JP2007202443A - Antibody enzyme to n-terminal region of chemokine receptor ccr5 - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antibody enzyme eliminating the function of a chemokine receptor CCR which is a coreceptor of an AIDS virus by degrading the chemokine receptor CCR5, and utilized for preventing the infection with the AIDS virus or treating the AIDS; and to provide an anti-HIV medicament utilizing the antibody enzyme. <P>SOLUTION: A monoclonal antibody is produced by using a peptide constituting the N-terminal region of the chemokine receptor CCR5 as an immunogen, and amino acid sequences and base sequences of variable regions of the heavy chain and the light chain of the monoclonal antibody are determined. The three-dimensional structure is estimated and the presence or absence of a triadic structure of catalyst residues is confirmed by carrying out molecular modeling as the amino acid sequence. Finally, it is found that the variable region of the light chain of the monoclonal antibody (5A2, 6A2, 2B8) having the triadic structure of the catalyst residues functions as the antibody enzyme. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an antibody enzyme against the N-terminal region of chemokine receptor CCR5, which is a co-receptor for AIDS virus (HIV), a gene encoding the same, a transformant into which the gene has been introduced, and an anti-HIV drug using them Is.

  AIDS (acquired immunodeficiency syndrome) is caused by infection with human immunodeficiency virus (HIV), the causative virus, resulting in opportunistic infections, malignant tumors, neurological symptoms, dementia, etc. It is a complicated condition. Infection with HIV progresses to AIDS through the acute phase, asymptomatic phase, and AIDS-related syndromes. Due to advances in HIV-related research and treatment methods, AIDS is becoming a treatable disease from an incurable disease, but at present, there is no reliable anti-HIV drug and no treatment with drugs has been established. is there.

  In 1984, CD4 was identified as a receptor for HIV in a study on the mechanism of HIV infection. HIV infection begins with the binding of the envelope protein (gp120) on the surface of the viral particle to the CD4 molecule expressed on the surface of human cells.

  However, CD4 alone does not cause fusion between the viral envelope and the cell membrane of the host cell, and HIV infection does not occur. Therefore, human cell membrane components other than CD4 molecules may act as cofactors for virus entry. The existence of a co-receptor (second receptor) was suggested. After more than 10 years from the discovery of CD4, the chemokine receptor CCR5 was discovered as one of the HIV co-receptors (see Non-Patent Document 1).

  The first step of HIV infection is the adsorption and invasion of helper T cells, macrophages and dendritic cells present in the blood and submucosa. The above described entry of HIV into the cell is caused by a multistep reaction between the viral envelope protein and the membrane protein of the target (host) cell. Details of the entry of the HIV into the target cell are as follows. First, the envelope protein (gp120) on the HIV particle surface binds to CD4 on the target cell surface. This gp120 binding to CD4 causes a structural (conformational) change in gp120, releasing the previously masked gp120 V3 loop region and allowing it to become a coreceptor (such as CCR5) on the target cell surface. Join. At this time, it is considered that the interaction between the V3 loop region and the co-receptor involves several extracellular regions including the amino-terminal region (N-terminal region) of the co-receptor. This interaction between the V3 loop region and the co-receptor further causes a structural change in the extracellular domain of the transmembrane protein gp41 of the HIV envelope protein, whereby the highly hydrophobic N-terminal part of gp41 is converted to lipid bilayers on the surface of the target cell membrane. It is considered that the protein is inserted into the stratification, and this triggers membrane fusion between the virus surface membrane and the target cell membrane, and the virus enters the cell (see Non-Patent Document 2).

  Subsequent studies have reported that when the chemokine receptor CCR5 is not normal in target cells, it is resistant to HIV infection and development (see Non-Patent Document 3). Therefore, many researchers and companies are now working on the development of drugs that target this co-receptor and inhibit HIV entry. The current state of research and development of such an anti-HIV drug targeting the HIV co-receptor is described in Non-Patent Document 4 shown below, for example. Non-Patent Document 4 reports a chemokine receptor antagonist as a candidate of an anti-HIV drug capable of preventing HIV infection by targeting the chemokine receptor CCR5.

However, anti-HIV drug candidates that have been studied so far are antagonists of the chemokine receptor CCR5 as described above, and no effective drug that can directly attack the chemokine receptor CCR5 and lose its function is proposed. Was not. Accordingly, the present inventors have started the development of an antibody enzyme that can degrade the chemokine receptor CCR5 to eliminate its function, and can be used for HIV infection prevention and AIDS treatment. As a result, the extracellular region of the chemokine receptor CCR5 is obtained from a monoclonal antibody obtained using the peptide constituting the extracellular region of the chemokine receptor CCR5, RSSHFPYSQYQFWKNFQTLK (SEQ ID NO: 13), or RSQKEGLHYTCS (SEQ ID NO: 14) as an immunogen. The antibody enzyme which can decompose | disassemble was discovered (refer patent document 1).
Samson, M., Libert, F., Doranz, B, J., et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. (1996) Nature.382: 722- 725. Lee, B., Sharron, M., Blancpain, C., et al. Epitope Mapping or CCR5 Reveals Multiple Conformational States and Distinct but Overlapping Structures Involved in Chemokine and Coreceptor Function. (1999) J. Biol. Chem. 274: 9617 -9626 "Kenmokine handbook" Osamu Yoshie, Naoyuki Nomiyama, supervised by Masayuki Miyasaka (Shujunsha), published on November 20, 200 Tsutomu Murakami, Naoki Yamamoto: Protein Nucleic Acid Enzyme, Vol. 43, pp. 677-685 (1998), “Development of New Anti-HIV Drugs—Drugs that Act on HIV Co-receptors (Second Receptors)” JP 2004-313166 A (publication date: November 11, 2004 (2004))

  However, as described above, there are several extracellular steps including the amino terminal region (N-terminal region) of the co-receptor for the binding of the V3 loop region and the co-receptor, which is an important step for HIV entry. The region is considered to be involved, and it was thought that the antibody enzyme described in Patent Document 1 alone may not sufficiently prevent and prevent HIV infection.

  It has also been reported that the N-terminal region of the CCR5 molecule is important for the binding of the HIV envelope protein and CCR5 (see Non-Patent Document 3).

  Accordingly, an object of the present invention is to provide an antibody enzyme that targets the N-terminal region of the chemokine receptor CCR5 and can eliminate the function of the chemokine receptor CCR5 and can be used for prevention of AIDS virus infection and treatment of AIDS. It was. Another object of the present invention is to provide a gene encoding the antibody enzyme, a transformant into which the gene has been introduced, and an anti-HIV drug using them.

  In order to solve the above problems, the inventors of the present application focused on an antibody enzyme that has an enzymatic action while being an antibody and can completely degrade the target protein, and an antibody against the N-terminal region of the chemokine receptor CCR5 Intensive study was conducted to obtain the enzyme. As a result, an antibody enzyme capable of specifically binding to the N-terminal region of the chemokine receptor CCR5 and decomposing the chemokine receptor CCR5 from a monoclonal antibody obtained using the N-terminal region of the chemokine receptor CCR5 as an immunogen was found. The invention has been completed.

  That is, the antibody enzyme according to the present invention is an antibody or antibody fragment against the N-terminal region of the human-derived chemokine receptor CCR5, specifically binds to the N-terminal region of the chemokine receptor CCR5, and degrades the chemokine receptor CCR5. It has the activity to do.

  Here, the “antibody fragment” means each peptide chain constituting an antibody having a peptide in the N-terminal region of the chemokine receptor CCR5 as an antigen, and further a peptide fragment in a partial region within each peptide chain. . The “antibody enzyme” is an immunoglobulin having an antigen-antibody reaction specifically with a target antigen and having enzyme activity. The antibody enzyme is not limited to a complete antibody molecule, and is an antibody fragment (for example, a light chain, heavy chain, light chain variable region) that can specifically bind to an antigen and has enzyme activity. , Heavy chain variable region, etc.). The enzyme activity is not particularly limited, but is preferably protease activity or peptidase activity.

  In particular, among the above-mentioned antibody enzymes, those exhibiting high degradation activity targeting the antigen protein are called “super antibody enzymes”. The above-mentioned “super antibody enzyme” is capable of completely degrading a target protein and has an activity close to that of a natural enzyme (Super Catalytic Antibody [I]: Decomposition of targeted protein by its antibody light chain. Hifumi, E , Okamoto, Y., Uda, T., J. Biosci. Bioeng., 88 (3), 323-327 (1999)). The antibody enzyme according to the present invention is an antibody having a peptide in the N-terminal region of the chemokine receptor CCR5 as an antigen, and is presumed to be capable of completely degrading the CCR5, and thus is included in “super antibody enzyme”.

  The antibody enzyme according to the present invention has the property of decomposing the N-terminal region of the chemokine receptor CCR5, which is its antigen, in a state in which the properties of the N-terminal region of the chemokine receptor CCR5 remain dark. Therefore, the above antibody enzyme has high specificity and can specifically degrade only the chemokine receptor CCR5. Since the chemokine receptor CCR5, particularly its N-terminal region, plays an important role in HIV infection as described above, the antibody enzyme capable of eliminating it is used for the prevention of HIV infection and the treatment of AIDS. It can be effectively used as an anti-HIV drug.

  The antibody enzyme according to the present invention preferably comprises a variable region of an antibody against the N-terminus of the chemokine receptor CCR5. This “variable region” refers to a portion consisting of about 110 amino acids from the N-terminus of the heavy chain (H chain) and light chain (L chain) constituting the antibody. The variable region has a variety of primary structures depending on the type of antibody, and when the antibody has an activity as an enzyme, it is highly likely that the active center is included. Therefore, if the antibody enzyme includes the variable region of an antibody, it can have high activity as an enzyme.

  The antibody enzyme according to the present invention preferably has a catalytic triad residue structure. Here, the “catalytic triad residue structure” refers to a structure presumed that at least three amino acid residues including serine are included in the active site to form an active center. A protease having this catalytic triad residue structure is called serine protease because serine is contained in the active site. Therefore, it can be said that the antibody enzyme is a kind of serine protease. If it has a structure presumed to be a catalyst triad residue, it can be predicted that it has high activity as a protease. As a result of detailed analysis of the properties and structural features of the mouse-derived antibody enzyme having an activity of cleaving and / or degrading peptides and antigen proteins, the inventors have cleaved and / or degraded peptides and antigen proteins. All of the antibody enzymes having the activity to clarify that serine residues, aspartic acid residues, histidine residues or glutamic acid residues are present in close proximity in the three-dimensional structure ( For example, refer to Japanese Unexamined Patent Application Publication No. 2004-97211 (published on April 2, 2004). Here, “existing in a three-dimensional structure” means that the distance between the serine residue, aspartic acid residue, histidine residue or glutamic acid residue is within the range of at least 3 to 20 mm, preferably It means that it exists in the range of 3-10cm.

  Specifically, as the antibody enzyme according to the present invention, one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1 or the amino acid sequence shown in SEQ ID NO: 1. And those comprising the amino acid sequence thus prepared.

  The amino acid sequence shown in SEQ ID NO: 1 is the variable region of the light chain (L chain) of monoclonal antibody “5A2” against the N-terminal region of chemokine receptor CCR5. The variable region of the 5A2 L chain specifically binds to the N-terminal region of the chemokine receptor CCR5 and has the degradation activity of the chemokine receptor CCR5, as shown in Examples described later.

  As another example of the antibody enzyme according to the present invention, one or several amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5. And those comprising an amino acid sequence.

  The amino acid sequence shown in SEQ ID NO: 5 is the variable region of the light chain (L chain) of the monoclonal antibody “6A2” of the chemokine receptor CCR5. The variable region of the 6A2 L chain also specifically binds to the N-terminal region of the chemokine receptor CCR5 and has the degradation activity of the chemokine receptor CCR5, as shown in the Examples described later.

  Furthermore, as another example of the antibody enzyme according to the present invention, one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 9 or the amino acid sequence shown in SEQ ID NO: 9. And those comprising an amino acid sequence.

  The amino acid sequence shown in SEQ ID NO: 9 is the variable region of the light chain (L chain) of the monoclonal antibody “2B8” of the chemokine receptor CCR5. The variable region of the L chain of 2B8 also specifically binds to the N-terminal region of the chemokine receptor CCR5 and has the degradation activity of the chemokine receptor CCR5, as shown in the examples described later.

  The above “one or several amino acids are substituted, deleted, inserted, or added” means substitution, deletion, insertion, or by a known mutant polypeptide production method such as site-directed mutagenesis. It means that as many amino acids as can be added (preferably 10 or less, more preferably 7 or less, most preferably 5 or less) are substituted, deleted, inserted or added. Such a mutant polypeptide is not limited to a polypeptide having a mutation artificially introduced by a known mutant polypeptide production method, and is a product obtained by isolating and purifying a naturally occurring polypeptide. Also good. Preferred mutations are conservative or non-conservative amino acid substitutions, deletions, or additions. More preferred are silent substitutions, additions and deletions, and particularly preferred are conservative substitutions. Thus, when the genetic engineering technique is used, one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in any one of SEQ ID NOs: 1, 5, and 9. In other words, the antibody enzyme comprising the amino acid sequence is a variant of the antibody enzyme comprising the amino acid sequence represented by any of SEQ ID NOs: 1, 5, and 9.

  The antibody enzyme according to the present invention specifically binds to the N-terminal region of the chemokine receptor CCR5, which plays an important role in HIV infection, and degrades the chemokine receptor CCR5 to eliminate its function. It can also be used as an inhibitory anti-HIV drug. That is, the anti-HIV drug according to the present invention comprises the above antibody enzyme.

  The anti-HIV drug according to the present invention can inhibit HIV infection. In addition, the anti-HIV drug according to the present invention can delay the onset of AIDS or prevent the progression of the disease in HIV-infected patients. That is, the anti-HIV drug according to the present invention can also function as a therapeutic drug for HIV-infected patients.

  The anti-HIV drug according to the present invention contains only the antibody enzyme according to the present invention and can be directly administered by intravenous injection or the like, but in addition to the antibody enzyme, pharmacologically It may further contain an acceptable carrier. Such an anti-HIV drug can be produced by a conventionally known production method. Since this anti-HIV drug specifically degrades only the N-terminal region of the chemokine receptor CCR5, it can be expected not only that the anti-HIV effect is remarkable but also that there are few side effects.

  The present invention also includes a gene encoding the antibody enzyme according to the present invention. If this gene is introduced so that it can be expressed in a suitable host (eg, bacteria, yeast), the antibody enzyme according to the present invention can be expressed in that host.

  The “gene” includes not only double-stranded DNA but also single-stranded DNA and RNA such as sense strand and antisense strand constituting the DNA. Furthermore, the “gene” may include sequences such as untranslated region (UTR) sequences and vector sequences (including expression vector sequences) in addition to the sequence encoding the antibody enzyme of the present invention.

  Specific examples of the gene according to the present invention include a gene consisting of the base sequence shown in SEQ ID NO: 2. The base sequence shown in SEQ ID NO: 2 is the base sequence of the gene encoding the variable region of the light chain (L chain) of monoclonal antibody 5A2 of chemokine receptor CCR5.

  Another example of the gene according to the present invention is a gene consisting of the base sequence shown in SEQ ID NO: 6. The base sequence shown in SEQ ID NO: 6 is the base sequence of the gene encoding the variable region of the 6A2 light chain (L chain) of the chemokine receptor CCR5 monoclonal antibody.

  Furthermore, as another example of the gene according to the present invention, a gene comprising the base sequence shown in SEQ ID NO: 10 can be mentioned. The base sequence shown in SEQ ID NO: 10 is the base sequence of the gene encoding the variable region of the light chain (L chain) of monoclonal antibody 2B8 of chemokine receptor CCR5.

  Furthermore, the present invention includes a transformant introduced with the above gene. This transformant is one in which the above gene is introduced so that it can be expressed in a suitable host (eg, bacteria, yeast), and the antibody enzyme according to the present invention can be expressed in its own body. Can be used for manufacturing. In addition, since the transformant accumulates the antibody enzyme according to the present invention in its own body, the transformant itself is also used as an anti-HIV drug for preventing HIV infection or an anti-HIV drug for AIDS treatment. Can be used.

  Since the antibody enzyme according to the present invention specifically binds to the N-terminal region of the chemokine receptor CCR5, which plays an important role in HIV infection, and can degrade the chemokine receptor, prevention of HIV infection, It can be effectively used as an anti-HIV drug used for the treatment of AIDS.

  Therefore, the present invention has an effect that it can contribute to prevention of HIV infection and AIDS treatment.

  The present invention will be described more specifically below, but the present invention is not limited to this description.

(1) Antibody Enzyme and Gene According to the Present Invention Here, as an example of the antibody enzyme according to the present invention, antibody fragments of monoclonal antibodies “5A2”, “6A2” and “2B8” against the N-terminal region of the chemokine receptor CCR5 Will be described as an example. In the following description, “monoclonal antibody 5A2 against the N-terminal region of chemokine receptor CCR5” is referred to as “5A2”, “monoclonal antibody 6A2 against the N-terminal region of chemokine receptor CCR5” as “6A2”, “ The “monoclonal antibody 2B8 against the N-terminal region of chemokine receptor CCR5” is referred to as “2B2.”

  More specifically, the antibody fragments of 5A2, 6A2, and 2B2 include the variable region of 5A2 light chain (hereinafter referred to as “5A2Lv”), the variable region of 6A2 light chain (hereinafter referred to as “6A2Lv”), and 2B8. It is the variable region of the light chain (hereinafter referred to as “2B8Lv”).

  The above three antibody enzymes are obtained from a monoclonal antibody obtained using a peptide (MDYQVSSPIYDINYYTSEPQQINVKQIAARLL: SEQ ID NO: 15) constituting the N-terminal region of the chemokine receptor CCR5 as an immunogen. As a result of molecular modeling of the amino acid sequence of the variable region of the monoclonal antibody, the antibody enzyme is an amino acid capable of constituting a catalytic triad residue consisting of serine, histidine (or glutamic acid), and aspartic acid. It was found as a sequence (antibody fragment).

  The antibody enzymes 5A2Lv, 6A2Lv, and 2B8Lv according to the present invention specifically bind to the N-terminal region of the chemokine receptor CCR5 and act as a degrading enzyme of the chemokine receptor CCR5. This is clear from the results shown in the examples described later.

  Here, the chemokine receptor CCR5 (hereinafter referred to as “CCR5”) will be briefly described.

  CCR5 is a co-receptor for AIDS virus (HIV) and is thought to bring about a conformational change in the HIV envelope and lead to membrane fusion between the virus and the host cell. Therefore, it is an important factor when HIV infects humans. It is. It is known that a human having a deficient mutant CCR5 gene is resistant to HIV infection and development.

  CCR5 is also the only one used as a co-receptor in the infection of HIV macrophage-directed strains involved in human-to-human infection. From this, it is expected that suppressing the function of CCR5 will lead to prevention of HIV infection and prevention of AIDS progression.

  The above-mentioned three antibody enzymes specifically bind to the CCR5, particularly the N-terminal region of CCR5, and can degrade CCR5 and abolish the function of CCR5. In humans having a deficient mutant CCR5 gene, there is no apparent immune deficiency or tissue lesion, and no health problems are seen. Therefore, the antibody enzyme that eliminates the function of CCR5 prevents HIV infection, It can be effectively used as an anti-HIV drug that suppresses the progression of AIDS symptoms.

  Here, the “N-terminal region” of CCR5 is one of the extracellular regions of CCR5, and is known to be important for the binding of HIV envelope protein and CCR5. Such an N-terminal region refers to a region from the N-terminal to the 30th position of the amino acid sequence of CCR5. In addition, the amino acid and base sequence information of human-derived CCR5 can be obtained from a known database or the like. For example, the amino acid sequence of human-derived CCR5 is shown in SEQ ID NO: 18, respectively.

  Next, the structure of 5A2Lv will be described in detail below. 5A2Lv is the variable region of the light chain of monoclonal antibody 5A2 that uses the peptide constituting the N-terminal region of CCR5 (referred to as “N-terminal peptide”) as an immunogen, and has the amino acid sequence shown in SEQ ID NO: 1 as the primary structure. ing.

  FIG. 1 schematically shows a three-dimensional structure estimated as a result of three-dimensional structure modeling (molecular modeling) of the variable region of 5A2 composed of a light chain and a heavy chain. As shown in FIG. 1, in the amino acid sequence shown in SEQ ID NO: 1, the 39th histidine (referred to as “His39” in FIG. 1), the 57th serine (referred to as “Ser57” in FIG. 1), the 75th 1st aspartic acid (referred to as “Asp75” in FIG. 1), 94th serine (referred to as “Ser94” in FIG. 1), and 96th serine (referred to as “Ser96” in FIG. 1) It is presumed that it constitutes a group. As shown in FIG. 2, the first aspartic acid (referred to as “Asp1” in FIG. 2), the 96th serine (referred to as “Ser96” in FIG. 2), and the 98th histidine (refer to FIG. 2). In this case, it is assumed that “His98” also constitutes a catalytic triad residue.

  The antibody enzyme according to the present invention comprises an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 1, that is, the 5A2Lv It may be a mutant that acts as a degrading enzyme for the N-terminal region of CCR5. Further, the antibody enzyme may be one in which the remaining amino sequence of the 5A2 L chain is appropriately added to the C-terminal side of the amino acid sequence shown in SEQ ID NO: 1. The range of “one or several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 3 means.

  The variable region of the heavy chain of 5A2 (hereinafter referred to as “5A2Hv”) has the amino acid sequence shown in SEQ ID NO: 3 as a primary structure, but this 5A2Hv has a structure presumed to be a catalytic triad residue. Did not exist. The base sequence of the gene encoding this 5A2Hv is shown in SEQ ID NO: 4.

  Next, the 6A2Lv structure will be described in detail below. 6A2Lv is the variable region of the light chain of monoclonal antibody 6A2 using the N-terminal peptide of CCR5 as an immunogen, and has the amino acid sequence shown in SEQ ID NO: 5 as the primary structure.

  FIG. 3 schematically shows the estimated three-dimensional structure as a result of the three-dimensional structure modeling (molecular modeling) of the 6A2 variable region composed of the light chain and the heavy chain. As shown in FIG. 3, in the amino acid sequence shown in SEQ ID NO: 5, the seventh serine (denoted as “Ser7” in FIG. 3), the 70th aspartic acid (denoted as “Asp70” in FIG. 3), the It is presumed that the 85th aspartic acid (referred to as “Asp85” in FIG. 3) and the eighth histidine (referred to as “His8” in FIG. 3) constitute a catalytic triad residue.

  The antibody enzyme of the present invention comprises an amino acid sequence in which one or more amino acids are substituted, deleted, inserted and / or added in the amino acid sequence shown in SEQ ID NO: 5, ie, the 6A2Lv mutation described above. And may act as a degrading enzyme for the N-terminal region of CCR5. Further, the antibody enzyme may be one in which the remaining amino sequence of the 6A2 L chain is appropriately added to the C-terminal side of the amino acid sequence shown in SEQ ID NO: 5. The range of “one or several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 3 means.

  Note that the variable region of the heavy chain of 6A2 (hereinafter referred to as “6A2Hv”) has the amino acid sequence shown in SEQ ID NO: 7 as a primary structure, but this 6A2Hv has a structure presumed to be a catalytic triad residue. Did not exist. The base sequence of the gene encoding 6A2Hv is shown in SEQ ID NO: 8.

  Next, the structure of 2B8Lv will be described in detail below. 2B8Lv is the variable region of the light chain of monoclonal antibody 2B8 using the N-terminal peptide of CCR5 as an immunogen, and has the amino acid sequence shown in SEQ ID NO: 9 as the primary structure.

  FIG. 4 schematically shows a three-dimensional structure estimated as a result of three-dimensional structure modeling (molecular modeling) of the variable region of 2B8 composed of a light chain and a heavy chain. As shown in FIG. 4, in the amino acid sequence shown in SEQ ID NO: 9, the first aspartic acid (referred to as “Asp1” in FIG. 4), the 28th serine (referred to as “Ser28” in FIG. 4), the first It is speculated that the 31st aspartic acid (referred to as “Asp31” in FIG. 4) and the 98th histidine (referred to as “His98” in FIG. 4) constitute the catalytic triad residue.

  The antibody enzyme of the present invention comprises an amino acid sequence in which one or more amino acids are substituted, deleted, inserted, and / or added in the amino acid sequence shown in SEQ ID NO: 9, that is, the 2B8Lv mutation described above. And may act as a degrading enzyme for the N-terminal region of CCR5. Furthermore, the antibody enzyme may be one in which the remaining amino sequence of the 2B8 L chain is appropriately added to the C-terminal side of the amino acid sequence shown in SEQ ID NO: 9. The range of “one or several” is not particularly limited. For example, 1 to 20, preferably 1 to 10, more preferably 1 to 7, more preferably 1 to 5, and particularly preferably 1 to 3 means.

  The variable region of the heavy chain of 2B8 (hereinafter referred to as “2B8Hv”) has the amino acid sequence shown in SEQ ID NO: 11 as a primary structure, but this 2B8Hv has a structure presumed to be a catalytic triad residue. Did not exist. The base sequence of the gene encoding 2B8Hv is shown in SEQ ID NO: 12.

  The gene according to the present invention is a gene encoding the above antibody enzyme. More specifically, the gene consisting of the base sequence shown in SEQ ID NO: 2, the base sequence shown in SEQ ID NO: 6, or SEQ ID NO: 10 The thing which consists of a base sequence to show can be mentioned. The gene consisting of the base sequence shown in SEQ ID NO: 2 is one of the base sequences of the gene encoding 5A2Lv, and the gene consisting of the base sequence shown in SEQ ID NO: 6 is one of the base sequences of the gene encoding 6A2Lv. The gene consisting of the base sequence shown in SEQ ID NO: 10 is one of the base sequences of the gene encoding 2B8Lv. The gene is not necessarily the same as the nucleotide sequence shown in SEQ ID NO: 2, 6, or 10, and any variant is included as long as it is a gene encoding the antibody enzyme or fragment thereof according to the present invention. . Examples of such mutants include mutants in which one or more bases are deleted, substituted, or added in the base sequence of the gene encoding the antibody enzyme or fragment thereof.

(2) Regarding the method for obtaining the antibody enzyme according to the present invention The antibody enzyme according to the present invention includes, for example, spleen cells of immunized animals such as mice immunized with a peptide constituting the N-terminal region of CCR5 (N-terminal peptide); It can be produced by producing a monoclonal antibody using a hybridoma obtained by fusing a fusion partner such as mouse myeloma cell. When obtaining the heavy chain or light chain of a monoclonal antibody, the obtained monoclonal antibody may be separated into a heavy chain and a light chain. When obtaining the antibody fragment of the present invention, the relevant monoclonal antibody is first obtained, and then the monoclonal antibody may be cleaved using a suitable protease so that the desired antibody fragment can be obtained. For example, when the antibody enzyme according to the present invention is the full length of the light chain of the antibody against the N-terminal region of CCR5, such as the full length of the light chain of 5A2, the full length of the light chain of 6A2, or the full length of the light chain of 2B8. May be obtained by using a conventionally known antibody obtaining method, obtaining a monoclonal antibody using the corresponding N-terminal peptide of CCR5 as an immunogen, and separating and obtaining the light chain. When the antibody enzyme according to the present invention is a CCR5 antibody fragment, the relevant monoclonal antibody is first obtained, and then the desired antibody fragment can be obtained using the above monoclonal antibody using an appropriate protease. Just cut it. Moreover, the antibody obtained by a phage display method may be sufficient. Monoclonal antibodies can be obtained by the usual hybridoma method (see Kohler, G. and Milstein, C., Nature 256, 495-497 (1975)), trioma method, human B-cell hybridoma method (Kozbor, Immunology Today 4, 72 (See 1983)), EBV-hybridoma method (see Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., 77-96 (1985)) and the like.

  By the way, when obtaining the above three antibody enzymes (5A2Lv, 6A2Lv, and 2B8Lv), a peptide having the amino acid sequence shown in SEQ ID NO: 15 is used as an immunogen, and the antibody enzyme according to the present invention is obtained. However, the method is not limited to this, and a peptide having an amino acid sequence different from SEQ ID NO: 15 may be used as the immunogen. That is, as long as it is a peptide having an amino acid sequence constituting the N-terminal region of CCR5, a peptide having any amino acid sequence may be used. The number of amino acid residues of the peptide is not particularly limited as long as it is an immunogenic number. For example, it is preferably 5 to 40 amino acids, and preferably 7 to 35 amino acids. Most preferably it is. From the above viewpoint, the peptide used as an immunogen when obtaining the antibody enzyme according to the present invention is, for example, the amino acid sequence from the 1st position to the 35th position of the amino acid sequence of human-derived CCR5 represented by SEQ ID NO: 18. And a peptide containing a continuous region of at least 10 amino acids or more. In addition, to the peptide used as the immunogen, a few amino acids may be added to improve the immune response, or “other proteins usually used to improve the immune response of low molecular peptides” May be combined. The “other protein” is not particularly limited, and examples thereof include human IgG, BSA (bovine serum albumin), HSA (human serum albumin), KLH (keyhole limpet hemocyanin) and the like. it can. The peptide used as the immunogen may be obtained using a conventionally known amino acid synthesizer or may be obtained using a genetic engineering technique.

  In addition, antibody enzymes whose amino acid sequences and gene sequences encoding them are known, such as 5A2Lv, 6A2Lv, 2B8Lv described above, can be obtained using conventionally known genetic recombination techniques. . In this case, a method in which a gene encoding the antibody enzyme is incorporated into a vector or the like and then introduced into a host cell so that it can be expressed, and a peptide expressed in the cell is purified can be employed. In addition, it is preferable to incorporate a gene encoding the above antibody enzyme together with an appropriate promoter capable of expressing the target protein in a large amount in the host because the target antibody enzyme can be efficiently obtained.

  If the amino acid sequence of the antibody enzyme is not clear, mRNA is first obtained from a monoclonal antibody-producing cell or a hybridoma thereof, cDNA is synthesized from the mRNA, and the gene sequence is read. Thereafter, an amino acid sequence is estimated from the gene sequence, and a three-dimensional structure is predicted by molecular modeling to confirm whether or not a catalytic triad residue-like structure is included. Then, an antibody fragment containing the catalytic triad residue-like structure can be obtained as an antibody enzyme.

  In the case of a gene encoding the antibody enzyme according to the present invention, if the nucleotide sequence is known, after obtaining cDNA (or genomic DNA) from a monoclonal antibody-producing cell or a hybridoma thereof, The gene encoding the antibody enzyme according to the present invention can be obtained by performing PCR using a suitable primer as a template and amplifying the corresponding region. In addition, if a suitable mutation is introduced into a gene consisting of the nucleotide sequence shown in SEQ ID NO: 2, 6, or 10 by using site-directed mutagenesis, Mutants of the antibody enzyme consisting of the amino acid sequence shown in 1, 5, or 9 are obtained as expression products.

(3) Regarding the method of using the antibody enzyme according to the present invention So far, anti-HIV drugs having an action of suppressing the action of CCR5 have been developed. However, the antibody enzyme according to the present invention can be used together with these anti-HIV drugs. Based on a completely different approach, CCR5 can be attacked directly to lose its function as a co-receptor. Therefore, this antibody enzyme can be used as an anti-HIV drug.

  Anti-HIV drugs containing the above-mentioned antibody enzymes inhibit HIV infection with a new mechanism of action, such as enzymatically completely decomposing the N-terminal region of CCR5 and losing its co-receptor function, or developing AIDS Can be suppressed. Such an anti-HIV drug specifically degrades CCR5, so that it can be expected to have a remarkable anti-HIV effect and has few side effects. In view of the reaction mechanism of the antibody enzyme, it can be expected that the therapeutic effect on AIDS is further improved by using it together with other anti-HIV drugs currently used.

  The anti-HIV drug containing the antibody enzyme according to the present invention may be constituted only by the antibody enzyme according to the present invention, or may be mixed with a pharmacologically acceptable carrier or the like. The anti-HIV drug can be produced according to a known method for producing a pharmaceutical composition.

  Here, as the pharmacologically acceptable carrier, various organic or inorganic carrier substances that can be used as a pharmaceutical material are used, and an excipient, a lubricant, a binder, a disintegrant, or a liquid formulation in a solid formulation is used. In this case, it is added as a solvent, a solubilizing agent, a suspending agent, a tonicity agent, a buffering agent, a soothing agent, etc.

  Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose. Examples of the lubricant include magnesium stearate, calcium stearate, talc, colloidal silica, and the like. Is mentioned.

  Examples of the binder include pregelatinized starch, methylcellulose, crystalline cellulose, sucrose, D-mannitol, trehalose, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone.

  Examples of the disintegrant include starch, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, and carboxymethyl starch sodium.

  Examples of the solvent include water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and tricaprylin.

  Examples of the solubilizer include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

  Examples of the suspension include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glyceryl monostearate, or polyvinyl alcohol, polyvinylpyrrolidone, Examples include hydrophilic polymers such as sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, and hydroxypropyl cellulose.

  Examples of the isotonic agent include sodium chloride, glycerin, D-mannitol and the like. Examples of the buffer include buffer solutions of phosphate, acetate, carbonate, citrate and the like.

  Examples of the soothing agent include benzyl alcohol.

  Examples of the preservative include paraoxybenzoates, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.

  Examples of the antioxidant include sulfite and ascorbic acid.

  The content of the antibody enzyme in the anti-HIV drug is not particularly limited, and may be adopted after considering the optimum amount depending on the administration method.

  The anti-HIV drug can be produced by a method commonly used in the pharmaceutical technical field. Examples of dosage forms of anti-HIV drugs include tablets, capsules (including soft capsules and microcapsules), powders, granules, syrups, and other oral preparations, injections, suppositories, pellets, drops, etc. Oral agents are mentioned, and these are also less toxic and can be administered orally or parenterally, respectively.

  The dosage of the anti-HIV drug according to the present invention varies depending on the administration subject, administration route, symptoms and the like. Therefore, the above dose may be adopted after considering the optimum conditions as appropriate.

  Furthermore, the antibody enzyme according to the present invention can produce a transformant by introducing a gene encoding it into an appropriate host. That is, the transformant according to the present invention is a transformant into which a gene encoding the antibody enzyme has been introduced. Here, “gene introduced” means that the gene is introduced into a target cell (host cell) so as to be expressed by a known genetic engineering technique (gene manipulation technique). This transformant can express the antibody enzyme in its own body.

  More specifically, examples of the transformant according to the present invention include those obtained by introducing a gene consisting of the base sequence shown in SEQ ID NO: 2, 6, or 10 into a host cell. As said host cell, what is normally used, such as colon_bacillus | E._coli, yeast, baculovirus, can be used suitably.

  Since the above transformant specifically recognizes CCR5 in its own body and can completely decompose it and lose its function, it can be used as an anti-HIV drug. In addition, it is considered that a transformant capable of expressing a large amount of the above antibody enzyme can efficiently prevent HIV infection and treat AIDS symptoms, and obtain a more effective anti-HIV drug. May be possible.

  Embodiments will be described below in more detail with reference to the accompanying drawings. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  Moreover, all the academic literatures and patent literatures described in this specification are incorporated herein by reference.

(1. Production method of monoclonal antibody)
<Immunity>
A conjugate of CCR5 N-terminal region peptide (hereinafter “NC5 peptide”; MDYQVSSPIYDINYYTSEPQQINVKQIAARLL; SEQ ID NO: 15) and KLH (Pierce, USA) was prepared as follows. NC5 peptide was purchased from Peptide & Protein Research Cons. Devon, UK.

  To KLH (10 mg / ml), 0.63 mg of a crosslinking reagent MBS (m-maleimido-benzoyl-N-hydrosuccinimide, source: Peerce, IL, USA) was added with stirring and allowed to react for 1 hour. After removing unreacted MBS using a column (Sephadex G-25, manufactured by Parmacia), NC5 peptide (2 mg) and KLH (2 mg) were reacted in 50 mM phosphate buffer (pH 7.2 to 7.4). I let you. At this time, KLH reacts with cysteine in the amino acid sequence of the NC5 peptide to become a conjigate. Using this as an antigen for immunization, Balb / c mice (6 weeks old female) were immunized as follows.

  The above conjugate was mixed with FCA (Freund's complete adjuvant; Mitsubishi Yatron Co., Ltd., Code RM606-1) in a clean bench, and 100 μl each was immunized 3 times in a mouse foot pad.

  The increase in the antibody titer was confirmed by collecting blood from the mouse orbital venous plexus and measuring the antibody titer against the NC5 peptide by the usual ELISA method. The ELISA method was as follows. NC5 peptide was prepared at 5 μg / ml in PBS. 50 μl of the NC5 peptide was injected into a 96-well plate. The well plate was allowed to stand at 4 ° C. overnight, and the plate was washed 3 times with PBS containing 0.05% Tween 20 (“PBS-T”). 150 μl of PBS containing 2% gelatin was added to all wells of the well plate. The well plate was allowed to stand at room temperature for 1 hour for blocking. The collected serum was diluted 1/100 with PBS, and serial dilution from 1/100 to 4 times was repeated 7 times to prepare 8 types of antiserum dilutions per sample. As a negative control, serum from unimmunized mice was also used in the test. 50 μl of serially diluted serum was added to each well after blocking and allowed to stand at room temperature for 1 hour (primary reaction). After washing with PBS-T, 50 μl of peroxidase (POD) -labeled anti-mouse IgG (Fc) (ICN code 67429) diluted 1/1000 in PBS-T was added to each well. The mixture was allowed to stand at room temperature for 1 hour (secondary reaction). After washing the well plate with PBS-T, 100 μl of TMB substrate (3,3 ′, 5,5′-tetramethylbenzidine, source: KPL, Gaithersburg, MD, USA) was added to each well, and the plate was allowed to stand for 30 minutes. Absorbance was measured with a reader (wavelength 450 nm).

<Cell fusion>
The spleen was removed from the mouse with increased antibody titer, and cell fusion between mouse lymph node cells (1 × 10 ⁸ cells) and mouse myeloma cells (P3U1; 2 × 10 ⁷ cells) was performed. The cell fusion method was carried out by a conventional method using polyethylene glycol (PEG 4000, Wako Pure Chemical Industries, Ltd. Code 162-09115). Thereafter, HAT selection, screening, and cloning were repeated to establish 8 anti-NC5 monoclonal antibody-producing cells shown in Table 1.

  The isotype of the established monoclonal antibody was determined using an isotyping kit (Iso Strip ™ mouse monoclonal antibody isotyping kit (Roche 1493027)). The method was performed according to the attached manual. The results are shown in Table 1.

<Cross-reactivity test with peptides and proteins other than NC5 peptide>
Using the culture supernatant of 5 clones (5A2, 6A2, 2B8, 3F4, and 4H6) of the anti-NC5 monoclonal antibody-producing cells obtained above, various peptides and proteins other than NC5 peptide The cross-reactivity with was examined by ELISA. Peptides and proteins include NC5 peptide, Keyhole Lympet Hemocyanine (indicated as “KLH” in FIGS. 15 and 16), human serum albumin (indicated as “HSA” in FIGS. 15 and 16), bovine serum albumin (indicated in FIG. 15). 15 and FIG. 16 are referred to as “BSA”), human-γ-globulin (referred to as “Human-γ-globlin” in FIGS. 15 and 16), and human hemoglobin (“Human-hemoglobin” in FIGS. 15 and 16). ), TP41-1 peptide (TPRGGPDRPEGIEEGGERDRD: 21mer: SEQ ID NO: 16, referred to as “TP41-1” in FIGS. 15 and 16), RT-1 peptide (KLLLGTKALTFVIPLTEEAE: SEQ ID NO: 17, FIG. 15 and FIG. 15) 16 is expressed as “RT-1”). The RT-1 peptide is a partial peptide of reverse transcriptase of AIDS virus.

  The ELISA method was performed as follows. Samples were prepared so that the peptide and protein were each 5 μg / ml in PBS, and 200 μl each was placed in a 96-well plate and coated. The case where the ELISA method was performed without coating the peptide and protein was used as a negative control (indicated as “(−)” in FIGS. 15 and 16). The cell culture supernatant was diluted 1/2 with PBS-T and used for the primary reaction. As a secondary antibody, AP-F (ab ′) 2 Rabbit anti Mouse IgGAM (H + L) (source: Zymed 61-6322 Lot. 11167841R) was used after being diluted 500 times. Other operations were performed according to the ELISA method described above.

  The results are shown in FIGS. FIG. 15 shows 5A2 strain (indicated by “5A2” in the figure), 6A2 strain (indicated by “6A2” in the figure), and a culture medium (in the same figure) used for culturing each monoclonal antibody as a negative control. The results of the cross-reactivity test were shown for “negative control (medium)”. FIG. 16 also shows a cross-reactivity test for 2B8 strain (indicated by “2B8” in the figure), 3F4 strain (indicated by “3F4” in the figure), and 4H6 strain (indicated by “4H6” in the figure). The result of having performed was shown. As a result of the above cross-reactivity test, some non-specific binding to a substrate other than NC5 peptide was observed, but any monoclonal antibody produced by anti-NC5 monoclonal antibody-producing cells was higher than NC5 peptide. It was found to have specificity.

<Culture of anti-NC5 monoclonal antibody-producing cells>
Anti-NC5 monoclonal antibody-producing cells (hybridomas) were used in an IMDM medium (SIGMA, I-2510) containing 20% FCS, at 37 ° C. and CO ₂ concentration of 5.5%, 1 to 5 × 10 ⁷ cells. Until it was obtained. The cells obtained by the above culture were transferred to a 50 ml centrifuge tube and centrifuged at 1,400 rpm for 6 minutes to collect the cells. After removing the culture medium with an aspirator, the cells were suspended in 10 ml of PBS, and the cells were combined into one tube and centrifuged again at 1,400 rpm for 6 minutes. The supernatant was aspirated and removed with an aspirator and then suspended again with 10 ml of PBS, and the number of cells was counted using a hemocytometer.

After counting, a cell suspension of 1 to 5 × 10 ⁷ cells was taken, centrifuged again at 1,400 rpm for 6 minutes, and the supernatant was removed. Furthermore, it was inverted on a paper towel and PBS was removed completely. At this time, if the aspirate was insufficient, centrifugation was performed again, and the remaining PBS was removed with a micropipette.

<MRNA extraction and purification>
Extraction of mRNA was performed using QuickPrep ^™ mRNA purirfcation Kit (manufactured by Amersham Pharmacia Biotech Co., Ltd.) according to the recommended protocol of the kit. The method is shown below.

  For extraction of mRNA, 1.5 ml of an extraction buffer in which the crystals were completely dissolved by standing in an incubator at 37 ° C. for about 30 minutes was added to the cells (pellet) collected above, and a 21 G injection needle was added. The cells were disrupted by passing them through several times. To the cell disruption solution, 3 ml of Elution buffer was added, and the cells were further disrupted. The obtained cell disruption solution was dispensed into four 1.5 ml microtubes of RNase free, centrifuged at 18,000 × g for 20 minutes at room temperature (25 ° C.), and oligo (dT) -cellulose. A sample for use in a spin column was prepared.

  To isolate mRNA, first, suspend the oligo (dT) -cellulose spin column resin, remove the top and bottom caps, and place in a 15 ml centrifuge tube at 1,300 rpm for 2 minutes (resin should not be too dry) The centrifuge was stopped at 1 minute 45 seconds), and the stock solution in the column was removed by centrifugation. Thereafter, the lower cap was attached to the column. Add 4 ml of the sample (supernatant) that has been subjected to protein removal and DNA removal to the column, attach the top cap to the column, suspend the resin, and mix by inverting for 10 to 15 minutes. Was adsorbed. Then, after centrifuging at 1,400 rpm for 1 to 2 minutes with the upper and lower caps attached to the column, the upper cap was removed, and the supernatant was removed using an RNase free micropipette. 3 ml of High-salt buffer was added thereto, and after gently mixing for about 2 minutes, an upper cap was attached to the column, and centrifugation was performed at 1,400 rpm for 2 minutes to remove the supernatant. This operation was repeated two more times.

  Next, 3 ml of low-salt buffer is added to the column and suspended gently for about 1 to 2 minutes. Then, an upper cap is attached, placed in a 15 ml centrifuge tube, and centrifuged at 1,400 rpm for 2 minutes. Was removed. Then remove the lower cap and add 3 ml of low-salt buffer along the wall so that the resin surface is flat, then place the column in a 15 ml centrifuge tube and centrifuge at 1,300 rpm for 2 minutes. It was.

Next, a new 15 ml centrifuge tube was prepared, and the lids of two 1.5 ml microtubes for recovering mRNA were cut with scissors and then put over the 15 ml centrifuge tube to prepare an elution tube. A column was placed in an elution tube, 0.25 ml of Elution buffer heated to 65 ° C. was added thereto, and centrifuged at 1,300 rpm for 2 minutes. This operation was repeated twice, and the eluate was collected as an mRNA fraction. The recovered mRNA fraction was allowed to stand on ice. To 10 μl of the mRNA fraction, 70 μl of Elution buffer was added and diluted 8-fold, and A ₂₆₀ (RNA) and A ₂₈₀ (protein) were measured using the Elution Buffer as an absorbance blank. For the measurement of absorbance, a cell washed with DEPC-treated water (Dietyl Pyrocarbonate-treated water) after being RNase-free by immersing in a solution of hydrochloric acid: methanol = 1: 1 for about 1 hour was used. The RNA concentration was calculated as A ₂₆₀ × dilution rate 40 μg / ml.

  Further, 1/10 amount of 3M potassium acetate, 1/50 amount of glycogen, and 2.5 times amount of 95% ethanol were added to the recovered mRNA fraction and mixed. The mixed solution was placed at −30 ° C. for about 40 minutes, centrifuged at 4 ° C. and 15,000 rpm for 5 minutes, and then stored at −80 ° C.

  Glass and metals that had been sterilized by dry heat at 240 ° C. for 2 hours, and RNase and DNase free plastics were used in the preparation of the mRNA.

<Synthesis of first strand cDNA from mRNA>
The mRNA that had been stored in the ethanol-precipitated state at −130 ° C. was taken out, centrifuged at 15,000 rpm for 10 minutes at 4 ° C., and the supernatant was removed with a micropipette. Thereafter, 1 ml of cold 75% ethanol (the cold 75% ethanol prepared in advance with DEPC-treated water and stored at −30 ° C.) was gently added to the microtube from the side without the pellet to perform rinsing. It was. Next, the microtube was centrifuged at 15,000 rpm for 10 minutes at 4 ° C., and then the supernatant was removed with a micropipette. Then, vacuum drying was performed for 15 minutes, DEPC-treated water was added so that the RNA concentration was 0.2 μg / μl, and the mixture was allowed to stand for 1 hour to dissolve RNA. After dissolution, for confirmation, the mRNA solution was diluted with DEPC-treated water so that A ₂₆₀ = 1, and the absorbance was measured.

cDNA synthesis was performed using AMV Reverse Transcriptase First-strand cDNA Synthesis Kit (Invitrogen) according to the protocol of the kit. The method is shown below. The kit was removed from −80 ° C., and each reagent was dissolved on ice and mixed well. An mRNA solution corresponding to 2 μg of mRNA was added to an RNase-free 0.2 ml microtube. 2 ml of pd (T) _12-18 primer (0.5 μg / ml) was added to the microtube, and the volume was increased with DEPC-treated water to 17 μl. After mixing the above solutions, annealing treatment was performed at 70 ° C. for 10 minutes. Thereafter, the microtube was placed on ice, and 1 μl of 0.25M DTT, 1 μl of Rnase inhibitor, and 5 μl of 5 × Reaction buffer were added to the microtube and mixed well. Furthermore, 1 μl of AMV-RT was added to the microtube and mixed gently, and then an extension reaction was performed at 41 ° C. for 60 minutes. After 60 minutes, the extension reaction was stopped by placing the microtube on ice, and then the microtube was stored at −30 ° C.

<Amplification of antibody variable region gene by PCR>
Using the Mouse Ig primer Kit (Novagen) and NovaTaq ^™ Hot Start DNA Polymerase Kit (Novagen), the variable region of the antibody gene was amplified from the cDNA obtained above.

First, for amplification of genes encoding antibody heavy and light chains, Master mix (37.25 μl sterile ultrapure water, 0.5 μl 3′-Primer (final concentration 5 pmol), 3 μl 25 mM MgCl ₂ (final concentration 1.5 mM) ) Prepare a solution containing 1 μl 10 mM dNTPs (final concentration 0.2 mM), 5 μl 10 × NovaTaq Hot Start Buffer (final concentration 1 ×), and 0.25 μl NovaTaq Hot Start DNA Polymerase (final concentration 1.25 U). 47 μl of the solution was dispensed into PCR tubes prepared for the number of positive controls, negative controls, and primers, and 1 ml 5′-Primer (final concentration 5 pmol) and 2 ml cDNA solution were added to the PCR tubes. In addition, the thing attached to Mouse Ig primer Kit was used as said positive control, and the sterilized ultrapure water was used as a negative control.

  The solution in the PCR tube was mixed well, and PCR was performed using Biometra T-GRADIENT and TaKaRa PCR Thermal cycler PERSONAL. As PCR reaction conditions, the light chain is preheated at 95 ° C. for 7 minutes, heat denaturation is 98 ° C. for 15 seconds, extension reaction is 74 ° C. for 15 seconds, and annealing is 70 ° C. → 66 ° C. → 62 ° C. → 58 ° C. → 54 ° C. → Adopted the Step Down method, which reduces the temperature by 4 ° C in 6 steps at 50 ° C for 15 seconds. The above steps of heat denaturation, extension reaction, and annealing were performed 3 cycles at a time until the annealing temperature reached 54 ° C., and the reaction at the annealing temperature of 50 ° C. was performed 10 cycles. On the other hand, the heavy chain is preheated at 95 ° C. for 10 minutes, heat-denatured at 94 ° C. for 1 minute, annealing temperature at 50 ° C. for 2 minutes, and subjected to extension reaction for 40 cycles of 72 ° C. and 2 minutes, followed by 72 ° C. extension reaction. For 10 minutes. After completion of the PCR, amplification of each antibody variable region gene was confirmed by agarose gel electrophoresis.

<Cloning with TOPO TA Cloning Kit (Invitrogen)>
Cloning was performed as follows.

  PCR product 2 μl, Salt Solution 1 μl, and Sterile Water 2 μl, which were confirmed to be amplified by the antibody variable region by agarose gel electrophoresis, were dispensed into PCR tubes on ice and mixed. Further, 1 μl of TOPO vector was added to the PCR tube and mixed gently, and then the solution in the PCR tube was ligated at room temperature for 30 minutes. After 30 minutes, the PCR tube was immediately placed on ice to stop the reaction.

  Just before use, One Shot TOP10 Chemically Competent E. coli (manufactured by Invitrogen) was taken out from a freezer at −80 ° C. and dissolved on ice. To the above-mentioned One Shot TOP10 Chemically Competent E. coli, 2 μl of the ligation reaction solution that had been placed on ice was added and incubated on ice for 30 minutes to diffuse the DNA between the competent cells. After completion of the reaction, heat shock was applied at 42 ° C. for 45 seconds to incorporate the DNA, and then returned to ice and allowed to stand for 2 minutes. 250 μl of SOC medium, which had been returned to room temperature in advance, was added to the DNA solution in a clean bench and cultured at 37 ° C. for 1 hour with shaking (200 rpm).

  During this time, 100 μl of 100 mM IPTG stock and 20 μl of X-Gal 50 mg / ml were spread on one previously prepared Ampicillin / LB plate and dried for 15 to 30 minutes. After completion of the impression, the E. coli solution (50 μl or 100 μl) was spread on the Ampicillin / IPTG / X-Gal / LB plate and cultured overnight at 37 ° C.

  Based on the blue / white determination after transformation, about 12 colonies of white colonies considered to have a DNA fragment inserted were selected and further streaked culture was performed to eliminate false positives. In streaked culture, Ampicillin / IPTG / X-Gal / LB plate was used as in the transformation, streaked so that a single colony was obtained with a platinum loop, and cultured at 37 ° C. overnight.

  After confirming that the DNA fragment was inserted into each colony, plasmid DNA was prepared as follows.

  0.75 ml of the culture solution is taken from the culture solution of the transformant, mixed with 0.2 ml of 80% glycerol, stocked at −80 ° C., and the remaining 5.25 ml of the culture solution is added to a 1.5 ml microtube. Then, the cells were collected by centrifugation at 12,000 rpm for 1 minute, and the supernatant was carefully removed by aspiration. 100 μl of Solution I (50 mM D-glucose, 25 mM Tris-HCl, 10 mM EDTA, pH 8.0) was added to the microtube, and the mixture was well suspended by vortex mixing.

Next, 200 μl of Solution II (0.2N NaOH, 1% (w / v) SDS) was added to the above microtube and mixed by inverting, and then the microtube was placed on ice for 5 minutes. Solution III (3M CH ₃ COOK, After adding 150 μl of 2MCH ₃ COOH) and mixing gently, the microtube was placed on ice for 5 minutes.

  Then, after centrifugation at 15,000 rpm for 10 minutes at 4 ° C, the supernatant is taken with care not to allow precipitation (keep the amount collected at this time), and a new 1.5 ml microtube After adding an equal amount of phenol / chloroform and mixing, the mixture was centrifuged at 15,000 rpm for 2 minutes at 4 ° C. After repeating the above operations for a total of two phenol / chloroform treatments, the upper layer (aqueous layer) was transferred to another microtube, mixed with 1 ml of twice the amount of cold ethanol and shaken up and down. And allowed to stand at −70 ° C. for 5 minutes. Then, after centrifugation at 15,000 rpm for 10 minutes at 4 ° C., the supernatant was removed. Then, 1 ml of 70% cold ethanol was added to rinse the pellet, and the mixture was centrifuged at 15,000 rpm for 10 minutes at 4 ° C. After removing the supernatant, vacuum drying was performed for 15 minutes.

  After completion of vacuum drying, the pellet was dissolved with 45 μl of TE (10 mM Tris-HCl, 1 mM EDTA), and 5 μl of RNase A solution (5 mg / ml Rnase A, 10 mM Tris, 15 mM NaCl, pH 7.5) was added to the microtube ( Final concentration: 50 μg / ml). The solutions for two microtubes were collected in one microtube and reacted at 37 ° C. for 1 hour.

  After completion of the reaction, 2/3 equivalent of PEG (13% (w / v) PEG6000, 0.8 M NaCl) was added to the microtube, and the microtube was placed on ice for 1 hour or longer. Thereafter, centrifugation was performed at 15,000 rpm for 15 minutes at 4 ° C., and the supernatant was removed. The microtube was rinsed by adding 1 ml of 70% ethanol, and centrifuged at 15,000 rpm for 10 minutes at 4 ° C. After removing the centrifugation supernatant, vacuum drying was performed. After vacuum drying, 50 μl of sterilized water was added per sample to dissolve the sample to obtain a plasmid solution. Then, the DNA concentration of the sample was estimated by band comparison by agarose electrophoresis.

<Determination of nucleotide sequence>
The nucleotide sequence was determined using Thermo Sequenase ^™ Cy ^™ 5.5 Terminator Cycle Sequencing Kit (Pharmacia). Prepare PCR tubes for four types of bases, A (adenine), C (cytosine), G (guanine), and T (thymine), and add d (N) TP / Cy5.5-dd (N) TP. 1 μl was dispensed for each sample.

  Next, a DNA / primer mix was prepared. Mix 1.5 μg DNA prepared in the previous section, 3.5 μl Reaction Buffer, 1 μl TOPO13 Reverse (4 pmol / μl) (Invitrrogen), and 2 μl Thermo Sequenase (10 U / μl), and further sterilized ultrapure water Was added to a total volume of 31.5 μl. 7 μl of this was dispensed into the above PCR tube and immediately set on a Biometora thermal cycler, and 30 cycles of reaction cycles of 95 ° C. for 30 seconds, 58 ° C. for 30 seconds, and 72 ° C. for 120 seconds were performed. The reaction was stopped by cooling at 0 ° C.

After completion of the reaction, the sample was transferred from the PCR tube to a 1.5 ml microtube, and 1 μl of 20 mg / ml glycogen, 2 μl of 7.5 M ammonium acetate, and 30 μl of 98% ethanol were added and mixed well. After leaving still on ice for 10 minutes, it centrifuged at 12000 rpm for 20 minutes. Thereafter, the supernatant was removed by decantation on a paper towel, 200 μl of 70% ethanol was added, and the pellet and the inner wall of the tube were rinsed. Then, centrifugation was performed at 12000 rpm for 10 minutes, and then the supernatant was carefully removed using a micropipette. Then, the pellet is dried by shading, dissolved in 6 μl of Loading Dye, heat-treated at 72 ° C. for 3 minutes, and loaded with 2 μl each of LONG-READ TOWER ^™ System (Amersham Pharmacia Biotech.) For analysis. It was. The obtained base sequence was analyzed using DNASIS software and translated into an amino acid sequence.

<3D structure prediction of antibody variable region>
Based on the amino acid sequence of the antibody variable region deduced from the base sequence determined above, using the software AbM (Oxford Molecular, Oxford, UK), the CDR structure of the target antibody and the three-dimensional structure of the FR region. The structure was predicted. Based on the three-dimensional structure predicted by AbM, intermolecular force calculation was performed by software InsightII / Discover3 (Molecular Simulatoin, USA) to predict a thermodynamically stable three-dimensional structure.

  Furthermore, using the software PPC Protein AdviSer (Fujitsu Kyushu System Engineering), the Ser, His and Asp residue groups constituting the catalytic triad residues constituting the active site of the serine protease in the three-dimensional structure were searched. As a result, all three types of antibody light chains (L chains) of monoclonal antibodies 5A2, 6A2, and 2B8 had a catalytic triad residue structure (see FIGS. 1 to 3). Note that the way of viewing the figure is as already described in the section “Best Mode for Carrying Out the Invention”.

  Many previous studies by the inventors (Appl. Biochem. Biotech., 83, 209-220 (2000); J. Immunol. Methods, 269, 283-298 (2002); Immunol. Lett. 86, 249- 257 (2003); Biotechnol. Bioeng., 84 (7), 485-493 (2003); Chemical Industry, 54, 368-372 (2003); Biotechnol. Bioeng. 86 (2), 217-225 (2004); Science, 75 (11), 1254-1259 (2005).) Suggested that the anti-NC5 monoclonal antibodies 5A2, 6A2, and 2B8 may be antibody enzymes. Therefore, it was decided to examine the presence or absence of antigen resolution of the heavy chain (H chain) light chain (L chain) for the above three anti-NC5 monoclonal antibodies.

<Large acquisition and purification of anti-NC5 monoclonal antibody>
Large-scale preparation of the above three anti-NC5 monoclonal antibodies was performed by administering 0.5 × 10 ⁶ anti-NC5 monoclonal antibody-producing cells to Balb / c mice previously administered with pristane (2,6,10,14-tetramethylpentadecane). The ascites was collected from the mouse. Each anti-NC5 monoclonal antibody obtained as described above was purified by the following method and used in the intended experiment.

(1) Salting out About 8 ml of ascites was diluted with the same amount of PBS and then filtered to remove fibrin. This was divided into two high-speed cooling centrifuge tubes, and the same amount of saturated ammonium sulfate was added dropwise. This was left to stand in ice for 30 minutes, and then centrifuged at 4 ° C. and 10,000 rpm for 10 minutes. The supernatant was removed by decantation and the pellet was dissolved in 6 ml of PBS. Again, an equal amount of saturated ammonium sulfate was added for salting out, and the pellet was dissolved in 6 ml of PBS. This was combined into one tube and dialyzed twice against PBS.

(2) Column purification After completion of dialysis, the antibody was purified. The operation was performed at 4 ° C. according to the description of the MAPS-II kit (manufactured by BIO-RAD / purification kit using Protein A). As reagents to be used, 0.05% NaN ₃ / PBS, Binding buffer, Elution buffer, and 2M Tris-HCl (pH 8.0) were prepared as follows. 0.05% NaN ₃ / PBS was obtained by dissolving 0.1 g NaN ₃ in 200 ml PBS. The binding buffer was prepared by dissolving 47.1 g of binding buffer powder in distilled water and making up to 150 ml. At this time, using a pH meter, it was confirmed that the pH was 9 ± 0.2, and when it was out of the range, the pH was adjusted with HCl or NaOH. Elution buffer was prepared by dissolving 2.3 g of Elution buffer powder in distilled water and making up to 100 ml. At this time, using a pH meter, it was confirmed that the pH was 3 ± 0.2, and when it was out of the range, the pH was adjusted with HCl or NaOH. 2M Tris-HCl was prepared by dissolving 12.11 g of Tris in distilled water, adjusting the pH to 8.0 with HCl, and then making up to 50 ml with distilled water.

After the dialysis, the salted out ascites was diluted so that ascites: Binding buffer = 1: 1.2. When insoluble matter was observed, it was removed by filtration with filter paper. Further, before the purification operation, 0.05% NaN ₃ / PBS, Binding buffer, Elution buffer, and sample (the ascites filtrate) were degassed.

  Affigel protein A (BIO-RAD product name) included in the kit was packed, and the column was washed with the above binding buffer. The absorbance at UV 280 nm was monitored, and the gel was washed with the binding buffer until the baseline settled. The flow rate was adjusted to 0.2 ml / min, and the sample was applied to the column when the gel surface and the binding buffer liquid level almost coincided.

  Next, 50 ml or more of binding buffer was provided to remove contaminants other than antibodies. The peak portion detected here was collected as a pass-through fraction. When the baseline is stable after collecting the flow-through fraction, add 45 ml of Elution buffer when the surface of the gel and binding buffer are almost the same, and bind to Affigel Protein A (BIO-RAD). The eluted antibody was eluted, and the peak portion was fractionated as an antibody fraction. About each collect | recovered fraction, pH was measured with the pH test paper and neutralized with 2M Tris-HCl (pH8.0). The antibody fraction (antibody solution) was dialyzed twice against PBS, and the purity was confirmed by SDS-PAGE. Only the high-purity fraction was collected, the protein concentration was measured with a DC protein standard assay (manufactured by BIO-RAD), and was stored frozen at a concentration of 1 mg / ml or more (concentrate if less than 1 mg / ml). .

<Separation and purification of anti-NC5 antibody heavy chain (H chain) and light chain (L chain)>
(1) Ultrafiltration The purified antibody solution (for 5 mg of protein) was dialyzed twice against 50 mM Tris-HCl buffer (pH 8.0) containing 0.15 M NaCl. The antibody solution after completion of dialysis was centrifuged at 2,800 rpm at 4 ° C. using Centriprep-10 (manufactured by Millipore) and concentrated by ultrafiltration until about 1 ml. To the above antibody solution, 5 ml of 50 mM Tris-HCl + 0.15 M NaCl buffer (pH 8.0) was added, and ultrafiltration concentration was performed again until it reached about 1 ml. After performing the above operation again, the antibody solution was adjusted to 2.7 ml with 50 mM Tris-HCl + 0.15 M NaCl buffer (pH 8.0), and the antibody solution was put in a brown bottle. The antibody solution was stored at low temperature until used in the following experiments.

(2) Separation of heavy and light chain antibodies 50 mM Tris-HCl + 0.15 M NaCl buffer (pH 8.0), 1 M Tris solution, and 2.7 ml of antibody solution were degassed, respectively. 2-ME (mercaptoethanol) and 50 mM Tris-HCl + 0.15 M NaCl buffer (pH 8.0) were added to the antibody solution, and the solution was lightly mixed by pipetting and adjusted to pH 8.0 with 1 M Tris solution. Nitrogen was sealed in the reaction vessel. The reduction reaction was carried out in an incubator with stirring with a stirrer at 15 ° C. for 3 hours. After degassing, 600 μl of 0.6M iodoacetamide was added to the antibody solution and mixed. The pH of the antibody solution was adjusted to pH 8.0 with 1M Tris solution. The alkylation reaction was carried out with stirring at 15 ° C. for 15 minutes. Using a disk filter (pore size 0.2 μm), the antibody solution was departicled. The above antibody solution was subjected to ultrafiltration concentration using Centriprep-10 until the liquid volume became about 0.5 ml.

<Purification of anti-NC5 monoclonal antibody heavy and light chains by size exclusion HPLC>
Purification of heavy and light chains was performed using size exclusion chromatography (JASCO, PU-2080 Plus), column (Waters, Protein-Pak ^™ 300 SW, 7.5 × 300 mm Column). The column was equilibrated (about 120 minutes) at a flow rate of 0.15 ml / min with 6M guanidine hydrochloride (pH 6.5) used for the mobile phase. The antibody solution sample was injected into the column in two or three times. With reference to the chromatogram, the heavy chain and the light chain were separated, respectively. Each obtained heavy and light chain sample was dialyzed against PBS and the heavy and light chains were refolded. Samples were collected in a clean bench after changing the heavy and light chain sample buffer to 15 mM PB. The heavy chain and light chain samples were measured for concentration of heavy chain and light chain by DC protein standard assay (manufactured by BIO-RAD), purity was confirmed by SDS-PAGE, and the sample was stored at 4 ° C.

<Investigation of peptidase activity of anti-NC5 monoclonal antibody heavy chain and light chain>
A characteristic of a natural antibody enzyme is that the antibody itself specifically and completely degrades the target protein. However, it is known that when the degradation activity of an antibody enzyme is examined using a peptide substrate, it reacts nonspecifically. Therefore, it was examined whether the antibody has the peptide resolution by using the peptide TP41-1 peptide (TPRGPDRPEGIEEEGEGERDRD: 21mer: SEQ ID NO: 16), which has been often used for examining the peptidase activity of the antibody enzyme so far. Since the antigenic peptide used for immunization of mice was poorly soluble in water, it could not be used as a substrate for studying peptidase activity.

(1) Experimental method First, 240 μM TP41-1 peptide solution (15 mM PB (pH 6.5) was prepared in a clean bench and sterilized by filtration with a 0.2 μm filter. TP41-1 peptide solution in a test tube. And 15 mM PB (pH 6.5) were mixed at a ratio of 1: 1 as a control solution, and each reaction solution prepared as described above was as follows.
Reaction solution (1) 0.8 μM light chain + TP41-1 peptide solution ・ ・ ・ 600 μl Preparation reaction solution (2) 0.4 μM heavy chain + TP41-1 peptide solution ・ ・ ・ 600 μl preparation reaction solution (3) Control solution ・ ・ ・500 μl preparation The reaction was performed at 25 ° C.

  The reaction was analyzed using HPLC (puresil C18, Waters). As HPLC measurement conditions, a column oven was 40 ° C., and an eluent was 0.08% TFA, 13% acetonitrile, and ultrapure water. 30 μl of each reaction solution was collected in a clean bench, the reaction solution was passed through a 0.45 μm filter, and further centrifuged at 10,000 rpm for 1 minute as a sample and subjected to HPLC (sample injection amount 20 μl). ).

(2) Results In order to show the results of the above test, changes with time of the reaction substrate (TP41-1 peptide) are shown in FIGS. 5 shows the result of anti-NC5 monoclonal antibody 5A2 (Lot.1), FIG. 6 shows the result of anti-NC5 monoclonal antibody 5A2 (Lot.2), FIG. 7 shows the result of anti-NC5 monoclonal antibody 6A2, and FIG. 8 shows the result of the anti-NC5 monoclonal antibody 2B8. In each figure, the circle symbol indicates the result of the reaction solution (1), the square symbol indicates the result of the reaction solution (2), and the triangle symbol indicates the result of the reaction solution (3). Yes.

  According to FIG. 5, in the case of the reaction solution (1), degradation of TP41-1 peptide was confirmed after about 25 hours of induction period, and complete degradation of TP41-1 peptide was confirmed after about 96 hours. When the result of the reaction solution (3) (control solution) is compared with the result of the reaction solution (1), it can be seen that the TP41-1 peptide is clearly degraded. At this time, the result of tracing the decomposition reaction of TP41-1 peptide by HPLC is shown in FIG.

  Further, according to FIG. 6, in the case of the reaction solution (1), the degradation of TP41-1 peptide was confirmed after about 50 hours induction period, and the complete degradation of TP41-1 peptide was confirmed after about 120 hours. . When the result of the reaction solution (3) (control solution) is compared with the result of the reaction solution (1), it can be seen that the TP41-1 peptide is clearly degraded. At this time, the result of tracing the decomposition reaction of TP41-1 peptide by HPLC is shown in FIG.

  Therefore, from the results of FIGS. 5 and 6, it was found that the light chain of the anti-NC5 monoclonal antibody 5A2 can degrade the TP41-1 peptide with good reproducibility.

  Further, according to FIG. 7, in the case of the reaction solution (1), the degradation of TP41-1 peptide was confirmed after about 20 hours induction period, and the complete degradation of TP41-1 peptide was confirmed after about 70 hours. It was. In the case of the reaction solution (2), the degradation of TP41-1 peptide was slightly confirmed. When the result of the reaction solution (3) (control solution) is compared with the result of the reaction solution (1), it can be seen that the TP41-1 peptide is clearly degraded. At this time, the result of tracing the decomposition reaction of TP41-1 peptide by HPLC is shown in FIG.

  Therefore, from the results of FIG. 7, it was found that the light chain of the anti-NC5 monoclonal antibody 6A2 can degrade the TP41-1 peptide.

  Further, according to FIG. 8, in the case of the reaction solution (1), the degradation of TP41-1 peptide was confirmed after an induction period of about 50 hours, and the complete degradation of TP41-1 peptide was confirmed after about 142 hours. It was. When the result of the reaction solution (3) (control solution) is compared with the result of the reaction solution (1), it can be seen that the TP41-1 peptide is clearly degraded. At this time, the result of tracing the degradation reaction of TP41-1 peptide by HPLC is shown in FIG.

  Therefore, from the results of FIG. 8, it was found that the light chain of the anti-NC5 monoclonal antibody 2B8 can degrade the TP41-1 peptide.

  From the above results, it was found that the light chains of the anti-NC5 monoclonal antibodies 5A2, 6A2, and 2B8 all have peptidase activity.

<Kinetic analysis of anti-NC5 monoclonal antibody>
(1) Method In determining the kinetic parameters of an anti-NC5 monoclonal antibody, the light chain of the anti-NC5 monoclonal antibody degrades the substrate (TP41-1 peptide) while drawing a biphasic curve. It is difficult to find. Therefore, the kinetic parameters were analyzed under the condition that no induction period was present by adding the peptide again to the reaction solution once the substrate (TP41-1 peptide) was completely decomposed and reacting.

  Various kinetic parameters were determined using the initial decomposition rate. The initial degradation rate was calculated by changing the substrate concentration while keeping the concentration of the light chain of the anti-NC5 monoclonal antibody constant at 0.4 μM. The calculated initial decomposition rate was applied to the Michaelis-Menten equation, and the following kinetic parameters were calculated.

(2) Results FIG. 17 shows the kinetic analysis results (light chain of anti-NC5 monoclonal antibody 5A2) in the above enzymatic degradation experiment. FIG. 17A is a graph showing the relationship between the substrate (TP41-1 peptide) concentration and the degradation rate, and FIG. 17B is a graph showing the results of the Hanes-Woolf plot.

The light chain kinetic parameters of the anti-NC5 monoclonal antibody 5A2 calculated using the Hanes-Woolf plot were as follows.
Km = 1.7 × 10 ⁻⁶ (M)
kcat = 1.4 × 10 ⁻¹ (min ⁻¹ )
kcat / Km = 8.5 × 10 ⁴ (M ⁻¹ min ⁻¹ )
FIG. 18 shows the kinetic analysis result (light chain of anti-NC5 monoclonal antibody 2B8) in the above enzymatic degradation experiment. FIG. 18A is a graph showing the relationship between the substrate (TP41-1 peptide) concentration and the degradation rate, and FIG. 18B is a graph showing the results of the Hanes-Woolf plot.

The light chain kinetic parameters of anti-NC5 monoclonal antibody 2B8 calculated using the Hanes-Woolf plot were as follows.
Km = 1.1 × 10 ⁻⁵ (M)
kcat = 6.1 × 10 ⁻¹ (min ⁻¹ )
kcat / Km = 5.7 × 10 ⁴ (M ⁻¹ min ⁻¹ )
From the above results, it was found that the degradation reaction of the peptide substrate by the antibody light chain is an enzymatic reaction.

<Reactivity of anti-NC5 monoclonal antibody light chain with CCR5>
(1) Method After reacting the light chain of the anti-NC5 monoclonal antibody (5A2, 6A2, 2B8) with the reaction substrate (TP41-1 peptide) in advance, the MOLT-4 / CCR5 disruption solution (“MOLT-4 / CCR5”) About “MASANORI BABA, HIROSHI MIYAKE, MIKA OKAMOTO, YUJI IIZAWA, and KENJI OKONOGI” Establishment of a CCR5-Expressing T-Lymphoblastoid Cell Line Highly Susceptible to R5 HIV Type 1 ”AIDS RESEARCH AND HUMAN RETROVIRUSES Volume 16, Number 10,2000 , pp.9350941 ") was added to the above reaction solution so that the reaction solution: crushed solution = 4: 1 by volume ratio. The reaction solution was placed in an incubator at 25 ° C. to start the reaction. Reaction liquids were collected 0, 6, 12, 24, and 48 hours after the start of the reaction.

  All samples were subjected to SDS-PAGE (12% Running Gel, 3% Stacking Gel). During SDS-PAGE, 2 sheets of cold water and filter paper used in Western blotting, 1 Immobilion-P (Millipore, Cat.no IPVH0010 Lot.no K4DN9499W), 1 blotting buffer (25 mM Tris, 192 mM Glycine, 0) .1% SDS), and Immobilion-P and filter paper were cut into a gel size. Immobilion-P was immersed in methanol for 20 seconds 15 minutes before the end of SDS-PAGE, and then immersed in blotting buffer for 10 minutes or more. The pad and the filter paper were similarly immersed in the blotting buffer. After completion of SDS-PAGE, the gel was immersed in a blotting buffer for 5 minutes. Two pads, filter paper, Immobilion-P, gel, filter paper, two pads, and a positive electrode plate were stacked in this order on the negative electrode plate of the blotting apparatus and placed in the electrophoresis tank. Blotting buffer was poured between the anode plate and the cathode plate until the pad was soaked, and cold water was put on the outside from the upper end of the electrophoresis tank to about 1 cm. The blotting apparatus was energized (180 mA, 30 V) and blotted for 90 minutes.

  After completion of blotting, Immobilion-P was blocked with 3% skim milk / TBS-T for 1 hour. Immobilion-P was immersed in 3% skim milk / TBS-T to which RABBIT ANTI HUMAN CCR5 (manufactured by SEROTEC, Catalog Number AHP568 Lot.220501) was added to a final concentration of 1 μg / ml, and reacted for 1 hour. (Primary reaction). Immobilion-P after completion of the primary reaction was lightly washed with TBS-T, and then washed with 3% skim milk / TBS-T for 5 minutes × 3 times.

  Washed with a solution of alkaline phosphatase conjugated goat affinity purified F (ab ') 2 fragment to Rabbit IgG (whole molecule) (Cappel, Catalog Number 59306 Lot.00852) diluted 1000 times with 3% skim milk / TBS-T Of Immobilion-P was immersed and reacted for 1 hour (secondary reaction). Immobilion-P after the completion of the secondary reaction was lightly washed with TBS-T, and then washed with 3% skim milk / TBS-T for 5 minutes × 3 times. Positive bands were detected with BCIP / NBT (detection time 20 minutes). The intensity of the detected positive band was digitized by NIH Image (obtained from NIH, USA) and graphed.

(2) Results FIG. 9 shows the results of Western blotting. In the figure, “MOLT-4 / CCR5” at the top is the result when the light chain of the anti-NC5 monoclonal antibody (5A2, 2B8) is not allowed to act on MOLT-4 / CCR5 for comparison, Those with “5A2” are the results when the anti-NC5 monoclonal antibody 5A2 light chain is allowed to act on MOLT-4 / CCR5, and those with “2B8” are those with the anti-NC5 monoclonal antibody 2B8 light chain. It is a result at the time of making it act on MOLT-4 / CCR5. Also, in the figure, those with “0”, “6”, “12”, “24”, “48” at the top indicate “reaction solution after 0 hours of reaction start”, “reaction after 6 hours of reaction start”. “Reaction liquid”, “reaction liquid 12 hours after the start of reaction”, “reaction liquid 24 hours after the start of reaction”, and “reaction liquid 48 hours after the start of reaction” are shown. In the figure, “M” indicates a molecular weight marker.

  FIG. 10 is a graph of the band density in FIG. 9 quantified by NIH Image and graphed, and shows the change with time in the concentration (relative value) of MOLT-4 / CCR5 as a reaction substrate. In FIG. 10, the square symbol indicates the result when the anti-NC5 monoclonal antibody 5A2 light chain is allowed to act on MOLT-4 / CCR5, and the diamond symbol indicates the anti-NC5 monoclonal antibody 6A2 light chain MOLT-4 / CCR5. The circle symbol is the result when the light chain of the anti-NC5 monoclonal antibody 2B8 is allowed to act on MOLT-4 / CCR5, and the triangular symbol is the anti-NC5 monoclonal antibody (5A2) for comparison. , 6A2, 2B8) are the results when MOLT-4 / CCR5 is not allowed to act on the light chain.

  From the results of FIG. 9 and FIG. 10, the light chain of the anti-NC5 monoclonal antibody (5A2, 6A2, 2B8) and the light chain of the anti-NC5 monoclonal antibody (5A2, 6A2, 2B8) were observed. When compared with the case where the chain was allowed to act on MOLT-4 / CCR5, a decrease in MOLT-4 / CCR5 was confirmed in the latter case. In particular, when the light chain of the anti-NC5 monoclonal antibody 2B8 was allowed to act on MOLT-4 / CCR5, a marked decrease in MOLT-4 / CCR5 was confirmed.

  From the above results, it was found that the light chain of the anti-NC5 monoclonal antibody (5A2, 6A2, 2B8) is an antibody enzyme that cleaves the chemokine receptor CCR5.

  When AIDS virus infects humans, gp120 and gp41 present on the virus side, CD4 and chemokine receptor CCR5 present on the human cell side play an important role, and chemokine receptor CCR5 is not normal It is known that AIDS virus infection is not established.

  Since the antibody enzyme according to the present invention specifically acts on CCR5 as described above and can be decomposed to lose its function, anti-HIV for the prevention of HIV infection and the treatment of AIDS. Useful as a drug. The antibody enzyme of the present invention destroys CCR5 with a completely different mechanism of action from the conventional anti-HIV drug that suppresses the action of CCR5, so that no effective therapeutic method has been found at present. It is expected that it can be used effectively. It can also be used as a research tool for AIDS.

  Therefore, the present invention can be used in the medical, pharmaceutical industry, reagent industry, etc., and is very useful.

It is a figure which shows typically the three-dimensional structure estimated as a result of performing the three-dimensional structure modeling (molecular modeling) of the variable region of the anti-NC5 monoclonal antibody 5A2. It is a figure which shows typically the three-dimensional structure estimated as a result of performing the three-dimensional structure modeling (molecular modeling) of the variable region of the anti-NC5 monoclonal antibody 5A2. It is a figure which shows typically the three-dimensional structure estimated as a result of performing the three-dimensional structure modeling (molecular modeling) of the variable region of the anti-NC5 monoclonal antibody 6A2. It is a figure which shows typically the three-dimensional structure estimated as a result of performing the three-dimensional structure modeling (molecular modeling) of the variable region of the anti-NC5 monoclonal antibody 2B8. It is a line graph which shows the result of having examined the peptidase activity of the heavy chain and light chain of anti-NC5 monoclonal antibody 5A2 (Lot.1). It is a line graph which shows the result of having examined the peptidase activity of the heavy chain and light chain of anti-NC5 monoclonal antibody 5A2 (Lot.2). It is a line graph which shows the result of having examined the peptidase activity of the heavy chain and light chain of anti-NC5 monoclonal antibody 6A2. It is a line graph which shows the result of having examined the peptidase activity of the heavy chain and light chain of anti-NC5 monoclonal antibody 2B8. It is a figure which shows the result of the Western blot performed in order to investigate the reactivity of the light chain of anti- NC5 monoclonal antibody (5A2, 2B8) and CCR5. It is a line graph which shows the result of having examined the reactivity of the light chain and CCR5 of an anti-NC5 monoclonal antibody (5A2, 6A2, 2B8). It is a chart figure which shows the result which followed the degradation reaction of TP41-1 peptide when examining the peptidase activity of the light chain of anti-NC5 monoclonal antibody 5A2 (Lot.1) by HPLC. It is a chart figure which shows the result which followed the degradation reaction of TP41-1 peptide when examining the peptidase activity of the light chain of anti-NC5 monoclonal antibody 5A2 (Lot.2) by HPLC. It is a chart figure which shows the result of having tracked the degradation reaction of TP41-1 peptide by HPLC when examining the peptidase activity of the light chain of anti-NC5 monoclonal antibody 6A2. It is a chart which shows the result of having tracked the degradation reaction of TP41-1 peptide by HPLC when examining the peptidase activity of the light chain of anti-NC5 monoclonal antibody 2B8. It is a bar graph which shows the result of having investigated the cross-reactivity with various peptides and proteins other than NC5 peptide by the ELISA method using the culture supernatant of an anti-NC5 monoclonal antibody producing cell (5A2 and 6A2). It is a bar graph which shows the result of having investigated the cross-reactivity with various peptides and proteins other than NC5 peptide by the ELISA method using the culture supernatant of an anti-NC5 monoclonal antibody producing cell (2B8, 3F4, and 4H6). FIG. 17 (a) is a graph showing the relationship between the substrate concentration and the degradation rate, in which the kinetic analysis results in the substrate (TP41-1 peptide) enzymatic degradation experiment with the light chain of the anti-NC5 monoclonal antibody 5A2 are shown. 17 (b) is a graph showing the results of the Hanes-Woolf plot. FIG. 18 (a) is a graph showing the relationship between the substrate concentration and the degradation rate, in which the kinetic analysis results in the substrate (TP41-1 peptide) enzymatic degradation experiment with the light chain of the anti-NC5 monoclonal antibody 2B8 are shown. 18 (b) is a graph showing the results of the Hanes-Woolf plot.

Claims (11)

An antibody or antibody fragment against the N-terminal region of human-derived chemokine receptor CCR5,
Specifically binds to the N-terminal region of the chemokine receptor CCR5, and
An antibody enzyme having an activity of degrading the chemokine receptor CCR5.   2. The antibody enzyme according to claim 1, wherein the antibody enzyme comprises a variable region of an antibody against the N-terminal region of the chemokine receptor CCR5.   The antibody enzyme according to claim 1 or 2, wherein the antibody enzyme has a catalytic triad residue structure. The antibody enzyme comprises an amino acid sequence represented by SEQ ID NO: 1, or an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1. The antibody enzyme according to claim 3, wherein The antibody enzyme is an amino acid sequence shown in SEQ ID NO: 5, or
4. The antibody enzyme according to claim 3, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 5. The antibody enzyme is an amino acid sequence shown in SEQ ID NO: 9, or
4. The antibody enzyme according to claim 3, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 9.   A gene encoding the antibody enzyme according to any one of claims 1 to 6.   The gene according to claim 7, which comprises the base sequence shown in SEQ ID NO: 2.   8. The gene according to claim 7, comprising the base sequence shown in SEQ ID NO: 6.   The gene according to claim 7, comprising the base sequence shown in SEQ ID NO: 10.   A transformant into which the gene according to any one of claims 7 to 10 has been introduced.

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