Invention relating to a technique for modifying antibody subsites

Abstract

The object of this invention is to provide a novel modified antibody that improves effector activity. [Solution] The effector molecule is bound via the subsite and canonical site, The aforementioned subsite is an antibody in which the wild-type subsite has been modified, Compared to an antibody having the same structure as the antibody except for the presence of the wild-type subsite, an antibody exhibiting improved effector activity via binding to the effector molecule solves the above problem.

Description

[Technical Field]

[0001] This invention relates to a technique for modifying antibody subsites. [Background technology]

[0002] Antibodies activate effector functions, such as cytotoxic activity, through antigen recognition in the Fab region and subsequent binding to effector cells in the Fc region. Many effector molecules, such as complement and Fcγ receptors (FcγR), are known to bind to a common site (canonical site) on the Fc region. To date, attempts have been made to improve the performance of antibody drugs by focusing on the Fc region, the interaction site, and modifying the molecules to increase the affinity between the two and enhance effector activity. However, this approach targeting the canonical site has limitations in improving effector activity. On the other hand, it is known that each effector molecule can bind not only to the canonical site in the antibody molecule but also to other interaction sites (subsites) (Non-Patent Literature 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Rina Yogo et al. The Fab portion of immunoglobulin G contributes to its binding to Fcγreceptor III. Scientific Reports. 2019; 9: 11957 [Overview of the project] [Problems that the invention aims to solve]

[0004] The present invention aims to provide a novel modified antibody that binds to an effector molecule via a subsite and a canonical site, wherein the subsite is a modified wild-type subsite, and which exhibits improved effector activity via binding to the effector molecule compared to an antibody having the same structure as the antibody except for the presence of the wild-type subsite. [Means for solving the problem]

[0005] As a result of diligent research, the inventors discovered that subsites are involved in antibody activity, and by modifying the subsites molecularly, they developed a technology to improve effector activity, thus completing the present invention. In other words, the present invention includes the following aspects.

[0006] Item 1. It binds to the effector molecule via the subsite and canonical site, The aforementioned subsite is an antibody in which the wild-type subsite has been modified, An antibody having the same structure as the antibody except for having the aforementioned wild-type subsite, wherein effector activity via binding to the effector molecule is improved. Item 2. The antibody according to Item 1, wherein the effector molecule is FcγRI, FcγRII, FcγRIII, or C1q. Item 3. The aforementioned subsite is C H 1 area or C L The antibody described in item 1 or 2, which is the region. Item 4. The antibody according to any one of items 1 to 3, wherein the effector activity is ADCC activity, CDC activity, or phagocytic activity. Item 5. The antibody is a humanized anti-HER2 antibody, C L Amino acid sequence number Thr105-Glu124, C L Amino acid sequence numbers Thr163-Tyr177, C H Amino acid sequence number 1, Pro127-Leu142, C H Amino acid sequence numbers Asn159-Leu174 or C1 of the 1st region HThe antibody according to any one of items 1 to 4, wherein the amino acid residues of the subsite represented by amino acid sequence numbers Val186 - Thr197 in domain 1 are substituted with other amino acid residues. Item 6. The antibody according to any one of items 1 to 5, wherein the 192nd S, the 159th N, the 163rd L, and the 189th P of the subsite represented by SEQ ID NO: 1 of the humanized anti - HER2 antibody are each substituted with K, K, R, or K. Item 7. The antibody according to any one of items 1 to 6, wherein the heavy - chain constant region has an amino acid sequence selected from any of SEQ ID NOs: 2 to 5. Item 8. The antibody according to any one of items 1 to 7, wherein the canonical site (Fc region) is a modified wild - type canonical site. Item 9. The antibody according to any one of items 1 to 8, wherein the antibody is IgG. Item 10. An anti - cancer agent comprising the antibody according to any one of items 1 to 9. Item 11. A method for producing the antibody according to any one of items 1 to 9, comprising a step of modifying the subsite of the antibody.

Advantages of the Invention

[0007] According to the present invention, an antibody that binds to an effector molecule via a subsite and a canonical site, wherein the subsite is a modified wild - type subsite, and compared with an antibody having the same structure as the antibody except having the wild - type subsite, a novel modified antibody with improved effector activity via binding to the effector molecule can be obtained.

Brief Description of the Drawings

[0008] [[ID=@26]] [Figure 1] A figure schematically showing the subsite and the canonical site in the present invention. [Figure 2] A figure showing the effector activities of a humanized anti - HER2 antibody having a modified subsite against FcγIIIa, FcγRIIa, and FcγRI. [Figure 3]This is a figure showing the effector activities of a humanized anti-HER2 antibody having a modified subsite against FcγIIIa, FcγRIIa, and FcγRI (measured separately from Figure 2).

Mode for Carrying Out the Invention

[0009] As the antibody (immunoglobulin) of the present invention, an antibody having cytotoxic activity through immune functions such as antibody-dependent cell phagocytosis (ADCP) activity, suppression, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and complement-dependent cytotoxicity (CDC) activity is used. The antibody preferably has a determined amino acid sequence. As the class (isotype) of the immunoglobulin, any of IgG, IgA, IgM, IgD, and IgE can be used, and among these, IgG is preferred. The subclasses of IgG can all be used, and are appropriately selected according to the effector activity to be modulated. When the effector molecule is FcγRIII, it is preferable to use IgG1, but it is not limited thereto. Among IgG1, an anti-HER2 antibody (trastuzumab) is preferred. The origin of the antibody may be an animal-derived antibody such as a mouse, or it may be a human antibody, a chimeric antibody, or a humanized antibody. Among these, a humanized antibody is preferred.

[0010] The effector molecule of the present invention is a molecule that specifically binds to an antibody. As the effector molecule, for example, FcγRI, FcγRII, FcγRIII, C1q, etc. can be used, but it is not limited thereto, and any molecule can be used, and is appropriately selected according to the antibody to be used. In the present invention, FcγRIII and C1q, for which the interaction site with the subsite of the antibody has been determined, are preferred, and FcγRIII is most preferred.

[0011] The antibody and effector molecule used in the present invention specifically bind via a canonical site and a subsite. Examples of the combination of the antibody and effector molecule of the present invention include combinations such as a humanized anti-HER2 antibody and FcγRIII, the same antibody and FcγRI, the same antibody and FcγRII, and the same antibody and C1q. The effector activity induced varies depending on the combination of the antibody and effector molecule. For example, in the case of the combination of a humanized anti-HER2 antibody and FcγRIII, ADCC activity is induced; in the case of the combination of the same antibody and FcγRI, phagocytic activity is induced; in the case of the combination of the same antibody and FcγRII, inhibitory activity is induced; and in the case of the combination of the same antibody and C1q, CDC activity is induced.

[0012] In the present invention, the canonical site refers to a region that interacts with an effector molecule via the vicinity of C H near 2. The canonical site is a conserved interaction region regardless of the type of effector molecule.

[0013] In the present invention, the subsite refers to a new interaction region other than the canonical site among the interaction regions of the antibody and the effector molecule. The subsite was first discovered by the present inventors and exists, for example, in the C H 1 region or C L region of the Fab region, the C H 3 region of the Fc region, etc. The present inventors have found that the subsite varies depending on the combination of the effector molecule and the antibody. Examples of such combinations of the subsite and the antibody are as follows. The humanized anti-HER2 antibody interacts with FcγRIII using the C L region or C H 1 region of the Fab region as a subsite. Specifically, the amino acid sequence numbers Thr105-Glu124 and Thr163-Tyr177 of the C L region and the C HThe amino acid sequences Pro127-Leu142, Asn159-Leu174, and Val186-Thr197 in one region are subsites. In this invention, amino acid residue numbers were assigned according to Eu numbering, a numbering method for antibody sequences.

[0014] In the present invention, "modified" refers to a state in which the amino acids of the wild-type subsite are substituted, deleted, inserted, or chemically modified, or a combination thereof. In the present invention, a configuration in which at least one amino acid residue in the subsite is substituted with another amino acid residue is preferred, and a configuration in which one amino acid residue in the subsite is substituted with another amino acid residue is most preferred. Such substitutions include C L Amino acid sequence number Thr105-Glu124, C L Amino acid sequence numbers Thr163-Tyr177, C H Amino acid sequence number 1, Pro127-Leu142, C H Amino acid sequence numbers Asn159-Leu174 or C1 of the 1st region H One example is when an amino acid residue in a subsite indicated by amino acid sequence numbers Val186-Thr197 in a single region is substituted with another amino acid residue. Among these, for example, in the interaction between a humanized anti-HER2 antibody and FcγRIII, the antibody's C H A preferred embodiment is one in which the 192nd S in a subsite of one region is replaced with K, the 159th N is replaced with K, the 163rd L is replaced with R, and the 189th P is replaced with K.

[0015] In the present invention, in addition to subsite modification, the canonical site may also be modified. Canonical site modification includes the substitution, deletion, or insertion of amino acids in the wild-type canonical site, but also includes all previously reported canonical site modifications. Examples of canonical site modification include fucose residue knockout and modifications using GASDALI mutants to increase affinity for FcγRIIIA.

[0016] The antibody of the present invention exhibits improved effector activity via binding to the effector molecule compared to an antibody having the same structure as the antibody except for the presence of a wild-type subsite. Whether or not the effector activity is improved is determined by evaluating the effector activity of the antibody using a reporter assay system that detects effector activity. More specifically, this can be done by the method described in the examples.

[0017] 1. Method for producing modified antibodies The modified antibody of the present invention can be prepared as follows. (1) Obtaining antibodies The antibodies of the present invention can be obtained by expressing a vector encoding the antibody sequence in appropriate cells and purifying it. Known methods can be used for expression and purification. For example, trastuzumab can be expressed in CHO-HcD6 cells according to prior literature (Onitsuka, M. & Omasa, T. Rapid evaluation of N-glycosylation status of antibodies with chemiluminescent lectin-binding assay. J Biosci Bioeng 120, 107-110 (2015)), and cultured and purified according to Rina Yogo et al. The Fab portion of immunoglobulin G contributes to its binding to Fcγreceptor III. Scientific Reports. 2019; 9: 11957.

[0018] (2) Obtaining effector molecules The effector molecule of the present invention can be obtained by expressing a vector containing the gene sequence encoding the effector molecule in appropriate cultured cells and purifying it. Existing methods can be used for expression, culture, and production. For example, FcγRIIIa can be obtained by expressing a vector encoding sFcγRIIIa, a recombinant with histidine at the C-terminus, in CHO cells according to Rina Yogo et al. The Fab portion of immunoglobulin G contributes to its binding to Fcγreceptor III. Scientific Reports. 2019; 9: 11957, and purifying it by His-tagged column and gel filtration.

[0019] (3) Identification of interaction sites In this invention, first, the interaction region between the antibody and the effector molecule is determined. From the determined interaction region, subsites, which are interaction regions other than those mediated by the canonical site, are determined. For determining subsites, for example, crystal structure analysis of the antibody and effector molecule, cryo-electron microscopy, molecular dynamics simulation (MD simulation), etc., can be used, but are not limited to these, and any existing method can be used.

[0020] (4) Prediction of mutant amino acids For the canonical site determined in (3), molecular simulations using molecular dynamics or other methods predict the amino acid residues that modulate the interaction between the antibody and the effector molecule. It is preferable to introduce amino acid mutations that enhance the interaction between the antibody and the effector molecule. By substituting or modifying amino acids that are predicted to strengthen interactions such as electrostatic interactions, hydrophobic interactions, and hydrogen bonding, the interaction between the antibody and the effector molecule can be enhanced. For example, in the case of trastuzumab and FcγRIII, the mutant amino acids can be predicted as follows. In molecular simulation, a model of the complex of full-length IgG including the subsite in the Fab of trastuzumab and FcγRIIIa is constructed. For the full-length IgG of trastuzumab, a structure created using the IgG structure registered in the PDB as a template is created by molecular simulation. For FcγRIIIa, the crystal structure of PDB: 3AVE can be used, and molecular simulation of the complex with additional sugar residues can be performed using the AMBER package program or similar.

[0021] (5) Preparation of IgG variants The modified antibody of the present invention can be obtained by mutating specific amino acids in a subsite using molecular biological techniques. Mutations can be introduced according to existing methods. For example, they can be introduced by modifying a wild-type vector using PCR.

[0022] The modified antibody of the present invention can modulate effector activity through interaction with effector molecules via subsites. Modulation of effector activity includes enhancement and reduction of activity. It is preferable to improve effector activity by enhancing interaction with the Fc region.

[0023] The pre-modification humanized anti-HER2 antibody (trastuzumab) (hereinafter referred to as WT) is an existing sequence, and the heavy chain constant region (C) shown in SEQ ID NO: 1 H 1 area, C H 2 regions and C H It has 3 regions. Other light chain constant regions (CL Region) and variable region (V H Region, and V L The sequence of the region is an existing sequence and can be obtained, for example, from amino acid sequence information of biopharmaceuticals registered in DrugBank. The heavy chain constant region of the modified humanized anti-HER2 antibody preferably has the sequence shown in one of sequence numbers 2 to 5. The regions other than the heavy chain constant region of the modified humanized anti-HER2 antibody may have one or more amino acids deleted, substituted, added, inserted, or chemically modified, or a combination thereof, to the extent that it does not affect the effector activity.

[0024] C in the heavy chain constant region of a modified humanized anti-HER2 antibody H In the first region, it is preferable that the subsite, which is a subsite, has one of the following substitutions: the 192nd S is replaced with K, the 159th N is replaced with K, the 163rd L is replaced with R, and the 189th P is replaced with K. In this case, one or more amino acids may be deleted, substituted, or added to regions other than the above subsite, as long as it does not affect the effector activity. It is even more preferable that the heavy chain constant region of the modified humanized anti-HER2 antibody has one of the sequences of SEQ ID NOs: 2 to 5.

[0025] 2.Applications The modified antibody of the present invention can be used as an antibody drug, particularly as an anticancer agent, containing the modified antibody. The antibody drug may contain any other components besides the modified antibody of the present invention, such as surfactants, modifiers, preservatives, stabilizers, agglutination inhibitors, etc. The antibody drug is usually formulated using a pharmaceutically acceptable carrier by a method known to those skilled in the art and used as a pharmaceutical preparation. For formulation, commonly used excipients, bulking agents, binders, wetting agents, disintegrants, surfactants, lubricants, dispersants, buffers, preservatives, solubilizers, preservatives, colorants, flavoring agents, and stabilizers can be used. The modified antibody of the present invention can be administered orally or parenterally, such as by injection or intravenous infusion. Parenteral administration can be administered orally, intra-intramuscularly, intravenously, or intra-orally. Antibody drugs can be administered in various forms, including tablets, powders, granules, syrups, sprays, capsules, emulsions, suppositories, injections, intravenous infusions, ointments, or transdermal patches. [Examples]

[0026] The present invention is further illustrated by the following embodiments, which should not be construed as further limitations. Example 1. Preparation of a modified humanized anti-HER2 antibody (trastuzumab) We prepared modified trastuzumab using a humanized anti-HER2 antibody (trastuzumab) as the antibody and FcγRIIIa as the effector molecule, and then investigated the effector activity of the modified trastuzumab.

[0027] 1-1. Obtaining trastuzumab Trastuzumab (anti-HER2 humanized IgG1 (G1m17,1; Km3)) was expressed in the CHO-HcD6 cell line according to a previously reported method (Onitsuka, M. & Omasa, T. Rapid evaluation of N-glycosylation status of antibody with chemiluminescent lectin-binding assay. J Biosci Bioeng 120, 107-110 (2015)). CHO-HcD6 cells were cultured in BalanCD® CHO Growth A medium (Irvine Scientific) supplemented with 2 mM L-glutamine, 1% penicillin-streptomycin (Thermo Fisher Scientific), and 7.5 μg / ml puromycin (Nacalai Tesque). After cell proliferation, the supernatant of the culture medium was applied to an nProtein A Sepharose Fast Flow column (GE Healthcare), and then gel filtration was performed using a HiLoad 16 / 60 Superdex 200 pg column (GE Healthcare). IgG1 was further purified with a buffer containing 50 mM Tris-HCl, pH 8.0, and 150 mM NaCl.

[0028] 1-2. Obtaining FcγRIIIa Following prior literature (Shibata-Koyama, M. et al. The N-linked oligosaccharide at Fc gamma RIIIa Asn-45: an inhibitory element for high FcγRIIIa binding affinity to IgG glycoforms lacking core fucosylation. Glycobiology 19, 126-134 (2009)), the human FcγRIIIa gene was recombinantly prepared as a glycoprotein, retaining six histidine tags at the C-terminus, two N-linked glycosylation sites (Asn45 and Asn162), and substituting the remaining three N-linked glycosylation sites (Asn38, Asn74, and Asn169) with glutamine. In this invention, this recombinant water-soluble FcγRIIIa (sFcγRIIIa) glycoprotein is simply referred to as FcγRIIIa. A synthetic gene for FcγRIIIa containing an Igκ signaling sequence was purchased from FASMAC, subcloned into the pEHX1.2 vector (Toyobo), and the protein was produced by dihydrofolate reductase (dhFr)-mediated gene amplification. The expression vector was transfected into the dhFr-deficient CHO cell line, CHO / dhFr- (ATCC® CRL-9096). 48 hours after transfection, the transfected cells were planted in 6-well plates for methotrexate (MTX) pressure selection. The MTX concentration was gradually increased to 500 mM during multiple selection processes. MTX-resistant cells were subjected to monoclonal screening using limiting dilution to select clones with higher expression. High-expression clones were selected by ELISA using an anti-His antibody (GE Healthcare). High-expression CHO cells were cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum and 500 mM MTX. After 3 weeks of cell culture, the supernatant of the culture medium was applied to a cOmplete His-Tag Purification Resin column (Roche), and FcγRIIIa was purified using a buffer containing 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl by gel filtration using a HiLoad 16 / 60 Superdex 75 pg column.For desialylation, FcγRIIIa was incubated at 37°C for 12 hours in 50 mM sodium acetate (pH 5.5) and 150 mM NaCl in the presence of 1 unit of neuraminidase derived from Arthrobacter ureafaciens (Nacalai tesque) per 5 mg of FcγRIIIa.

[0029] 1-3. Identifying Subsites The subsite of trastuzumab interacting with FcγRIII was determined according to existing literature (Rina Yogo et al. The Fab portion of immunoglobulin G contributes to its binding to Fcγreceptor III. Scientific Reports. 2019; 9: 11957). Specifically, it was identified by hydrogen-deuterium mass spectrometry, C L The amino acid sequences in the region Thr105-Glu124, Thr163-Tyr177, and C H The amino acid sequences Pro127-Leu142, Asn159-Leu174, and Val186-Thr197 in one region were identified as subsites.

[0030] 1-4. Prediction of mutant amino acids using molecular simulations In molecular simulations, a model of the complex of trastuzumab IgG, including the subsite within the Fab, and FcγRIIIa was constructed. For the full-length trastuzumab IgG, the structure was created using the IgG structure registered in the PDB as a template and then simulated. For FcγRIIIa, the crystal structure of PDB: 3AVE was used, and molecular simulations of the complex with additional sugar residues were performed using the AMBER package program, etc. Based on the interaction model of wild-type trastuzumab and FcγRIII constructed above, amino acid residue substitutions that increase affinity with FcγRIII were predicted, targeting amino acid residues near the subsite and contact interface. In particular, modifications that strengthen interactions such as electrostatic interactions, hydrophobic interactions, and hydrogen bonding were made, and molecular simulations were performed.

[0031] The mode of interaction between IgG and FcγRIIIa via subsites is through the constant region of IgG. H 1 and C L Two models have been proposed in which IgG interacts with either of the following. In the verification of this invention, IgG's C H The results showed that FcγRIIIa interacts through one region. Based on the information of the interaction interface between IgG and FcγRIIIa obtained in the model, IgG variants that modulate the interaction were created in the following steps 1-5.

[0032] 1-5. Creation of Modified Body The C that interacts with FcγRIIIa obtained in 1-4 above H For a site presumed to be a subsite in one region, mutants (Table 1) were created by substituting a portion of the sequence shown in Sequence ID No. 1. Antibodies containing the original wild-type sequence were denoted as WT. The mutants were created by introducing amino acid mutations using primers in which the amino acid to be mutated was substituted with the target amino acid, according to existing methods.

[0033] [Table 1]

[0034] 1-6. Evaluation of Modified Forms Regarding the interaction subsite with FcγRIIIa (ADCC subsite), trastuzumab mutants designed to enhance the interaction were created, and the effector activity of the antibody was evaluated using a reporter assay system that detects effector activity mediated by FcγRIIIa. As a result, modified compounds with higher effector activity against FcγRIIIa compared to the wild type were observed (Figure 2 top, Figure 3 top). Among these modified compounds, modified compounds 4 (S192K), 8 (N159K), 9 (L163R), and 10 (P189K) were found to be preferable, as they showed approximately twice the degree of enhancement of ADCC activity compared to the wild type. On the other hand, no enhancement of effector activity was observed for FcγRIIa and FcγRI (Figure 2 bottom, Figure 3 bottom), indicating that this modified compound specifically enhances the activity of FcγRIIIa.

[0035] As described above, it was revealed that modifying the Fab region of IgG1 specifically enhances cytotoxic activity of the Fcγ receptor III, demonstrating that it is possible to enhance the functionality of the antibody as an antibody drug. Since these subsites are specific to their respective effector molecules, modifications can selectively increase or decrease affinity with specific effector molecules. Furthermore, synergistic effects can be expected when combined with modifications to the Fc region.

Claims

1. It binds to the effector molecule via the subsite and canonical site, The aforementioned subsite is an antibody in which the wild-type subsite has been modified, An antibody having the same structure as the antibody except for having the aforementioned wild-type subsite, wherein effector activity via binding to the effector molecule is improved.

2. The antibody according to claim 1, wherein the effector molecule is FcγRI, FcγRII, FcγRIII, or C1q.

3. The aforementioned subsite is C H 1 area or C L The antibody according to claim 1, which is a region.

4. The antibody according to claim 1, wherein the effector activity is ADCC activity, CDC activity, or phagocytic activity.

5. The aforementioned antibody is a humanized anti-HER2 antibody, C L Amino acid sequence number Thr105-Glu124, C L Amino acid sequence numbers Thr163-Tyr177, C H Amino acid sequence number 1, Pro127-Leu142, C H Amino acid sequence numbers Asn159-Leu174 or C1 of the 1st region H The antibody according to claim 1, wherein an amino acid residue in a subsite indicated by amino acid sequence numbers Val186-Thr197 of one region is substituted with another amino acid residue.

6. The antibody according to claim 5, wherein the subsite indicated by SEQ ID NO: 192 of the humanized anti-HER2 antibody is substituted with K, the N at 159 is substituted with K, the L at 163 is substituted with R, and the P at 189 is substituted with K.

7. The antibody according to claim 6, wherein the heavy chain constant region has an amino acid sequence selected from any of SEQ ID NOs: 2 to 5.

8. The antibody according to claim 1, wherein the canonical site (Fc region) is a modified wild-type canonical site.

9. The antibody according to claim 1, wherein the antibody is IgG.

10. An anticancer agent comprising the antibody described in claim 1.

11. A method for producing an antibody according to claim 1, comprising the step of modifying the antibody subsite.

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