Patent Application
20080176753
The present invention relates to a method of selecting a protein specifically binding to a specific ligand.
In vivo factors, such as a protein, nucleic acid, or lipid, play various roles in a living body. Many of the factors exhibit their functions by themselves, like enzymes. It has been known that such in vivo factors transmit information via the interaction between them, so as to induce or regulate various types of in vivo reactions. Accordingly, precise clarification of such an interaction between in vivo factors is important for understanding such in vivo reactions. Moreover, it is considered that accumulation of knowledge in such a reaction mechanism is extremely useful as a key for the development of a treatment method or a therapeutic agent for certain diseases and the like.
As a method of studying the interaction between in vivo factors, and in particular, a protein (for example, an antibody, a receptor, etc.) and a ligand specific therefor (for example, a protein, a low molecular weight compound, a lipid, a sugar chain, etc.), there is a method of selecting a protein specifically binding to a specific ligand from a diversifying library such as a phage display. In such a method, when a protein specifically binding to a specific ligand is identified, the specificity or affinity of the protein to the ligand is measured by ELISA (Enzyme-Linked ImmunoSorbent Assay) or RIA, so as to select a molecule specifically binding to the ligand of interest, in many cases (Non-Patent Publications 1 and 2).
Non-Patent Publication 1: Gram et al., Proc. Natl. Acad. Sci., 89: 3576-3580, 1992
Non-Patent Publication 2: Cumbers et al., Nat. Biotechnol., 20: 1129-1134, 2002
It has been confirmed that many in vivo factors generated from a diversifying library, such as a protein, are problematic in that such factors exhibit non-specific binding ability to factors other than a ligand of interest. Thus, it has been revealed that, if a first screening is carried out using only such binding ability to the ligand of interest as an indicator, a majority of factors become non specific binding factors, and thereby it becomes extremely difficult to obtain necessary factors that exhibit specific binding ability.
Under the aforementioned circumstances, the present inventors have conducted intensive studies directed towards achieving a method of selecting a protein exhibiting specific binding ability to a ligand of interest from a diversifying library. As a result, the inventors have discovered a method of easily and efficiently identifying a protein that specifically binds to the ligand of interest.
The present invention relates to a method of selecting a protein that specifically binds to a ligand of interest from a diversifying library.
With regard to the screening of a protein using affinity to a ligand as an indicator, the present inventors have completed a method of easily and efficiently identifying a desired protein, which comprises simultaneously measuring not only affinity to a ligand of interest, but also affinity to a control ligand, making a comparison between the two types of affinities, so as to eliminate proteins that non-specifically bind to the ligand of interest, thereby easily and efficiently identifying such a desired protein.
That is to say, the present invention relates to the following (1) to (11):
Using the method of the present invention, a protein specifically binding to a ligand of interest can be rapidly and efficiently selected from a diversifying library.
The term “diversifying library” is used in the present invention to mean a library capable of presenting various types of proteins. The method of the present invention can be applied to any type of library, as long as such a library is able to present various types of proteins. Thus, the type of the library is not limited. Examples of a library that can be used herein include a phage library capable of expressing various types of proteins, and a cell library capable of presenting various types of proteins on the surfaces of cells. A particularly suitable library is a cell library capable of presenting a variety of antibody molecules on the surfaces of cells. An example of the aforementioned “cell library” used herein is a library that is constituted with DT40 cells derived from chick, which is able to present various antibody molecules on the cell surface thereof (WO2004/011644).
The concept of the “protein” of the present invention includes molecules formed by allowing two or more amino acids to bind to one another via a covalent bond (for example, a peptide bond), such as a naturally-derived protein, a mutant thereof, a polypeptide constituting a portion thereof, or an artificially synthesized peptide.
The “protein expression library” of the present invention is included in the aforementioned “diversifying library,” and thus the protein expression library has a concept indicating a host population capable of expressing a variety of proteins. The term “host” is used herein to include all hosts that can be conceived of by persons skilled in the art based on common technical knowledge. For example, prokaryocytes, eukaryotes, phages, viruses, etc. are included in such hosts. In addition, a protein that is allowed to express by such a “host” may be allowed to express either in the cells of the above “host,” or on the surfaces of the cells. Otherwise, the above “host” may be cultured, so that the protein may also be allowed to express in a medium that is suitable for the expression of the above protein. As such a “protein expression library” that can be applied to the method of the present invention, a commercially available product can also be used.
The “antibody expression library” of the present invention is included in the aforementioned “protein expression library,” and thus the antibody expression library has a concept indicating a host population capable of expressing a variety of antibody molecules. The term “host” is used herein to include all hosts that can be conceived of by persons skilled in the art based on common technical knowledge. Examples of such a host include prokaryocytes, eukaryotes, phages, and viruses. Preferred examples include eukaryotes and phages. More preferred examples include animal cells and phages, and particularly preferred examples include DT40 cells and Ramos cells.
Any type of a “ligand of interest” can be used in the present invention, as long as such a ligand of interest binds to a protein. Thus, the type of the “ligand of interest” of the present invention is not limited. Examples of such a ligand of interest include all ligands that can be conceived of by persons skilled in the art, such as a protein, a nucleic acid, a lipid, a sugar chain, or a low molecular weight compound. Moreover, any type of a “control ligand” can be used in the present invention, as long as such a control ligand binds to a protein. Thus, the type of the “control ligand” of the present invention is not limited. Examples of such a control ligand include all ligands that can be conceived of by persons skilled in the art, such as a protein, a nucleic acid, a lipid, a sugar chain, or a low molecular weight compound. In terms of the relationship with the “ligand of interest,” such a control ligand preferably has low identity at the amino acid sequence level with the “ligand of interest” (approximately 30% or less, more preferably 20% or less, and further more preferably 10% or less), and further the “control ligand” itself does not have non-specific binding activity. The “control ligand” used in the present invention may be either of a single type, or of multiple types. Thus, the number of types is not limited. For example, the control ligand may be of two or less types, and preferably of one type. Moreover, taking into consideration the relationship of the control ligand with the “ligand of interest,” several types of proteins, lipids, carbohydrates, etc. contained in skim milk, such as ovalbumin or white-egg lysozyme, may be selected.
Furthermore, such a “ligand of interest” or “control ligand” may also be labeled. For such labeling, any type of labeling method and any type of labeling substance may be applied, as long as such a labeling method and a labeling substance are commonly used in the present technical field. Thus, the type of a labeling method and the type of a labeling substance are not limited. Fluorescent labeling can preferably be used.
3. Selection of Protein that Binds to Ligand of Interest
In the present invention, a protein binding to a ligand of interest can be selected based on common knowledge in the present technical field. For example, a ligand of interest is allowed to bind to a suitable carrier, and such a bound ligand of interest is then allowed to come into contact with proteins existing in a diversifying library. Thereafter, a mixture consisting of the ligand of interest and the protein is incubated under appropriate conditions, and the generated complex of the carrier-the ligand of interest-the protein is then recovered by centrifugation or the like, so as to select the protein binding to the ligand of interest. When such a protein binding to a ligand of interest is obtained, if a host that expresses the above protein is not a single clone, the single clone can be obtained by the limiting dilution method or subculture. Several proteins as obtained above bind to a ligand of interest via different affinity.
The type of a “carrier” used herein is not limited. Appropriate carriers such as sepharose beads, agarose beads, glass substrates, or magnetic beads, can be used. Magnetic beads are particularly preferable.
Conditions for allowing a ligand of interest to bind to a protein can be determined by persons skilled in the art, depending on the type of the ligand of interest. Such conditions are not particularly limited. When a protein to be selected is an antibody molecule for example, a mixture consisting of the ligand of interest and the protein may be incubated at a temperature between approximately 4° C. and 37° C. for approximately 15 minutes to 24 hours.
As a method of confirming the binding ability of the thus obtained protein to the ligand of interest, a method that is publicly known in the present technical field can be applied. Examples of such an applicable method include an ELISA method, an RIA method, a surface plasmon resonance (SPR) method, a blotting method, and a method using ligand beads. The ligand of interest used herein may be labeled. When a substance that specifically binds to a protein specifically binding to the ligand of interest (for example, a protein such as an antibody, a nucleic acid, a lipid, etc.) can be obtained, the binding specificity of the ligand of interest and the above protein can be confirmed by a common method using the above substance, which has been labeled or unlabeled.
4. Determination of Binding Ability between Protein Binding to Ligand of Interest and Control Ligand
As a method of determining the binding ability between the protein that has been selected using the binding ability thereof to the ligand of interest as an indicator and a control ligand, a method that is publicly known in the present technical field can be applied.
Examples of such an applicable method include an ELISA method, an RIA method, a surface plasmon resonance (SPR) method, a blotting method, and a method using ligand beads. The control ligand used herein may be labeled. When a substance that specifically binds to a protein specifically binding to the control ligand (for example, a protein such as an antibody, a nucleic acid, a lipid, etc.) can be obtained, the binding specificity of the control ligand and the above protein can be confirmed by a common method using the above substance, which has been labeled or unlabeled.
The degree of determination whether or not it is a protein that binds to a ligand of interest but does not bind to a control ligand is different depending on a method to be applied. For example, when the binding strength is expressed as an O.D. value (absorbance measured at a wavelength suitable for detection of the binding of a protein to a ligand) or the like, if the O.D. value showing the binding ability of the protein to the ligand of interest/the O.D. value showing the binding ability of the protein to the control ligand is at least 2, if such a value is at least 1.0 in the case of using ovalbumin as a control ligand, and if such a value is at least 0.7 in the case of using white-egg lysozyme as a control ligand, it can be determined that it is a protein that specifically binds to the ligand of interest but that does not bind to the control ligand.
It can be determined that the protein obtained via the aforementioned steps is a protein that specifically binds to the ligand of interest.
The examples will be given below. However, these examples are not intended to limit the scope of the present invention.
In the present example, a method, which comprises inducing somatic recombination in immunoglobulin locus and selecting an antibody molecule specifically binding to streptavidin from a DT40 cell population generating various immunoglobulin molecules, will be described. Please refer to WO2004/011644 for a method of preparing such a DT40 cell population.
DT40 cells were cultured at 39.5° C. in the presence of 5% CO2 in a 5% CO2 thermostatic bath. An IMDM medium (Invitrogen) was used as a medium. 10% FBS, 1% chick serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 55 μM 2-mercaptoethanol were added to the medium, and the obtained medium was used herein. In addition, Trichostatin A (Wako Pure Chemical Industries, Ltd.) was dissolved in methanol, resulting in a concentration of 5 mg/ml, and this mixture was used as a stock. The stock was diluted with a medium as appropriate, resulting in a final concentration of 2.5 ng/ml, and the thus diluted solution was then used. Culture was continued, while keeping a cell concentration of 105 to 106 cells/ml.
Dynabeads M-280 Tosylactivated (Dynal) was used as magnetic beads, and Dynal MPC (Dynal) was used as a magnetic stand. 200 μl of beads were washed with 500 μl of buffer A (0.1 M sodium phosphate: pH 7.4) three times. Thereafter, the resultant beads were reacted with 240 μg of streptavidin (Nacalai Tesque, Inc.) at 37° C. for 24 hours in 400 μl of buffer A, while stirring by rotation. Subsequently, the beads were washed with 500 μl of buffer C (10 mM sodium phosphate: pH 7.4; 150 mM NaCl; and 0.1% BSA) twice. Thereafter, 500 μl of buffer D (0.2 M Tris-HCl: pH 8.5; and 0.1% BSA) was added to the resultant beads, and the obtained mixture was then reacted at 37° C. for 4 hours, while stirring by rotation, so as to conduct blocking. Thereafter, the resultant was washed with 500 μl of buffer C twice, and it was then suspended in 400 μl of buffer C that contained 0.02% sodium azide.
Dynabeads M-280 Tosylactivated (Dynal) was used as magnetic beads, and Dynal MPC (Dynal) was used as a magnetic stand. 200 μl of beads were washed with 500 μl of buffer A (0.1 M sodium phosphate: pH 7.4) three times. Thereafter, the resultant beads were reacted with 120 μg of rabbit IgG (SIGMA) at 37° C. overnight in 200 μl of buffer A, while stirring by rotation. Subsequently, the beads were washed with 200 μl of buffer C (10 mM sodium phosphate: pH 7.4; 150 mM NaCl; and 0.1% BSA) twice. Thereafter, 200 μl of buffer D (0.2 M Tris-HCl: pH 8.5; and 0.1% BSA) was added to the resultant beads, and the obtained mixture was then reacted at 37° C. for 4 hours, while stirring by rotation, so as to conduct blocking. Thereafter, the resultant was washed with 500 μl of buffer C twice, and it was then suspended in 200 μl of buffer C that contained 0.02% sodium azide.
Selection with Streptavidin Magnetic Beads and Rabbit IgG Magnetic Beads:
Approximately 5×107 wild-type DT40 cells, which had been treated with 2.5 ng/ml Trichostatin A for 7 weeks, were washed with 10 ml of a washing buffer (1% BSA-containing PBS) once, and were then washed with 1 ml of the washing buffer once. Thereafter, the cells were mixed with 5×106 streptavidin magnetic beads (or with rabbit IgG magnetic beads in the case of selection with rabbit IgG magnetic beads) in 1 ml of the washing buffer. Thereafter, the obtained mixture was incubated at 4° C. for 30 minutes, while gently rotating it. Thereafter, the resultant was washed with 1 ml of the washing buffer 5 times. Finally, the cells, which had bound to the magnetic beads, were suspended in 500 μl. The obtained suspension was added to 30 ml of the medium, and 300 μl each of the obtained mixture was then dispensed into a 96-well plate, followed by culture at 39.5° C. 1 week later, ELISA was performed on the culture supernatant.
Two plates of 96-well Immunoplate Maxisorp (NaigeNunc) were prepared. 200 μl each of 5 μg/ml streptavidin (Nacalai Tesque, Inc.) or ovalbumin (Sigma) (both of which had been dissolved in PBS) was added to either one of the above two plates, and it was then left at room temperature overnight, so as to immobilize them on the plates (rabbit IgG (SIGMA) and white-egg lysozyme were used in the case of selection with the rabbit IgG magnetic beads). Thereafter, blocking was carried out with 200 μl of 0.5% skim milk at room temperature for 1 hour, and the resultant was then washed with 200 μl of an ELISA washing buffer (PBS that contained 0.05% Tween20) 3 times. Thereafter, 100 μl of the cell culture supernatant was added to the resultant, and the obtained mixture was then reacted at room temperature for 1 hour. 100 μl of the same type of culture supernatant was dispensed onto each of the streptavidin-immobilized plate and the ovalbumin-immobilized plate (in the case of selection of rabbit IgG magnetic beads, the rabbit IgG-immobilized plate and the egg-white lysozyme plate). Thereafter, the obtained mixture was washed with 200 μl of an ELISA washing buffer 5 times. Thereafter, a horse radish peroxidase-conjugated goat anti-chick IgM antibody (Bethyl) was diluted with PBS 2000 times, and 100 μl each of the thus diluted antibody was then added to the above resultant. The obtained mixture was reacted at room temperature for 45 minutes, and the reaction product was then washed with the ELISA washing buffer 5 times. Thereafter, 100 μl of TMB+ (Dako) was added to the reaction product, and the obtained mixture was then reacted at room temperature for 4 minutes. Thereafter, 100 μl of 1 N sulfuric acid was added to the reaction system, so as to terminate the reaction. Quantification was carried out by measuring the absorbance at 450 nm, using mQuant Biomolecular Spectrometer (Bio-Tek Instruments).
The results of ELISA are shown in Table 1 and
It was revealed that many of the total 28 clones that reacted with streptavidin reacted also with ovalbumin. It was shown that one of these clones (clone 22: named as clone SD-10) specifically reacted only with streptavidin (clone 22 shown in Table 1: refer to the bars indicated with the arrows in
In order to eliminate IgM derived from serum and the like, a medium as prepared below was used for a culture supernatant that was used to further analyze specificity by ELISA. Immunoglobulin was eliminated in the form of a precipitate from chick serum (Invitrogen), using 50% saturated ammonium sulfate, and the supernatant was then dialyzed to PBS. An increase in the volume generated as a result of the dialysis was corrected by concentration by Centri Prep (Amicon), so as to obtain antibody-eliminated chick serum. The thus obtained serum was added in a concentration of 6% to the AIM-V serum free medium (Invitrogen). Thereafter, cells were added in a concentration of approximately 106/ml to the resultant medium. The obtained mixture was cultured for 2 days, and the culture supernatant was collected, followed by performing ELISA.
200 μl each of 5 μg/ml streptavidin, ovalbumin, human IgG (Sigma), or 0.5% skim milk (Difco) (all of which had been dissolved in PBS) was added to a 96-well Immunoplate Maxisorp, and it was then left at room temperature overnight, so as to immobilize them on the plate. Thereafter, blocking was carried out with 200 μl of 0.5% skim milk at room temperature for 1 hour, and the resultant was then washed with 200 μl of an ELISA washing buffer 3 times. Thereafter, 100 μl of the cell culture supernatant, which had been diluted every 5 times on a scale of 1 to 6, that is, from 1 to 3,125 times, was added to the resultant. The obtained mixture was then reacted at room temperature for 1 hour. Clone SD10-1 obtained by limiting dilution of the previous SD10 was used as a clone herein. Thereafter, the reaction product was washed with 200 μl of the ELISA washing buffer 5 times. Thereafter, a horse radish peroxidase-conjugated goat anti-chick IgM antibody was diluted with PBS 2000 times, and 100 μl each of the thus diluted antibody was then added to the above resultant. The obtained mixture was reacted at room temperature for 45 minutes, and the reaction product was then washed with the ELISA washing buffer 5 times. Thereafter, 100 μl of TMB+ was added to the reaction product, and the obtained mixture was then reacted at room temperature for 4 minutes. Thereafter, 100 μl of 1 N sulfuric acid was added to the reaction system, so as to terminate the reaction. Quantification was carried out by measuring the absorbance at 450 nm, using mQuant Biomolecular Spectrometer.
The results of ELISA performed to study specificity are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 2004-324217 | Nov 2004 | JP | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/JP2005/020346 | 11/7/2005 | WO | 00 | 10/29/2007 |
