JP2012158534A - ANTI-Her2 HUMAN MONOCLONAL ANTIBODY - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a human heavy chain variable part antibody which binds with Her2, a method of measurement using the antibody, and a pharmaceutical composition or a reagent containing the antibody.SOLUTION: The human heavy chain variable part antibody which is active to bind with Her2 is successfully obtained by repeating a step of selecting a phage for expressing the human heavy chain variable part antibody a plurality of times which binds with Her2-expressing human cultured cell. Thus provided are the human heavy chain variable part antibody which binds with Her2 in human cell or human tissue, a composition for detecting Her2 using the antibody, and a Her2-binding pharmaceutical composition.

Description

  The present invention relates to a human monoclonal antibody against human Her2, a measurement method using the antibody, and a pharmaceutical composition. More specifically, a novel heavy chain variable region antibody that binds to Her2 is selected from a human heavy chain variable region antibody phage library, and an immunoassay and pharmaceutical composition using the selected anti-Her2 heavy chain variable region antibody About.

  Her2 is a cell membrane protein. When an anti-Her2 antibody that binds to its extracellular region is administered to the body, it can inhibit signal transmission related to Her2, or can be expected to inhibit cancer cell growth due to signal transmission (non- Patent document 1) etc. can be expected to have a therapeutic effect on solid cancers such as breast cancer, and the blood concentration of the polypeptide in the Her2 extracellular region increases as the solid cancer grows (Non-patent document 3). It can also be expected as a method for evaluating the degree of progression of the disease state, such as diagnosis of solid cancer such as PET.

  Based on a mouse monoclonal antibody (epitope name: 4D5) that binds to Her2 extracellular domain IV and inhibits the physiological activity of Her2, human framework region (FR) leaving non-human complementarity determining region (CDR) residues An antibody (Herceptin; Trastuzumab) (Non-patent Document 1, Patent Document 1) that has been humanized by introducing residues and developed as a pharmaceutical, mouse monoclonal that binds to Her2 extracellular domain I and inhibits the physiological activity of Her2 There is a description of an antibody (Patent Document 2) that is humanized based on an antibody (epitope name: 7C2 / 7F3), leaving its CDR portion.

  Moreover, scFv (single chain Fv), its dimer, etc. have been reported as a low molecular antibody which recognizes epitope 4D5 couple | bonded with Her2 extracellular domain IV (epitope name: 4D5) (nonpatent literature 2). Among the low-molecular-weight antibodies, heavy chain variable region antibodies (VH domain antibodies) have a molecular weight of about 25 kDa, whereas the molecular weight of immunoglobulin (IgG) is about 150 kDa. Production using yeast or other microorganisms as the host, 2) Reduction in drug substance production cost because the host is a microorganism, 3) Improvement in cell or tissue permeability, and 4) improvement in formulation stability can be expected.

  In obtaining a heavy chain variable region antibody which is a low molecular weight antibody, a method called a phage display method is currently mainly used (Non-patent Documents 4 and 5). In this method, a phage displaying a heavy chain variable region antibody in E. coli is produced, and a phage having binding activity to the target antigen is selected.

  Prior art document information related to the invention of the present application is shown below.

Special Table 2002-544238 JP 2008-188013 A

Novel therapeutic strategies targeting the epidermal growth factor receptor (EGFR) family and its downstream effectors in breast cancer., G. Atalay, F. Cardoso, A. Awada & M. J. Piccart, Annals of Oncology, 2003; 14: 1346-1363. PEGylation and multimerization of the anti-p185HER-2 singlechain Fv FRagment 4D5: Effects on tumor targeting., S. Kubetzko, E. Balic, R. Waibel, U. Zangemeister-Wittke, and A. Pluckthun, J Biol Chem. 2006 Nov 17; 281 (46): 35186-201. Potential clinical utility of serum HER-2 / neu oncoprotein concentrations in patients with breast cancer., Carney WP, Neumann R, Lipton A, Leitzel K, Ali S, Price CP., Clin Chem. 2003 Oct; 49 (10): 1579 -98. Selection of human antibody FRagments by phage display., Lee CM, Iorno N, Sierro F, Christ D., Nat Protoc. 2007; 2 (11): 3001-8. Sequence determinants of protein aggregation in human VH domains., Dudgeon K, Famm K, Christ D., Protein Eng Des Sel. 2009 Mar; 22 (3): 217-20.

  The present invention uses an animal cell forcibly expressing Her2 as an antigen, selects a functional antibody against Her2 from a human heavy chain variable region antibody phage library by a cell panning method, and measures using the antibody It is an object of the present invention to provide a method and a pharmaceutical composition or reagent containing the antibody.

  Positive clones could be obtained from the human heavy chain variable region antibody phage library using the cell panning method for Her2 highly expressing cells. The obtained positive clones were confirmed to be capable of binding to Her2 by immunostaining. Further, by analyzing the nucleotide sequence, the entire nucleotide sequence of the obtained heavy chain variable region antibody clone was determined, and the entire amino acid sequence was determined based on the information.

That is, the present invention relates to a human monoclonal antibody against anti-Her2, a measurement method using the antibody, and a pharmaceutical composition or reagent containing the antibody, more specifically,
[1] Her2 having FRVSAQYMS (SEQ ID NO: 1) as the amino acid sequence of heavy chain CDR1, SIASEG (SEQ ID NO: 2) as the amino acid sequence of heavy chain CDR2, and ANSTRRNAKLGY (SEQ ID NO: 3) as the amino acid sequence of heavy chain CDR3 Heavy chain variable region antibody (VH domain antibody) that binds to
[2] In each amino acid sequence of the heavy chain CDRs 1 to 3 described in [1], the amino acid sequence in which one or two amino acids are substituted, deleted, or added to the heavy chain CDRs 1 to 3, respectively. A human heavy chain variable region antibody that binds;
[3] The human heavy chain variable region antibody that binds to Her2, wherein the antibody according to any one of [1] to [2] has a framework region (FR) derived from a human,
[4] A human heavy chain variable region antibody that binds to Her2, comprising heavy chain CDRs 1 to 3 in the amino acid sequence of the human heavy chain variable region antibody described in SEQ ID NO: 4.
[5] A Her2 detection reagent comprising as an active ingredient the human heavy chain variable region antibody that binds to Her2 according to any one of [1] to [4],
[6] A Her2 binding pharmaceutical composition comprising the human heavy chain variable region antibody that binds to Her2 according to any one of [1] to [4] as an active ingredient.

  The inventors obtained a novel human heavy chain variable region antibody clone from the human heavy chain variable region antibody gene library by cell panning by the phage display method.

Thereby, it is possible to prepare a composition for detecting Her2 using an antibody that binds to Her2. In addition, a method for detecting Her2 using the composition and method or a method for diagnosing various diseases, and a measurement kit can be supplied.

The structure of the Her2 animal cell expression vector pIRES2-EGFP is shown. Her2 gene expression vector for animal cells. An animal cell expression vector comprising a replication origin sequence for animal cells such as SV40ori, a CMV-derived promoter, an SV40-derived polyA addition sequence, and Kan / Neo as a selection marker. This vector further includes a pUC-derived replication origin sequence and the aforementioned Kan / Neo as a selection marker, and is a shuttle vector that can be amplified in E. coli. This vector is inserted with a fluorescent protein marker (FP: AcGFP1) gene linked to an IRES (ribosome entry site in mRNA) sequence for the purpose of expressing a fluorescent protein marker (FP) in animal cells. The photomicrograph of the cell staining which shows the Her2 binding activity by the human heavy chain variable region antibody expression phage (clone number 220-16) is shown. Her2 forced expression cells prepared by transfecting HEK293 cells with pIRES2-EGFP were evaluated, and the antibody binding strength was evaluated by cell staining with a human heavy chain variable region antibody expression phage of clone number 220-16. The upper micrograph is a Her2 / EGFP forced expression cell, and the lower micrograph is a neurotrophic factor receptor p75 / EGFP forced expression cell used as a negative control. The first from the left is the result of Her2 staining using the selected phage (clone number 220-16), the second from the left is the result of fluorescence development of the transfected vector-derived EGFP, and the third from the left is the DNA-binding fluorescent dye. As a result of staining of cell nuclei with Hoechst33342, the fourth from the left shows a micrograph in which the three images on the left are superimposed. These micrographs show that the selected phage (clone number 220-16) binds specifically to the Her2-expressing cell membrane. The bar shows a length of 80 μm in the micrograph. The base sequence and amino acid sequence of the anti-Her2 human heavy chain variable region antibody (clone number 220-16) are shown. The amino acid sequence surrounded by a square frame indicates the location of heavy chain CDR1, heavy chain CDR2, heavy chain CDR3 and the amino acid sequence thereof in order from the top.

  The present invention provides an anti-Her2 human heavy chain variable region antibody (VH domain antibody) having binding activity against Her2. The antibody of the present invention is preferably an isolated human heavy chain variable region antibody or a purified human heavy chain variable region antibody.

Her2 (also known as ErbB2, UniProt ACCESSION number: P04626) is a transmembrane glycoprotein with a molecular weight of about 185 kDa consisting of a total length of 1,225 amino acids, its extracellular region is composed of about 630 amino acids, and from the N-terminal of its Her2 extracellular region Domains I, II, III, and IV are recognized in order (Patent Documents 1 and 2). Antibodies against Her2 are expected to alter signal transduction through Her2.

  The “human heavy chain variable region antibody” in the present invention refers to, for example, heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, which are complementarity determining regions (CDRs) present in the heavy chain variable domain (VH) sequence. Examples thereof include an antibody containing each amino acid sequence (for example, the sequences shown in SEQ ID NOs: 1 to 3). In addition, the human heavy chain variable region antibody of the present invention includes an antibody having an altered amino acid sequence, a modified antibody to which another molecule (for example, a polymer such as polyethylene glycol) is bound, or a human heavy chain having an altered sugar chain. Chain variable region antibodies and the like are included.

  The antibody of the present invention includes an antibody in which the heavy chain CDR is appropriately modified within a range not affecting the binding property to Her2. For example, a human heavy chain variable region antibody that binds to Her2 having heavy chain CDRs 1 to 3 as amino acid sequences in which one or two amino acids are substituted, deleted, or added in each of the heavy chain CDRs 1 to 3 Is mentioned. As long as the antibody having these modified heavy chain CDRs has the same binding activity to Her2 as the above-described modified human heavy chain variable region antibody, the human heavy chain variable region antibody of the present invention is used. included.

  Here, “equivalent activity” means that the antibody of interest has the same biological or biochemical activity as the antibody of the present invention. The “activity” specifically refers to an activity that binds to Her2 expressed in animal cells such as humans.

  In the present invention, “equivalent” does not necessarily have the same level of activity, and the activity may be enhanced, or the activity may be decreased as long as it has activity.

  A method well known to those skilled in the art for preparing a polypeptide having an activity equivalent to that of a certain polypeptide includes a method for introducing a mutation into the polypeptide. For example, those skilled in the art can obtain an antibody having an activity equivalent to that of the antibody by appropriately introducing mutation into the antibody of the present invention (for example, heavy chain CDR region) using site-directed mutagenesis. Can be prepared. Amino acid mutations can also occur in nature. Thus, an antibody having an amino acid sequence in which one or two amino acids are mutated in the amino acid sequence of the antibody of the present invention and having an activity equivalent to that of the antibody is also included in the human heavy chain variable region antibody of the present invention. .

  For example, usually 75 amino acid sequences for the heavy chain CDR sequences of the present invention, ie heavy chain CDR1 (SEQ ID NO: 1), heavy chain CDR2 (SEQ ID NO: 2) and heavy chain CDR3 (SEQ ID NO: 3). Antibodies or antibody fragments having a homology (identity) of at least% (eg, 77.8% or 83.3% or more), preferably 90% or more (eg, 91.7% or more) are included in the antibody of the present invention.

  The amino acid residue to be mutated is preferably mutated to another amino acid in which the properties of the amino acid side chain are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, V), amino acids with two carboxyl groups in the structure (D, E), and 2 in the structure. An amino group having an amino acid base-containing side chain having one or more amino groups (R, K, H) and an amino acid having an aromatic-containing side chain (H, F, Y, W) can be mentioned (in parentheses) Each represents a single letter of amino acid).

  In addition to the above-described modifications, the antibody of the present invention may be further linked to other substances as long as it retains activity. Examples of other substances include peptides, lipids, sugars and sugar chains, acetyl groups, natural and synthetic polymers, and the like. These modifications can be made to confer additional functions or to stabilize the antibody.

  The human heavy chain variable region antibody of the present invention includes a human heavy chain that binds to Her2 specified by the heavy chain CDR contained in the full-length sequence of the single-chain variable region antibody fragment (VH) (the full-length sequence of the heavy chain variable domain). A chain variable region antibody can be preferably indicated.

Examples include human heavy chain variable region antibodies that bind to Her2 described below;
A human heavy chain variable region antibody that binds to Her2, comprising heavy chain CDRs 1 to 3 in the amino acid sequence of the single chain variable region antibody fragment (VH) described in SEQ ID NO: 4.

  The human heavy chain variable region antibody that binds to the epitope to which the antibody of the present invention binds can be obtained by methods known to those skilled in the art. As a known method, for example, an epitope to which the antibody of the present invention binds is determined by a usual method, and a human heavy chain variable region antibody is produced using a polypeptide having an amino acid sequence contained in the epitope as an immunogen. Is mentioned.

  The antibody of the present invention includes the above-described human heavy chain variable region antibody, an antibody with a modified amino acid sequence, a non-human animal chimeric antibody, a modified antibody to which another molecule (for example, a polymer such as polyethylene glycol) is bound, Examples include antibodies with modified sugar chains.

  As one of the preferred embodiments of the antibody of the present invention, four framework regions surrounding three heavy chain CDRs (heavy chain CDR1 to heavy chain CDR3), that is, heavy chain FR (heavy chain FR1 to heavy chain FR4) are substituted with other Examples include modified antibodies such as mutant human antibodies to which an amino acid sequence derived from a human heavy chain gene is applied.

  In addition, the human heavy chain variable region CDR (heavy chain CDR1 to heavy chain CDR3) comprises a human-derived amino acid sequence that specifically binds to Her2, and surrounds three CDRs (heavy chain CDR1 to heavy chain CDR3). Examples thereof include a chimeric antibody characterized by being a heavy chain FR derived from a non-human animal. These modified antibodies can be produced using known methods.

  Human antibodies are considered to have low antigenicity in the human body and are useful when administered to humans for therapeutic purposes. A chimeric antibody between a non-human animal and a human is useful for use in measuring Her2 concentration, such as in vitro diagnosis, as long as it can impart good properties such as antibody stability.

  In addition, after producing the chimeric antibody, amino acids may be substituted, deleted, added and / or inserted with other amino acids. The human heavy chain variable region antibodies of the present invention also include human heavy chain variable region antibodies having such amino acid substitutions.

  The binding activity of the antibody of the present invention to the antigen (Her2) is, for example, dot blotting, Western blotting, enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA). ), Immunoprecipitation, intermolecular interaction measurement using surface plasmon resonance, flow cytometry, or cell staining.

  For details of general technical means relating to these measurement / detection methods, reference can be made to reviews and books. For example, procedures using various antibodies are described in `` Methods in ENZYMOLOGY '' Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73 (Immunochemical Techniques (Part B)), ibid Vol. 74 (Immunochemical Techniques (Part C)). ), Ibid Vol. 84 (Immunochemical Techniques (Part D: Selected Immunoassays)) ibid Vol. 92 (Immunochemical Techniques (Part E: Monoclonal Antibodies and General Immunoassay Methods)), ibid Vol. 121 (Immunochemical Techniques (Part I: Hybridoma Technology) and Monoclonal Antibodies)) (published by Academic Press).

  The human heavy chain variable region antibody in the present invention is an antibody lacking other than the human heavy chain variable region antibody portion of the whole antibody such as IgG (whole antibody), and is expressed in Her2 expressed in animal cells such as humans. As long as it has the binding activity, it is not particularly limited.

  Since the human heavy chain variable region antibodies alone in the present invention are assembled and multimers (dimers, trimers, tetramers, etc.) may be formed naturally or artificially, these multimers are also human heavy chain variable region antibodies. include.

  As long as the amino acid sequence of the heavy chain variable region (heavy chain CDR) contained in the human heavy chain variable region antibody has antigen binding activity (binding activity with Her2), substitution, deletion or addition of one or two amino acids And / or may be inserted.

  A gene encoding a human heavy chain variable region antibody that binds to Her2 is isolated from a human heavy chain variable region antibody expression phage, and the human heavy chain variable region antibody that binds to Her2 is transformed into Escherichia coli or This includes the step of synthesizing a desired human heavy chain variable region antibody in a recombinant cell using animal cells or the like as a host.

  Furthermore, the present invention provides a Her2 detection reagent (test agent) containing the antibody or antibody fragment of the present invention as an active ingredient.

  A test / measurement kit containing the reagent as a constituent component is also included in the present invention.

  In addition, a composition for detecting Her2 containing the human heavy chain variable region antibody of the present invention is provided. A method for detecting Her2 using the composition or the above reagent and a method for diagnosing various diseases are also included in the present invention.

  It should be noted that all prior art documents cited in the present specification are incorporated herein by reference.

  The present invention will be described more specifically with reference to the following examples, but the present invention is not limited thereto. Various gene manipulations in the examples were performed according to the methods described in Molecular cloning third. Ed. (Cold Spring Harbor Lab. Press, 2001).

(1) Construction of a cultured human cell that expresses Her2 The cultured cell expression vector pIRES2 shown in FIG. 1 shows the full-length gene of Her2 for the purpose of expressing Her2 using a human cultured cell (HEK293 strain: Human Embryo Kidney Cell) as a host. -EGFP was inserted into the multicloning site using the SalI cleavage site and the NruI / samI cleavage site to prepare a Her2 full-length expression vector (FIG. 1). The HEK293 cells were transfected using Lipofectamin (Invitrogen) and plus reagent (Invitrogen)) according to the method indicated by Invitrogen.

(2) Concentration of positive phages by cell panning method and positive clone isolation control cells (non-specific phage adsorption cells: HEK293 strain itself) and Her2 antigen-expressing cells at 1 × 10 ⁷ cells / mL each Dilute with PBS. First, 1 × 10 ¹¹ pfu of a commercially available human heavy chain variable region antibody phage library (Danaform; domain antibody phagemid library) was added to the control cells and incubated at 4 ° C. By this operation, a group of phages that bind to the HEK293 cell surface in advance can be removed. After incubation for 30 minutes, the supernatant phage solution was recovered. Next, the recovered phage solution was added to Her2 antigen-expressing cells and incubated at 4 ° C. for 30 minutes. After incubating for 30 minutes, the cells were washed three times with 1 mL of PBS and subjected to acid elution treatment at room temperature using 1 mL of 100 mM Glycine-HCl (pH 2.0), 500 mM NaCl solution. After 5 minutes of acid elution treatment, the supernatant phage solution was recovered. The recovered phage solution was immediately neutralized by adding 20 μL of 2M Tris buffer. The collected phage solution is mixed with E. coli TG1 for infection (37 ° C., 30 minutes), seeded on LB agar medium (10 cm ² × 15 sheets) containing 100 μg / mL ampicillin and 4% glucose, and incubated at 37 ° C. Cultured overnight. In addition, the phage rescue operation from the infected Escherichia coli followed the standard method of a commercial kit. The cell panning method described above was performed 3 rounds, and the concentration operation was repeated to a level where positive phages could be isolated.

(3) Selection of positive phage by cell staining evaluation
After three rounds of selection, 48 single clone phages were prepared according to the standard method of a commercial kit, and evaluation by cell staining using the single clone phages was performed. As a result, it was confirmed that the 220-16 clone bound specifically to Her2. (Figure 2)

(4) Analysis of nucleotide sequence of 220-16 clone Phagemid was isolated from 220-16 clone that was positive in cell staining, and nucleotide sequence analysis was performed by a known method. Further, the base sequence of the obtained human heavy chain variable region antibody (VH domain antibody) is shown in SEQ ID NO: 5, and the amino acid sequence of the human heavy chain variable region antibody is shown in SEQ ID NO: 4. The base sequence and amino acid sequence of the 220-16 clone are shown together in FIG. In FIG. 3, the sequences in the square frames indicate heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. The amino acid sequences of heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 were FRVSAQYMS (SEQ ID NO: 1), SIASEG (SEQ ID NO: 2), and ANSTRRNAKLGY (SEQ ID NO: 3), respectively.

Claims (7)

It binds to Her2 having FRVSAQYMS (SEQ ID NO: 1) as the amino acid sequence of heavy chain CDR1, SIASEG (SEQ ID NO: 2) as the amino acid sequence of heavy chain CDR2, and ANSTRRNAKLGY (SEQ ID NO: 3) as the amino acid sequence of heavy chain CDR3. Human heavy chain variable region antibody (VH domain antibody).   A human that binds to Her2 having each of the amino acid sequences of heavy chain CDRs 1 to 3 described in claim 1, wherein one or two amino acids are substituted, deleted, or added, respectively, as heavy chain CDRs 1 to 3. Heavy chain variable region antibody. A human heavy chain variable region antibody that binds to Her2, wherein the antibody according to any one of claims 1 to 2 has a framework region (FR) derived from a human.   The human heavy chain variable region antibody that binds to Her2 according to any one of claims 1 to 3, wherein the antibody is a chimeric antibody or a humanized antibody.   A human heavy chain variable region antibody that binds to Her2, comprising heavy chain CDRs 1 to 3 in the amino acid sequence of the human heavy chain variable region antibody (VH domain antibody) described in SEQ ID NO: 4.   A reagent for detecting Her2 comprising the human heavy chain variable region antibody that binds to Her2 according to any one of claims 1 to 5 as an active ingredient. A Her2 binding pharmaceutical composition comprising as an active ingredient the human heavy chain variable region antibody that binds to Her2 according to any one of claims 1 to 5.