;
(B) the dissociation constant between the immobilized recognition molecule and the target protein molecule is 1 μM or less,
(C) Supplying a target protein molecule solution having a concentration C (M) of 50 to 5000 times the dissociation constant concentration to the immobilized recognition molecule at a flow rate v (L / second) for a time t (second). In this case, the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the target protein molecule is supplied,
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of target protein molecule binding sites of the recognition molecule is in a state of being bound to the target protein molecule.
A blood processing substrate characterized by the above. The blood processing substrate according to claim 1, wherein the recognition molecule is immobilized on the substrate surface through a covalent bond. A blood processing device in which the blood processing substrate according to claim 1 or 2 is filled in a container having an inlet and an outlet. A blood processing method comprising passing blood containing a target substance through a blood processing device in which a blood processing substrate according to claim 1 or 2 is filled in a container having an inlet and an outlet. A recognition molecule immobilized on the surface of a substrate for blood treatment that adsorbs or removes at least one target substance selected from harmful substances, etiological cells, viruses, virus-infected cells in blood,
(A) at least one molecule having a molecular weight of 130 kDa or less, which is artificially prepared and has a binding affinity for a target protein molecule present on the surface of the target substance,
(B) the dissociation constant with the target protein molecule is 1 μM or less,
(C) When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied for a time t (second) at a flow rate v (L / second), the target protein molecule is supplied. The molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² )
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of target protein molecule binding sites of the recognition molecule is in a state of being bound to the target protein molecule.
A recognition molecule characterized by that. (A) an artificially produced molecule having binding affinity for a target protein molecule present on the surface of the target substance, (b) a molecular weight of 130 kDa or less, and (c) a target protein molecule From candidate recognition molecules whose dissociation constant is 1 μM or less,
When a target protein molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / second) for a time t (second), the substrate to which the target protein molecule is supplied Between the molar surface density d (mol / mm ² ) of the candidate recognition molecule immobilized on the surface area S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Including selecting a target protein molecule in which 50% or more of the target protein molecule binding sites of the candidate recognition molecule are bound to the target protein molecule.
A method for selecting a recognition molecule for immobilizing a surface of a blood treatment substrate that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in blood. A method for producing a substrate for blood treatment that adsorbs or removes at least one target substance selected from harmful substances, pathogenic cells, viruses, and virus-infected cells in blood, the target for immobilized recognition molecules When a protein molecule solution is supplied at a concentration C (M) that is 50 to 5000 times the dissociation constant concentration and at a flow rate v (L / second) for a time t (second), the substrate surface area to which the target protein molecule is supplied Between the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Is set so that 50% or more of the number of binding sites of the target protein molecule possessed by the recognition molecule is bound to the target protein molecule, and the binding affinity for the target protein molecule is set. A method for producing a blood processing substrate, comprising immobilizing at least one recognition molecule having a molecular weight of 130 kDa or less on the surface of the substrate. A blood processing substrate for removing 90% or more of CD4 positive cells in blood,
(A) An artificially prepared at least one recognition molecule having a molecular weight of 130 kDa or less having binding affinity for CD4 molecules on the cell surface is immobilized on the substrate surface,
(B) the dissociation constant between the immobilized recognition molecule and the CD4 molecule is 1 μM or less,
(C) A CD4 molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied to the immobilized recognition molecule at a flow rate v (L / sec) for a time t (second). The molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the CD4 molecule is supplied,
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of CD4 molecule binding sites of the recognition molecule is in a state of being bound to the CD4 molecule.
A blood processing substrate characterized by the above. The blood processing substrate according to claim 8, wherein the recognition molecule is immobilized on the surface of the substrate through a covalent bond. A blood processing device in which the blood processing substrate according to claim 8 or 9 is filled in a container having an inlet and an outlet. 10. A blood processing method, wherein blood containing a target substance is passed through a blood processing device in which a blood processing substrate according to claim 8 or 9 is filled in a container having an inlet and an outlet. A recognition molecule immobilized on the surface of a blood processing substrate that removes 90% or more of CD4-positive cells in blood,
(A) at least one molecule having a molecular weight of 130 kDa or less, which is artificially prepared and has binding affinity for CD4 molecules on the cell surface;
(B) the dissociation constant with the CD4 molecule is 1 μM or less,
(C) When a CD4 molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / sec) for a time t (seconds), the group to which CD4 molecules are supplied Between the molar surface density d (mol / mm ² ) of the recognition molecule immobilized on the material surface area S (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
When it is supplied so that the above relationship is established, 50% or more of the number of CD4 molecule binding sites of the recognition molecule is in a state of being bound to the CD4 molecule.
A recognition molecule characterized by that. (A) an artificially produced molecule having binding affinity for CD4 molecules on the cell surface, (b) a molecular weight of 130 kDa or less, and (c) a dissociation constant of 1 μM or less with CD4 molecules From the candidate recognition molecule
When a CD4 molecule solution having a concentration C (M) that is 50 to 5000 times the dissociation constant concentration is supplied at a flow rate v (L / sec) for a time t (seconds), the substrate surface area S to which the CD4 molecules are supplied Between the molar surface density d (mol / mm ² ) of the candidate recognition molecule immobilized on (mm ² ),
100 <Cvt / Sd <2 × 10 ⁵
Including selecting a substance in which 50% or more of the number of CD4 molecule binding sites of the candidate recognition molecule is bound to the CD4 molecule when supplied so that the relationship of
A method for selecting a recognition molecule for surface immobilization of a blood processing substrate that removes 90% or more of CD4-positive cells in blood. A method for producing a blood processing substrate for removing 90% or more of CD4 positive cells in a solution, wherein the concentration of the CD4 molecule solution is 50 to 5000 times the dissociation constant concentration relative to the immobilized recognition molecule. When supplying a time t (second) at a flow rate v (L / second) at C (M), the molar surface density d of the recognition molecule immobilized on the substrate surface area S (mm ² ) to which the CD4 molecule is supplied. (Mol / mm ² )
100 <Cvt / Sd <2 × 10 ⁵
Is set so that 50% or more of the number of binding sites of CD4 molecules possessed by the recognition molecule is in a state of binding to CD4 molecules, and the molecular weight of 130 kDa having binding affinity for CD4 molecules. The manufacturing method of the base material for blood treatment including fix | immobilizing the following at least 1 type of recognition molecule on the base-material surface.