JP2006149383A - Technique for efficiently selecting protein specifically binding to ligand - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for selecting a protein that specifically binds to a specific ligand. <P>SOLUTION: The method for selecting a protein that specifically binds to a target ligand from a diversified library is provided, comprising the step of bringing proteins in the library into contact with the target ligand to select proteins that bind to the target ligand, the step of bringing the proteins thus obtained into contact with a control ligand to judge the presence/absence of the bindability between the proteins and the control ligand, and the step of selecting the objective protein judged as having no bindability to the control ligand. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for selecting a protein that specifically binds to a specific ligand.

  Many in vivo factors, such as proteins, nucleic acids, and lipids, that play various roles in the body, such as enzymes, perform their functions alone, but information can be obtained through interactions between in vivo factors. It is known that various in vivo reactions are induced or controlled through transmission. Therefore, accurately elucidating the interaction between factors in vivo is important for understanding in vivo reactions, and by accumulating knowledge about such reaction mechanisms, treatment of specific diseases, etc. It is considered to be extremely useful as a clue to development of a method or a therapeutic agent.

  Phage display is a method for studying interactions between factors in vivo, especially proteins (eg, antibodies, receptors, etc.) and their specific ligands (eg, proteins, low molecular weight compounds, lipids, sugar chains, etc.). And a method for selecting a protein that specifically binds to a specific ligand from a diversified library. When identifying a protein that specifically binds to a specific ligand in these methods, the specificity and affinity for the ligand are measured by ELISA (enzyme immunoassay) or RIA, and specifically bound to the target ligand. In many cases, molecules are selected (Non-Patent Document 1, Non-Patent Document 2).

Gram et al., Proc. Natl. Acad. Sci. 89: 3576-3580, 1992. Cumbers et al., Nat. Biotechnol. , 20: 1129-1134, 2002.

It has been confirmed that many in vivo factors such as proteins produced by a diversified library exhibit non-specific binding to factors other than the target ligand. As a result, when primary screening is performed using only the binding to the target ligand as an index, the majority becomes a non-specific binding factor, making it difficult to obtain a factor exhibiting the required specific binding. It has been found.
In view of the above circumstances, the present inventors conducted extensive research on a method for selecting a protein exhibiting specific binding to a target ligand from a diversified library, and as a result, identified a protein that specifically binds to the target ligand. We have found a method that can be made easier and more efficient.
The present invention relates to a method for selecting a protein that specifically binds to a target ligand from a diversified library.

The present inventors non-specifically bind to a target ligand by simultaneously measuring and comparing the affinity for not only the target ligand but also the control ligand when screening proteins using the affinity for the ligand as an index. Thus, a method for easily and efficiently identifying a desired protein has been completed.
That is, the present invention relates to the following (1) to (11).
(1) The invention according to the first embodiment of the present invention is “a method for selecting at least one protein that specifically binds to a target ligand from a diversified library,
(A) A step of contacting a variety of proteins present in the library with a target ligand, incubating a mixture of the protein group and the target ligand, and recovering a complex of at least one protein and the target ligand (b) A step of contacting the target ligand with at least several proteins selected in (a), incubating a mixture of the protein and the target ligand, and confirming the binding between the protein and the target ligand;
(C) contacting at least several proteins selected in step (a) with one or two specific control ligands, incubating a mixture of the protein and control ligand, and binding of the protein and control ligand Determining whether or not, and
(D) selecting a protein that has been confirmed to be binding to the target ligand in step (b) and has not bound to the control ligand in step (c);
A method comprising:
(2) The invention according to the second embodiment of the present invention is the above (1), wherein the protein to be contacted with the target ligand in the step (a) is presented on the surface of the host. Is the method described in ”.
(3) The invention according to the third embodiment of the present invention is “the method according to (1) or (2) above, wherein the target ligand and / or control ligand is bound to a carrier”. It is.
(4) The invention according to the fourth embodiment of the present invention is “the method according to (3) above, wherein the carrier is a magnetic bead”.
(5) The invention according to the fifth embodiment of the present invention is as follows: “Determining the presence or absence of binding in the step (b) by ELISA method, RIA method, surface plasmon resonance (SPR) method, blotting method, ligand bead The method according to any one of (1) to (4) above, which is carried out by a method selected from the group consisting of methods using
(6) The invention according to the sixth embodiment of the present invention is “the method according to (5) above, wherein the target ligand and / or the control ligand are labeled”.
(7) The method according to any one of (1) to (6) above, wherein the diversification library is a protein expression library. Is.
(8) The invention according to the eighth embodiment of the present invention is “the method according to (7) above, wherein the protein expression library is an antibody expression library”.
(9) The invention according to the ninth embodiment of the present invention ”,“ the antibody expression library is a library composed of cells displaying an antibody on a cell surface ”(8 ) Described method.
(10) The invention according to the tenth embodiment of the present invention is “the method according to (9) above, wherein the cell is a DT40 cell”.
(11) The invention according to the eleventh embodiment of the present invention is “a protein selected by the method according to any one of (1) to (10) above”.

  By using the method according to the present invention, proteins that specifically bind to the target ligand can be rapidly and efficiently selected from the diversified library.

1. Diversified library The “diversified library” in the present invention means a library capable of presenting a wide variety of proteins. The method according to the present invention can be applied to any library that can display a wide variety of proteins. Although not limited, for example, a phage library capable of expressing various proteins, a library of cells capable of displaying various proteins on the cell surface, and the like can be used. A library of cells capable of presenting antibody molecules is suitable. Here, as the “cell library”, a library composed of chicken-derived DT40 cells capable of presenting various antibody molecules on the cell surface can be used (WO2004 / 011644).
In the “protein” of the present invention, in addition to naturally-occurring proteins, two or more amino acids such as mutants thereof, polypeptides constituting a part thereof, and artificially synthesized peptides are covalently bonded (for example, It is a concept that includes molecules linked by peptide bonds.
The “protein expression library” in the present invention is included in the above-mentioned “diversification library” and is a concept representing a host population capable of expressing a wide variety of proteins. As used herein, “host” includes all those that can be assumed by those skilled in the art based on common general technical knowledge, and examples include prokaryotic cells, eukaryotic cells, phages, and viruses. In addition, the protein expressed by the “host” may be expressed in the cell of the “host”, may be expressed on the cell surface, or the “host” is cultured to express the protein. And may be expressed in a suitable medium. A “protein expression library” that can be applied to the method according to the present invention can be purchased commercially.

The “antibody expression library” in the present invention is a concept included in the above-mentioned “protein expression library” and represents a host population capable of expressing various antibody molecules. The term “host” includes all those that can be assumed by those skilled in the art based on common general technical knowledge. For example, prokaryotic cells, eukaryotic cells, phages or viruses are included, and preferably, eukaryotic cells and phages are used. More preferably animal cells and phages, and particularly preferably DT40 cells and Ramos cells.

2. Target Ligand and Control Ligand “Target Ligand” and “Control Ligand” in the present invention may be any one as long as it binds to a protein, for example, protein, nucleic acid, lipid, carbohydrate. And all those that can be envisioned by those skilled in the art, such as small molecule compounds. In addition, the “control ligand” in the present invention may be any as long as it binds to a protein, and is not limited. For example, a protein, nucleic acid, lipid, carbohydrate, low molecular weight compound, etc. Includes all that can be envisaged, but preferably in the relationship with the “target ligand”, the amino acid identity with the amino acid sequence of the “target ligand” is low (approximately 30% or less, more preferably, 20% or less, more preferably 10% or less), and the “control ligand” itself has no non-specific binding activity. The “control ligand” used in the present invention may be one type or a plurality of types, for example, two types or less, preferably one type, but not limited to ovalbumin or egg white lysozyme. It is possible to select a specific factor in relation to the “target ligand” such as protein, lipid, carbohydrate and the like.
Further, the “target ligand” or “control ligand” may be labeled, and any method and substance may be used as long as the label is a method and a labeling compound that are usually used in the art. Although not limited, a fluorescent label etc. can be used conveniently.

3. Selection of a protein that binds to a target ligand In the present invention, a protein that binds to a target ligand can be selected based on ordinary knowledge in the art. For example, the target ligand is bound to an appropriate carrier, the bound target ligand is brought into contact with a protein present in the diversified library, incubated under appropriate conditions, and the resulting carrier-target ligand-protein complex is then obtained. A protein that is recovered by centrifugation or the like and that binds to the target ligand can be selected. When a protein that binds to the target ligand is obtained, if the host that expresses the protein is not a single clone, a single clone can be obtained by the limiting dilution method and subculture. Some of the proteins thus obtained bind with a different affinity to the target ligand.
The “carrier” used here is not limited, and suitable ones such as sepharose beads, agarose beads, glass substrates, magnetic beads and the like can be used, but magnetic beads are particularly preferable.
Conditions for allowing the target ligand to bind to the protein can be set by those skilled in the art according to the target ligand. Although there is no particular limitation, for example, when the protein desired to be selected is an antibody molecule, conditions for incubating at a temperature of about 4 ° C. to 37 ° C. for about 15 minutes to 24 hours can be set. .
Furthermore, as a method for confirming that the protein obtained as binding to the target ligand binds to the target ligand, it can be performed by a method well known in the art, for example, ELISA method, RIA method, surface A plasmon resonance (SPR) method, a blotting method, a method using ligand beads, and the like can be used. The target ligand used here may be labeled. Alternatively, when a substance that specifically binds to a protein that specifically binds to a target ligand (for example, a protein such as an antibody, a nucleic acid, a lipid, etc.) is available, the labeled or not labeled The binding specificity between the target ligand and the protein can be confirmed by a usual method using the substance.

4). Determination of the binding property between the protein that binds to the target ligand and the control ligand As a method for determining the binding property between the selected protein and the control ligand using the binding property to the target ligand as an index, a method well known in the art Can be performed.
For example, an ELISA method, an RIA method, a surface plasmon resonance (SPR) method, a blot method, a method using ligand beads, and the like can be used. The control ligand used here may be labeled. Alternatively, if a substance that specifically binds to a protein that specifically binds to a control ligand (eg, a protein such as an antibody, a nucleic acid, a lipid, etc.) is available, the labeled or not Using the substance, the binding specificity between the control ligand and the protein can be confirmed by an ordinary method.
Here, the determination of whether or not a protein binds to a target ligand but does not bind to a control ligand depends on the method used. D. O. which shows the binding to the target ligand when expressed by a value (absorbance measured at a wavelength suitable for detecting the binding between the protein and the ligand). D. Value / O. D. When the value is at least 2 or more, at least 1.0 or more when ovalbumin is used as the control ligand, and at least 0.7 or more when egg white lysozyme is used as the control ligand, it specifically binds to the target ligand. The control ligand can be determined to be a protein having no binding property.
The protein obtained through the above steps can be determined to be a protein that specifically binds to the target ligand.

  Examples are shown below, but the present invention is not limited thereto.

  In this example, a method for selecting antibody molecules that specifically bind to streptavidin from a population of DT40 cells that induces somatic cell recombination at an immunoglobulin locus and produces various immunoglobulin molecules. Will be described. See WO2004 / 011644 for methods for preparing the DT40 cell population.

1. Selection of antibodies that specifically bind to streptavidin
Cultured cells:
DT40 cells were cultured at 59.5% CO ₂ at 39.5 ° C. in a 5% CO ₂ thermostat. The medium was IMDM medium (Invitrogen), and 10% FBS, 1% chicken serum, penicillin 100 units / ml, streptomycin 100 μg / ml, 2-mercaptoethanol 55 μM were used. Trichostatin A (Wako Pure Chemical Industries) was dissolved in methanol at 5 mg / ml as a stock, and diluted with a medium as appropriate so that the final concentration was 2.5 ng / ml. The culture was continued while the cell concentration was maintained at 10 ⁵ to 10 ⁶ cells / ml.

Preparation of streptavidin magnetic beads:
Dynabeads M-280 Tosylactivated (Dynal) was used for the magnetic beads, and Dynal MPC (Dynal) was used for the magnetic stand. 200 μl of beads were washed 3 times with 500 μl of buffer A (0.1 M sodium phosphate, pH 7.4) and then rotated with 240 μg of streptavidin (Nacalai Tesque) in buffer A, 400 μl for 24 hours at 37 ° C. The reaction was carried out with stirring. The beads were then washed twice with 500 μl of buffer C (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.1% BSA). Thereafter, 500 μl of buffer D (0.2 M Tris-HCl, pH 8.5, 0.1% BSA) was added, and the mixture was allowed to react at 37 ° C. for 4 hours with stirring by rotation to perform blocking. Thereafter, the cells were washed twice with 500 μl of buffer C and then suspended in 400 μl of buffer C containing 0.02% sodium azide.

Production of rabbit IgG magnetic beads:
Dynabeads M-280 Tosylactivated (Dynal) was used for the magnetic beads, and Dynal MPC (Dynal) was used for the magnetic stand. 200 μl of beads were washed 3 times with 500 μl of buffer A (0.1 M sodium phosphate, pH 7.4), then stirred with 120 μg rabbit IgG (SIGMA) in buffer A, 200 μl at 37 ° C. overnight by rotation. While reacting. The beads were then washed twice with 200 μl of buffer C (10 mM sodium phosphate, pH 7.4, 150 mM NaCl, 0.1% BSA). Thereafter, 200 μl of buffer D (0.2 M Tris-HCl, pH 8.5, 0.1% BSA) was added, and the mixture was reacted by stirring at 37 ° C. for 4 hours while rotating, thereby blocking. Thereafter, the cells were washed twice with 500 μl of buffer C and then suspended in 200 μl of buffer C containing 0.02% sodium azide.

Selection with streptavidin and rabbit IgG magnetic beads:
About 5 × 10 ⁷ wild-type DT40 cells treated with trichostatin A, 2.5 ng / ml for 7 weeks were washed once with 10 ml of a washing buffer (PBS containing 1% BSA) and once with 1 ml. Were mixed with 5 × 10 ⁶ streptavidin magnetic beads (rabbit IgG magnetic beads in the case of selection with rabbit IgG magnetic beads) in the washing buffer of 4 and incubated at 4 ° C. for 30 minutes with gentle rotation. Thereafter, it was washed 5 times with 1 ml of washing buffer. Finally, the cells bound to the magnetic beads were suspended in 500 μl, added to 30 ml of medium, dispensed 300 μl in 96-well plates, and cultured at 39.5 ° C. One week later, ELISA was performed on the culture supernatant.

Two plate ELISA:
Two 96-well immunoplate maxisorp (NaigeNunc) were prepared, and 200 μl each of 5 μg / ml streptavidin (Nacalai Tesque) and ovalbumin (Sigma) (both dissolved in PBS) were added at room temperature. It was left to stand overnight and fixed to the plate (in the case of selection with rabbit IgG magnetic beads, rabbit IgG (SIGMA) and egg white lysozyme (SIGMA) were used). Thereafter, the mixture was blocked with 200 μl of 0.5% skim luc at room temperature for 1 hour, and washed 3 times with 200 μl of ELISA washing buffer (PBS supplemented with 0.05% Tween 20). To this was added 100 μl of cell culture supernatant and allowed to react at room temperature for 1 hour. Here, 100 μl of the same culture supernatant was dispensed to both the streptavidin-fixed plate and the ovalbumin-fixed plate (in the case of rabbit IgG magnetic bead selection, rabbit IgG-fixed plate and egg white lysozyme-fixed plate). Thereafter, the plate was washed 5 times with 200 µl of ELISA washing buffer, and horseradish peroxidase-conjugated goat anti-chicken IgM antibody (Bethyl) was diluted 2000 times with PBS and added in 100 µl portions. After reacting at room temperature for 45 minutes, the plate was washed 5 times with an ELISA washing buffer, and then 100 μl of TMB + (Dako) was added and reacted at room temperature for 4 minutes, and then the reaction was stopped with 100 μl of 1N sulfuric acid. Quantification was performed by measuring the absorbance at 450 nm with an mQuant Biomolecular Spectrometer (Bio-Tek Instruments).

The results of ELISA are shown in Table 1 and FIG. Table 1 shows the ELISA results of antibodies reacting with streptavidin (28 clones in total). D. The ELISA results for the reactivity with ovalbumin of the same clone are also shown by O.D. D. Shown by value. FIG. 1 is a graph of Table 1.
Of all 28 clones that reacted with streptavidin, it was revealed that many of them also reacted with ovalbumin. One of these clones (clone 22: named clone SD-10) was shown to specifically react only with streptstreptavidin (in Table 1, clone 22, in FIG. 1, the graph indicated by the arrow). reference). Since all clones showing high affinity for streptavidin react strongly with ovalbumin, selection using only the affinity for streptavidin as an index is nonspecific for any ligand. It has been experimentally found that the probability of selecting antibodies that bind automatically is very high. On the other hand, it was confirmed that such a non-specific antibody can be efficiently excluded from the beginning by using the method of the present invention. In fact, when assaying with streptavidin alone, 28 clones are selected as candidates, but most of them also bind strongly to ovalbumin. By excluding such clones, it was possible to immediately limit to one clone. In this experiment, the target ligand is streptavidin and the control ligand is ovalbumin, but this method is also effective in combination with other specific proteins (for example, target ligand: control ligand = rabbit IgG: skim milk, egg white lysozyme: skim milk) It has been confirmed that. The results of actually using egg white lysozyme are also shown in FIG. The presence of cells was observed in 53 wells of a 96-well plate. Of these, 0. D. There were 6 clones that reacted with a value of 1 or more (clone 3, 13, 31, 35, 38, 47). By simultaneously examining the reactivity to lysozyme, it was possible to immediately determine whether these 6 clones were specific for rabbit IgG.

2. Check for specificity
Preparation of culture supernatant for ELISA for verification of streptavidin specificity:
The culture supernatant used for analyzing the further specificity by ELISA was a medium prepared as follows in order to remove serum-derived IgM and the like. Immunoglobulin was removed from chicken serum (Invitrogen) with 50% saturated ammonium sulfate as a precipitate, and the supernatant was dialyzed against PBS. Volume increase caused by dialysis was corrected by concentration with Centri Prep (Amicon) to obtain antibody-removed chicken serum. This was added to AIM-V serum-free medium (Invitrogen) at a concentration of 6%. Cells were added thereto at a concentration of about 10 ⁶ / ml, cultured for 2 days, and the culture supernatant was removed and subjected to ELISA.

ELISA for verification of streptavidin specificity:
Add 200 μl of 5 μg / ml streptavidin, ovalbumin, human IgG (Sigma) and 0.5% skim milk (Difco) (both dissolved in PBS) to 96-well immunoplate maxi soap, and overnight at room temperature It was left to fix to the plate. Thereafter, the mixture was blocked with 200 μl of 0.5% skim luc at room temperature for 1 hour and washed 3 times with 200 μl of ELISA washing buffer. 100 μl of the cell culture supernatant diluted 1-fold to 3125-fold in 5-fold increments was added to this and allowed to react at room temperature for 1 hour. The clone used here was clone SD10-1, which was obtained by limiting dilution of the previous SD10 once. Thereafter, the plate was washed 5 times with 200 μl of ELISA washing buffer, and horseradish peroxidase-conjugated goat anti-chicken IgM antibody was diluted 2,000 times with PBS and added in 100 μl aliquots. After reacting at room temperature for 45 minutes, the plate was washed 5 times with an ELISA washing buffer. Thereafter, 100 μl of TMB + was added and reacted at room temperature for 4 minutes, and then the reaction was stopped with 100 μl of 1N sulfuric acid. Quantification was performed by measuring the absorbance at 450 nm using an mQuant Biomolecular Spectrometer.

  The result of ELISA for specificity verification is shown in FIG. It was confirmed that SD-10-1 obtained by limiting dilution of SD-10 shown in FIG. 1 reacts strongly only with the originally intended streptavidin. In the case where the present invention is not applied, it is necessary to perform the same assay for 28 clones, so it has been found that there is at least a 28-fold cost reduction effect.

FIG. 1 shows a method for selecting cells that produce antibodies specifically binding to streptavidin by streptavidin magnetic beads from a diversified library of chicken DT40 cells each displaying various antibody molecules on the cell surface. The result of having performed ELISA using a culture supernatant is shown. The 22nd clone (arrow) is SD-10. FIG. 2 shows the results of performing ELISA using an egg white lysozyme as a target antigen instead of ovalbumin when selecting anti-rabbit IgG antibodies. FIG. 3 shows the results of detailed examination of the specificity of the antibodies finally selected in FIG. 1 for various ligands (streptavidin, ovalbumin, hIgG (human IgG), skim milk) by ELISA.

Claims (11)

A method of selecting at least one protein that specifically binds to a target ligand from a diversified library,
(A) A step of contacting a variety of proteins present in the library with a target ligand, incubating a mixture of the protein group and the target ligand, and recovering a complex of at least one protein and the target ligand (b) A step of contacting the target ligand with at least several proteins selected in (a), incubating a mixture of the protein and the target ligand, and confirming the binding between the protein and the target ligand;
(C) contacting at least several proteins selected in step (a) with one or two specific control ligands, incubating a mixture of the protein and control ligand, and binding of the protein and control ligand Determining whether or not, and
(D) selecting a protein that has been confirmed to be binding to the target ligand in step (b) and has not bound to the control ligand in step (c);
Comprising a method.   The method according to claim 1, wherein the protein to be contacted with the target ligand in the step (a) is displayed on the surface of a host.   The method according to claim 1 or 2, wherein the target ligand and / or the control ligand are bound to a carrier.   The method of claim 3, wherein the carrier is a magnetic bead.   The determination of the presence or absence of binding in the step (b) is performed by a method selected from the group consisting of ELISA method, RIA method, surface plasmon resonance (SPR) method, blotting method, and method using ligand beads. The method according to claim 1, wherein:   6. The method of claim 5, wherein the target ligand and / or control ligand is labeled.   The method according to any one of claims 1 to 6, wherein the diversification library is a protein expression library.   The method according to claim 7, wherein the protein expression library is an antibody expression library.   9. The method according to claim 8, wherein the antibody expression library is a library composed of cells displaying antibodies on the cell surface.   The method according to claim 9, wherein the cell is a DT40 cell. A protein selected by the method according to any one of claims 1 to 10.

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