JPWO2015190439A1 - Screening method for low molecular weight compounds that bind to antibodies - Google Patents

Abstract

An object of the present invention is to provide a method for simply screening in silico a low molecular weight compound capable of binding to an antibody. The method for screening for a low molecular weight compound that binds to an antibody according to the present invention comprises the step of: combining the first binding target site contained in the framework region having amino acid sequence 1 in the variable region of the light chain of the antibody; The step of selecting a compound exhibiting a low docking score in the docking simulation for both of the second binding target sites contained in the framework region having amino acid sequence 2 in the variable region.

Description

  The present invention relates to a method for simply screening in silico a low molecular weight compound capable of binding to an antibody.

  In recent years, antibody drugs are one of medical proteins that have been actively developed. An antibody drug is a drug that uses an antibody as an active ingredient and uses the function of an antibody, and has reduced side effects that a conventional drug has because it works specifically with a target molecule, and High therapeutic effect can be expected. Antibody drugs actually contribute to the improvement of various pathological conditions. On the other hand, since it is administered in a large amount to a living body, it is said that purity has a great influence on quality when compared with other medical proteins.

  Conventionally, the molecular form of immunoglobulin G (IgG) has been mainly used for antibody drugs. However, in recent years, research has been actively conducted on low molecular weight antibodies that eliminate the Fc region as next-generation antibodies that can be produced at low cost. One reason for this is that low-molecular-weight antibodies can reduce production costs compared to IgG antibodies. Specifically, since a sugar chain is generally bound to the Fc region of an antibody, it was necessary to use Chinese hamster ovary cells (CHO cells) for the production of IgG antibodies. However, even when manufactured using CHO cells, the sugar chain of IgG antibody is heterogeneous, and it may cause an antigen-antibody reaction in the human body due to contamination with something different from the sugar chain of human antibody. It was necessary. On the other hand, low-molecular-weight antibodies do not need to consider the sugar chain added to the Fc region, and therefore can be produced inexpensively using Escherichia coli or yeast.

  However, since low molecular weight antibodies do not have the Fc region present in IgG antibodies, affinity chromatography using protein A as a ligand often used for IgG antibody purification cannot be used for purification. Therefore, affinity chromatography columns using a carrier on which a low molecular weight compound having binding properties to the Fab region or Fv region of the antibody is immobilized have been developed for the purification of low molecular weight antibodies (Patent Document 1, Prometic). -"Fabsorbent (trademark)" manufactured by Bioscience. These low molecular weight compounds have a nucleotide binding site existing between the light chain and heavy chain of an antibody and a depression (pocket) between two light chain domains of an antibody as a binding target site (Patent Document 1). Non-patent document 1).

Thus, in recent years, antibody pharmaceuticals that do not have an Fc region and affinity chromatography corresponding thereto have been actively developed. However, in addition to these, the molecular forms of antibody drugs include those using only the variable region of the light chain of the antibody (V _L ) or the variable region of the heavy chain (V _H ), or the variable of the heavy chain of a camel. There are various molecular forms such as those using regions (nanobody), and the development of affinity chromatography using these as target sites is still insufficient.

International Publication No. 2012/0999949 Pamphlet

S. Williams, IBC's BioProcess International Conference & Exhibition (2009)

  As described above, the low molecular weight compounds that have been developed so far for affinity chromatography for purifying low molecular weight antibodies include nucleotide bonds existing between the light chain and heavy chain of the antibody and the two light chains of the antibody. A depression between domains is used as a binding target site. However, various antibody drugs have been developed, and in order to easily purify each antibody, a compound that binds to a carrier used in affinity chromatography for purification and that has a high affinity for the antibody can be easily screened. There is a need for a way to do this.

  Accordingly, an object of the present invention is to provide a method for simply screening in silico a low molecular weight compound capable of binding to an antibody.

  The inventors of the present invention have made extensive studies to solve the above problems. As a result, the specific amino acid sequence of the framework region with relatively few mutations in the variable region of the antibody is conserved in common among the antibodies. By using this sequence, low molecular weight compounds having high affinity for various antibodies can be obtained. The present invention was completed by finding that it can be easily screened in silico.

  Hereinafter, the present invention will be described.

[1] A method for screening a low molecular weight compound that binds to an antibody,
In the variable region of the light chain of the antibody, the first binding target site contained in the framework region having the following amino acid sequence 1, and in the variable region of the heavy chain of the antibody, in the framework region having the amino acid sequence 2 Selecting a compound that exhibits a low docking score in a docking simulation for both of the included second binding target sites.
Amino acid sequence 1: In the amino acid sequence shown in SEQ ID NO: 1, Xaa at the 9th position is serine, phenylalanine, leucine, glycine, alanine or aspartic acid, and Xaa at the 15th position is proline, valine, threonine or leucine Xaa at positions 24 to 31 is any amino acid or deletion, Xaa at position 39 is glutamine, lysine, leucine or glutamic acid, and Xaa at positions 79 and 80 is any amino acid or deletion In the amino acid sequence shown in SEQ ID NO: 2, Xaa at the 9th position is glycine, alanine, proline or serine, Xaa at the 14th position is any amino acid or deletion, Xaa at position 17 is glycine, alanine, serine, threonine, glutami , Glutamic acid, arginine or aspartic acid, Xaa at positions 41 to 43 is any amino acid or deletion, Xaa at position 47 is valine, methionine, isoleucine or leucine, and Xaa at position 50 is valine. , Leucine, phenylalanine or isoleucine, Xaa at position 56 is threonine, arginine, methionine, glutamic acid, lysine, asparagine or aspartic acid, Xaa at position 61 is leucine, alanine, valine or phenylalanine, Amino acid sequence wherein Xaa at position is leucine, methionine, tryptophan or isoleucine, and Xaa at position 66 is serine, arginine, threonine, asparagine, glycine or cysteine

[2] The method according to [1] above, wherein the first binding target site comprises the following amino acid sequence 3 and amino acid sequence 4.
Amino acid sequence 3: In the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence in which the fourth position Xaa is glutamine, lysine, leucine or glutamic acid. Amino acid sequence 4: Amino acid sequence shown in SEQ ID NO: 4

[3] The method according to [1] or [2] above, wherein the second binding target site comprises the following amino acid sequence 5 and amino acid sequence 6.
Amino acid sequence 5: In the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence in which Xaa at position 6 is an arbitrary amino acid or a deletion. Amino acid sequence 6: Amino acid sequence shown in SEQ ID NO: 6

  The screening method according to the present invention involves simple screening in silico of low molecular weight compounds that bind to a carrier of affinity chromatography used for purification of various antibodies, for example, low molecular weight antibodies that do not have an Fc region. Can do. In addition, the low molecular weight compounds screened by the method of the present invention are general-purpose and diverse ones showing affinity for various antibodies, so that they can be used not only for low molecular weight antibodies but also for the industrial production of immunoglobulins. It can also be used. The low molecular weight compound can be used for antibody characterization, identification, quantification and the like in addition to antibody purification. Furthermore, conventionally, when purifying an immunoglobulin containing an Fc region, a low molecular weight compound (number of binding target sites: 2) having a nucleotide binding site existing between a light chain and a heavy chain as a binding target site, A low molecular weight compound (number of binding target sites: 2) having a light chain domain as a target site has been used. That is, the number of binding target sites for the antibody of the conventional low molecular weight compound is 2. On the other hand, the low molecular weight compounds screened by the method of the present invention are present in the heavy chain and light chain constituting the antibody, respectively, and can bind to the antibody at a total of four binding target sites per antibody molecule. . Therefore, it can be said that the low molecular weight compound according to the present invention is very suitable for antibody purification and the like because it can bind to an antibody with higher affinity.

FIG. 1 is a diagram showing the alignment results of the _VL region of the kappa chain of human antibodies. FIG. 2 is a diagram showing alignment results of _VH regions of human antibodies. In FIG. 1 and FIG. 2, “FR” indicates a framework region, and “CDR” indicates a complementary strand determining region.

  A screening method for a low molecular weight compound that binds to an antibody according to the present invention includes a first binding target site contained in a framework region having amino acid sequence 1 in a variable region of a light chain of the antibody, and a heavy chain of the antibody The step of selecting a compound exhibiting a low docking score in the docking simulation for both of the second binding target sites contained in the framework region having amino acid sequence 2 in the variable region.

  In the method of the present invention, a depression (pocket) contained in the framework region is used as a binding target site for which the binding ability of a low molecular compound to an antibody, particularly a human antibody, should be evaluated. The framework region refers to a region with relatively few mutations other than the complementarity determining region (CDR) in direct contact with the antigen in the variable region (V region) which is the tip of the Fab region of the antibody. It is said that the framework region has relatively few amino acid sequence variations among the variable regions, and that the three-dimensional structure is similar between antibodies. In the method of the present invention, a compound having a high affinity for a specific amino acid sequence in the framework region is selected. Therefore, the compound exhibits a relatively high affinity for various antibodies in common.

  In the method of the present invention, a compound having an affinity for a binding target site having a specific amino acid sequence contained in each framework region in both the light chain and the heavy chain is selected.

  The specific amino acid sequence used in the method of the present invention aligns the amino acid sequences of the framework regions of human antibodies, and forms a recess (pocket) that is relatively high in homology and into which a low molecular compound can enter. .

  In the present invention, the term “alignment” refers to pairwise alignment and multiple alignment, and refers to aligning the amino acid sequences of proteins so that corresponding portions are aligned between two or more sequences. Here, “pair-wise alignment” refers to a sequence comparison in which the amino acid sequence to be compared and the amino acid sequence stored in the database are compared one-to-one, and “multiple alignment” is a one-to-three or more sequence comparison. A multiple sequence comparison performed in large numbers. In addition, sequence homology in two or more amino acid sequences and the like is easily calculated by software for alignment.

  In the amino acid sequence used in the method of the present invention, “any amino acid or deletion” represents a so-called “gap” corresponding to insertion / deletion of an amino acid in the course of evolution. As “any amino acid”, 20 kinds of amino acids constituting a protein are preferable. Further, “deletion” indicates a single bond between amino acids before and after the chemical structure of the protein.

  The term “protein” includes any molecule having a polypeptide structure, and also includes fragmented polypeptide chains and two or more polypeptide chains linked by peptide bonds. Accordingly, in the present specification, “peptide”, “polypeptide” and “protein” are used interchangeably. A “domain” is a unit of a protein's higher order structure, consisting of a sequence of tens to hundreds of amino acid residues, and sufficient for expressing any physicochemical or biochemical function. A unit.

  As a result of the alignment, the ratio of the number of matching amino acids divided by the total number of aligned amino acids is called “sequence homology”. It is expected that the higher the sequence homology, the less the mutation and the similar three-dimensional structure. In general, if there is a sequence homology of 50% or more by deletion, addition and / or substitution of one or more amino acids with respect to a certain amino acid sequence, it is expected that the three-dimensional structure is similar. If it is 40% or more, it is said that it can be used as a template of a structure for docking simulation of a low molecular compound (D. Baker & A. Sali, Science, 294, pp. 93-96 (2001)). In the case of antibodies, when the amino acid sequences of the light chain framework regions and heavy chain framework regions are aligned, the sequence homology is as high as 50% or more, and the three-dimensional structures are very similar. On the other hand, when the amino acid sequences of the framework regions of the light chain and heavy chain are aligned, the sequence homology is as low as less than 30%, and generally the three-dimensional structure is not similar. However, the sequence homology between the amino acid sequences of the binding target sites in the light chain and heavy chain framework regions of the human antibody specified in the present invention is as high as 42%, and the three-dimensional structure is likely to be similar. Accordingly, screening with the same low molecular weight compound can be performed on these binding target sites.

  The amino acid sequence of the binding target site in the present invention is preferably the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 4 for the light chain and SEQ ID NO: 5 and SEQ ID NO: 6 for the heavy chain. It is preferably 40% or more, more preferably 45% or more, and even more preferably 50% or more. The amino acid sequence shown in the SEQ ID NO of the binding target site is the amino acid sequence of κ chain, λ chain, and two types of κ chain reported as human antibodies for the light chain. Since the same low molecular weight compound is expected to bind if a binding target site having an amino acid sequence having a high sequence homology of 40% or more with the amino acid sequence of the binding target site of the chain is expected, the screening method in that case It is obvious that the present invention is also included in the present invention. Similarly, the present invention includes a screening method in which a binding target site is specified that increases the sequence homology of the amino acid sequence between the light and heavy chains, even if it is not a human-derived antibody.

  In the present invention, a compound is selected that exhibits a low docking score in a docking simulation for both two binding target sites having a specific amino acid sequence.

  The computer software for performing the docking simulation for screening the low molecular weight compound is not particularly limited, but, for example, software commonly used in in silico drug discovery (http: //www.jbic.or). .Jp / activity / st_pr_pj / mypresto / index_mypr.html).

  In the method of the present invention, a compound docking score is obtained by docking simulation, the compounds are ranked, and a compound showing a low docking score is selected. In the present invention, the “docking score” is a value corresponding to the binding energy between the compound and the binding target site according to the present invention. The smaller the value, the higher the affinity of the compound for the binding target site.

  Specifically, in the docking simulation using the above software sievgene, it is preferable to select a compound having a docking score of less than −2.0 for both the first binding target site and the second binding target site. The docking score is more preferably less than −3.0. On the other hand, if the docking score is too low, it is expected that the antibody used in the docking simulation will be overfitted and the affinity with other antibodies will decrease, so that the docking score is preferably −5.5 or more.

  The screening method according to the present invention is a simple method using a computer, but if an unlimited compound is evaluated, the efficiency is naturally poor. Therefore, it is preferable to select the compounds to be evaluated to some extent in advance. For example, it can be selected from compounds sold by general reagent manufacturers based on solubility and the like.

  The compound selected by the method of the present invention can be immobilized, for example, on a carrier comprising a water-insoluble substrate and used as an affinity ligand. Here, the term “affinity ligand” is a term that refers to a substance or functional group that selectively binds a target molecule from a set of molecules based on the affinity between specific molecules represented by the binding between an antigen and an antibody. In the present invention, it refers to a small molecule compound that specifically binds to a small molecule antibody and an immunoglobulin.

  In the present invention, the term “small molecule antibody” refers to an antibody consisting only of a partial fragment produced from an immunoglobulin by protease degradation or genetic engineering. Typical low molecular weight antibodies include Fab, single chain antibody (scFv), Diabody, and the like. Fab is a molecule in which a light chain and a heavy chain are disulfide-bonded. Molecular weight is about 60 kDa, scFv is a light chain and heavy chain variable region linked by a short linker, molecular weight is about 30 kDa, Diabody is a scFv linker. Is a molecule in which two molecules of scFv are covalently associated by adjusting the length of the protein, and has two antigen-binding sites.

The molecules targeted by the affinity ligand in the present invention are all molecules to which the target molecule-binding domain can bind. For example, the light chain variable region (V _L ) and heavy chain variable region (V _H ) of an antibody. Fragmented low-molecular-weight antibodies, low-molecular-weight antibody derivatives, immunoglobulin G (IgG) with various molecular forms, such as those that use only the domain (domain antibody) and those that use the variable region of the heavy chain of a camel (nanobody) And antibody derivatives. Here, examples of the “small molecule antibody derivative” include an artificial antibody obtained by fusing the Fv region and the Fc region of human IgG, and examples of the “antibody derivative” include a part of the domain of human IgG. Examples include chimeric antibodies that are fused by substituting the domain of an IgG antibody of a biological species, and humanized antibodies that are fused by substituting CDRs of other IgG species (Complementarity-Determining Regions) with the CDRs of an antibody of another species. It is done.

  The affinity for low molecular weight antibodies and immunoglobulins with various molecular forms detects biosensors such as the Biacore system (GE Healthcare) using the surface plasmon resonance principle, and exothermic / endothermic changes caused by binding. It can be evaluated by a biomolecule interaction detection / analysis apparatus such as an isothermal titration calorimeter (ITC).

  The water-insoluble carrier used in the present invention preferably has a large surface area in view of the purpose and method of use of the affinity separation matrix, and is preferably a porous material having a large number of pores of an appropriate size. The form of the carrier can be any of beads, monoliths, fibers, membranes (including hollow fibers), and any form can be selected. Examples of the method for immobilizing a ligand include a method in which a reactive group is introduced into a carrier and a low molecular compound is bound by a known coupling method via a linker group.

  Examples of the reactive group include an amino group, a carboxy group, an ether group, and a thioether group. The reactive group may be introduced by introducing a compound called “spacer”. As a coupling method, for example, the carrier is reacted with cyanogen bromide, epichlorohydrin, diglycidyl ether, tosyl chloride, tresyl chloride, hydrazine, sodium periodate, etc., or the carrier is activated. A method in which a reactive group is introduced on the surface and a low molecular compound to be immobilized as a ligand is coupled and immobilized, or a system in which a low molecular compound to be immobilized as a carrier and a ligand exists is a condensation such as carbodiimide. Examples of the immobilization method include adding a reagent or a reagent having a plurality of functional groups in the molecule such as glutaraldehyde, followed by condensation and crosslinking.

  The low molecular compound of the present invention used as a ligand may be immobilized by chemically modifying the carrier, or a spacer molecule useful for immobilization may be introduced. The essence of the present invention is that the function of the low molecular weight compound is similarly imparted to the matrix in which the low molecular weight compound is immobilized as a ligand, and how the low molecular weight compound is modified / modified for immobilization. And within the scope of the present invention.

  Separation and separation of immunoglobulins, antibody derivatives, low molecular antibodies and low molecular antibody derivatives by affinity chromatography using the above affinity separation matrix in which the low molecular weight compound found by the present invention is immobilized on a water-insoluble carrier. It becomes possible to refine and characterize.

  Purification methods for these antibodies, antibody derivatives, low-molecular-weight antibodies, and low-molecular-weight antibody derivatives can be achieved by procedures according to affinity column / chromatography purification methods using protein A columns that are already available as commercial products. (Roque ACA et al., Journal of Chromatography A, 1160, 44-55 (2007)). That is, an antibody, an antibody derivative, a low molecular antibody, and a buffer containing a low molecular antibody derivative are adjusted to be neutral, and then the solution is passed through an affinity column packed with the affinity separation matrix of the present invention. Adsorbing antibody derivatives, low molecular weight antibodies and low molecular weight antibody derivatives. Next, an appropriate amount of pure buffer is passed through the affinity column, and the inside of the column is washed. At this point, the desired antibody, antibody derivative, low molecular antibody and low molecular antibody derivative are adsorbed to the affinity separation matrix of the present invention in the column. Then, an acidic buffer adjusted to an appropriate pH is passed through the column to elute the desired antibody, antibody derivative, low molecular antibody and low molecular antibody derivative, thereby achieving high purity purification. A substance for promoting dissociation of the desired antibody from the matrix may be added to the acidic buffer used for elution. In the same manner, the antibody, antibody derivative, low molecular antibody and low molecular antibody derivative adsorbed on the affinity separation matrix of the present invention can be characterized as having the binding target site.

  The affinity separation matrix of the present invention should be washed by passing it through a pure buffer having a suitable strong acidity or strong alkalinity to such an extent that the low molecular weight compound as a ligand and the substrate of the carrier do not completely impair the function. Can be reused. An appropriate denaturant or organic solvent may be added to the buffer solution. When the low molecular weight compound of the present invention is used as a ligand, the chemical stability of the ligand is expected to be higher than when a protein such as protein A is used as a ligand. Here, chemical stability means the property of retaining the function of a low molecular compound. In the present invention, “retaining the function of a low molecular weight compound” refers to retaining affinity for a binding target site (pocket) existing in the framework region of the variable region of an immunoglobulin light chain or heavy chain. . That is, the higher the “chemical stability” of the ligand, the less the affinity to the target site decreases even when various chemical treatments are performed.

  This application claims the benefit of priority based on Japanese Patent Application No. 2014-118466 filed on June 9, 2014. The entire content of the specification of Japanese Patent Application No. 2014-118466 filed on June 9, 2014 is incorporated herein by reference.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited by the following examples, but may be appropriately modified within a range that can meet the purpose described above and below. Of course, it is possible to implement them, and they are all included in the technical scope of the present invention.

Example 1
Results of alignment of representative amino acid sequences of known human antibody light chain (κ chain) and heavy chain (http://www2.mrc-lmb.cam.ac.uk/vbase/alignments2.php; FIG. 1 and 2), amino acid sequences having high sequence homology in each chain were extracted and used as common amino acid sequences. Alignment of the amino acid sequences of the framework regions of the common amino acid sequences of the obtained light and heavy chains was performed, and the sequence of SEQ ID NO: 1 as the sequence common to the light chain and SEQ ID NO: 2 as the sequence common to the heavy chain Derived. Furthermore, in each of the sequences of SEQ ID NO: 1 and SEQ ID NO: 2, sequences that are exposed on the surface due to the three-dimensional structure of the antibody and that have a relatively high sequence homology were extracted. The sequences in the light chain are shown in SEQ ID NO: 3 and SEQ ID NO: 4, and the sequences in the heavy chain are shown in SEQ ID NO: 5 and SEQ ID NO: 6. The sequence homology between the entire sequence including SEQ ID NO: 3 and SEQ ID NO: 4 and the entire sequence including SEQ ID NO: 5 and SEQ ID NO: 6 was 42%.

  From the data registered in PDB (Protein Data Bank, http://pdbj.org), which is a database of known human antibodies whose steric structure has been clarified, the above common amino acid sequence (SEQ ID NO: 1 and sequence) Those having an amino acid sequence having a sequence homology of 50% or more with No. 2) in the framework region were searched, and the three-dimensional structure of a human antibody (PDB code: 2xa8) was downloaded from the extracted one. The sequence homology between the amino acid sequence of the light chain framework region of the human antibody 2xa8 and SEQ ID NO: 1 was 94%, and the sequence homology between the amino acid sequence of the heavy chain framework region and SEQ ID NO: 2 was 82%. It was. Look at the three-dimensional structure of the framework region of the light and heavy chains, search for cavities (pockets) to which low molecular weight compounds can bind, and sequence homology in alignment of common amino acid sequences of the framework regions of the light and heavy chains Was selected as a binding target site, which contains an amino acid sequence corresponding to an amino acid sequence as high as 42% (light chain: SEQ ID NO: 3 and SEQ ID NO: 4, heavy chain: SEQ ID NO: 5 and SEQ ID NO: 6). At this time, the sequence homology between the amino acid sequence of the binding target site in the framework region of the light chain of the selected human antibody 2xa8 and the common amino acid sequence (SEQ ID NOs: 3 and 4) in the binding target site of the framework region of the light chain The sequence homology between the amino acid sequence of the binding target site in the heavy chain framework region and the common amino acid sequence (SEQ ID NOs: 5 and 6) in the binding target site of the heavy chain framework region was 100%. It was. In addition, the sequence homology between the amino acid sequences of the light chain and heavy chain binding target sites in human antibody 2xa8 was 50%.

Docking simulation is performed for each of the two binding target sites of human antibody 2xa8 using docking simulation software sievgene (http://www.jbic.or.jp/activity/st_pr_pj/mypresto/index_mypr.html) did. In order to limit the number of compounds by using a stock library (http://www.namiki-s.co.jp/service/screening) of Namiki Shoji Co., Ltd. as a compound library, the following five conditions were selected as selection conditions. Determined.
1. 1. An easily available compound made by ENAMINE 2. Molecular weight is 250 or more. 3. The number of hydrogen bond donors is 5 or less and the number of hydrogen bond acceptors is 10 or less. Water soluble (less than clogP5)
5). Excluding highly reactive structures

  A docking simulation was conducted using 1.24 million selected compounds. The compounds were ranked according to the obtained docking scores, and the top 10,000 docking scores were selected for each binding target site. Table 1 shows the docking scores and compound number distributions for the light chain and heavy chain target sites. In Table 1, the docking score corresponds to the binding energy between the compound and the binding target site, and the smaller the value, the higher the affinity with the binding target site.

  The average docking score for both light chain and heavy chain binding target sites is within the top 10% out of a total of 20000 compounds with low docking score values for light or heavy chain binding target sites Were selected as candidate compounds. Their chemical structures are shown below.

Example 2
From the data registered in PDB (http://pdbj.org), which is a database of known human antibodies whose steric structure has been clarified, the above common amino acid sequence ( Search for those having 50% or more of amino acid sequence in the framework region between SEQ ID NO: 1 and SEQ ID NO: 2), and extract the three-dimensional structure of the human antibody (PDB code: 2hwz) from the extracted one downloaded. The sequence homology between the amino acid sequence of the light chain framework region of the human antibody 2hwz and SEQ ID NO: 1 was 91%, and the sequence homology between the amino acid sequence of the heavy chain framework region and SEQ ID NO: 2 was 57%. It was. In the human antibody 2hwz, a depression (pocket) corresponding to the binding target site selected in Example 1 was selected as the binding target site. At this time, the sequence homology between the amino acid sequence of the binding target site in the framework region of the light chain of the selected human antibody 2hwz and the common amino acid sequence (SEQ ID NOs: 3 and 4) in the binding target site of the framework region of the light chain 83%, the sequence homology between the amino acid sequence of the binding target site in the framework region of the heavy chain and the common amino acid sequence (SEQ ID NO: 5 and 6) in the binding target site of the framework region of the heavy chain was 58%. It was. In addition, the sequence homology between the amino acid sequences of the light chain and heavy chain binding target sites in human antibody 2hwz was 58%.

  For each of the two binding target sites of human antibody 2hwz, docking simulations were performed using 10,000 compounds with the highest docking score for each of the two binding target sites of human antibody 2xa8 in Example 1 above. Table 2 shows the distribution of the docking score and the number of compounds with respect to the binding target site of each of the light and heavy chains.

  The docking scores of the five compounds finally selected in Example 1 above to the binding target sites of the light chain and heavy chain of human antibody 2hwz were examined. The results are shown in Table 3. In Table 3, compound names beginning with “NS-” are code numbers assigned to each compound by Namiki Shoji Co., Ltd., and “p1_2xa8”, “p2_2xa8”, “p1_2hwz” and “p2_2hwz” are human antibody 2xa8 and 2 hwz shows the docking score of each compound for each binding target site of light chain and heavy chain, “avg — 2xa8” and “avg — 2hwz” mean the docking score of each compound for each binding target site of human antibodies 2xa8 and 2 hwz, respectively Indicates.

  The results shown in Table 3 confirmed that the five compounds with an average docking score for the 2xa8 binding target site were in the top 10% also had an average docking score for the 2 hwz binding target site in the top 10%. This result is considered to result from the fact that the structure of the binding target site according to the present invention is similar between the light chain and the heavy chain among human antibodies.

Comparative Example 1
An example of a compound that gives a low docking score to only one of the two binding target sites of the light and heavy chains is shown in Table 4. In Table 4, “N / A” indicates that there is no score value because the docking scores for the binding target sites of the light and heavy chains were not included in the top 10,000.

  As shown in Table 4, the compound having an ID of NS-01599272 gave a good score for the light chain binding target site, but did not give a good score for the heavy chain target site. On the other hand, the compound with ID NS-02992114 gave a good score for the binding target site of the heavy chain, but did not give a good score for the light chain target site. Therefore, these compounds are considered to have a lower affinity for human antibodies than the compounds selected in Examples 1 and 2 above.

Claims (3)

A method of screening for low molecular weight compounds that bind to an antibody,
In the variable region of the light chain of the antibody, the first binding target site contained in the framework region having the following amino acid sequence 1, and in the variable region of the heavy chain of the antibody, in the framework region having the amino acid sequence 2 Selecting a compound that exhibits a low docking score in a docking simulation for both of the included second binding target sites.
Amino acid sequence 1: In the amino acid sequence shown in SEQ ID NO: 1, Xaa at the 9th position is serine, phenylalanine, leucine, glycine, alanine or aspartic acid, and Xaa at the 15th position is proline, valine, threonine or leucine Xaa at positions 24 to 31 is any amino acid or deletion, Xaa at position 39 is glutamine, lysine, leucine or glutamic acid, and Xaa at positions 79 and 80 is any amino acid or deletion In the amino acid sequence shown in SEQ ID NO: 2, Xaa at the 9th position is glycine, alanine, proline or serine, Xaa at the 14th position is any amino acid or deletion, Xaa at position 17 is glycine, alanine, serine, threonine, glutami , Glutamic acid, arginine or aspartic acid, Xaa at positions 41 to 43 is any amino acid or deletion, Xaa at position 47 is valine, methionine, isoleucine or leucine, and Xaa at position 50 is valine. , Leucine, phenylalanine or isoleucine, Xaa at position 56 is threonine, arginine, methionine, glutamic acid, lysine, asparagine or aspartic acid, Xaa at position 61 is leucine, alanine, valine or phenylalanine, Amino acid sequence wherein Xaa at position is leucine, methionine, tryptophan or isoleucine, and Xaa at position 66 is serine, arginine, threonine, asparagine, glycine or cysteine The method according to claim 1, wherein the first binding target site comprises the following amino acid sequence 3 and amino acid sequence 4.
Amino acid sequence 3: In the amino acid sequence shown in SEQ ID NO: 3, the amino acid sequence in which the fourth position Xaa is glutamine, lysine, leucine or glutamic acid. Amino acid sequence 4: Amino acid sequence shown in SEQ ID NO: 4 The method according to claim 1 or 2, wherein the second binding target site comprises the following amino acid sequence 5 and amino acid sequence 6.
Amino acid sequence 5: In the amino acid sequence shown in SEQ ID NO: 5, the amino acid sequence in which Xaa at position 6 is an arbitrary amino acid or a deletion. Amino acid sequence 6: Amino acid sequence shown in SEQ ID NO: 6