WO2018135501A1 - 抗gpr20抗体及び抗gpr20抗体-薬物コンジュゲート - Google Patents
抗gpr20抗体及び抗gpr20抗体-薬物コンジュゲート Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/77—Internalization into the cell
Definitions
- the present invention relates to an anti-GPR20 antibody that binds to GPR20 and has an internalizing action, a method for producing the anti-GPR20 antibody, an antibody-drug conjugate containing the antibody, an antitumor agent containing the antibody-drug conjugate, and the like .
- Cancer is the top cause of death, and the number of affected cases is expected to increase with the aging of the population, but the treatment needs are not yet fully met.
- Conventional chemotherapeutic agents are sufficiently selective for their side effects due to their toxicity to not only tumor cells but also normal cells due to their low selectivity. The problem is that it cannot be done. For this reason, in recent years, more selective molecular targeting drugs and antibody drugs have been developed, targeting molecules that exhibit mutations and high expression characteristic of cancer cells, and specific molecules involved in canceration of cells. It has been broken.
- Gastrointestinal stromal tumor is a mesenchymal tumor that develops in the gastrointestinal tract and mesentery from the esophagus to the rectum, and its incidence is estimated to be 1-2 per 100,000.
- Non-Patent Document 1 Approximately 86% of GISTs have activating mutations in the receptor tyrosine kinases KIT or PDGFRA, which contribute to the growth of tumor cells.
- GIST treatment is based on surgical resection, tyrosine kinase inhibitors (Tyrosine kinase inhibitor, TKI) such as Imatinib, Sunitinib, and Regorafenib are prescribed for unresectable, progressive, and metastatic GIST.
- Non-patent document 2 These TKIs are often markedly effective against GISTs with the above mutations, but require continuous administration and cannot completely eliminate GIST and are ultimately targeted KIT or PDGFRA secondary mutations, activating mutations such as RAS and BRAF, and activation of other signal transduction pathways make the drug unresponsive and the pathology progresses. In addition, for a wild type GIST that does not recognize a mutation in KIT and PDGFRA, these TKIs show little therapeutic effect (Non-patent Document 3). For this reason, development of an effective treatment method for TKI resistant GIST has been desired.
- GPR20 G Protein-coupled receptor 20
- GPCR G protein-coupled receptor
- the N-terminal side is extracellular and the C-terminal side.
- the human GPR20 gene was first cloned in 1997 (Non-Patent Document 4), but subsequently partly deduced in amino acid sequence compared to the human GPR20 gene cloned by another researcher in 2008 (Non-Patent Document 4). Reference 5).
- the latter sequence which is identical to the sequence registered in the NCBI human genome-wide sequence analysis database, is currently published on the public database as a DNA sequence and amino acid sequence encoding human GPR20.
- NM_005293, NP_005284 (NCBI ) And other accession numbers.
- GPR20 has an amino acid sequence similar to GPCR that recognizes nucleotides or lipids, but its physiological function and ligand in vivo have not been identified. From an experiment in which GPR20 was expressed in HEK293 cells, it was reported that GPR20 constitutively activates a Gi-type trimer G protein under conditions without stimulation by a ligand (Non-patent Document 5).
- GPR20 has been confirmed to express messenger RNA (mRNA) in the heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, rectum, and white blood cell, especially in the small intestine. High expression was reported (Non-patent Document 5). In the brain, expression in the thalamus, putamen and caudate nucleus has been reported (Non-patent Document 4). GPR20-deficient mice show a hyperactivity disorder phenotype characterized by an increase in total distance traveled in the open field test, suggesting that GPR20 is associated with spontaneous activity in the central nervous system ( Patent Document 1).
- mRNA messenger RNA
- Non-patent Document 6 GPR20 expression is highly expressed in GIST (Non-patent Document 6), and GPR20 expression is controlled by ets variant 1 (ETV1), which is a major transcriptional regulator of GIST.
- ETV1 ets variant 1
- Antibody has high blood stability and is expected to reduce side effects because it binds specifically to the target antigen, and many antibody drugs against molecules highly expressed on the surface of cancer cells have been developed.
- the mechanism of action of antibody drugs targeting tumor cells directly includes antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), signal blocking of receptors involved in tumor growth and induction of apoptosis Etc.
- an antibody-drug conjugate can be mentioned.
- An ADC for cancer is obtained by binding a drug having cytotoxic activity to an antibody that binds to an antigen expressed on the surface of a cancer cell and internalizes the antigen by the binding.
- the ADC for cancer can be expected to cause the cancer cell to die by accumulating the drug in the cancer cell by efficiently delivering the drug to the cancer cell (Non-patent Document 8).
- ADCETRIS (brentuximab vedotin) in which monomethyl auristatin E is bound to an anti-CD30 monoclonal antibody is approved as a therapeutic agent for Hodgkin lymphoma and anaplastic large cell lymphoma.
- Kadcyla (trastuzumab emtansine) obtained by binding emtansine to an anti-HER2 monoclonal antibody is used for the treatment of HER2-positive progression and recurrent breast cancer.
- the characteristics of the target antigen suitable for ADC as an antitumor drug are that it is specifically expressed at high levels on the surface of cancer cells, is low or not expressed in normal cells, can be internalized in cells, and antigens are cells For example, it is not secreted from the surface.
- An important feature of an antibody suitable for ADC is that it has high internalization ability in addition to specifically binding to a target antigen.
- the internalization ability of an antibody depends on the properties of both the target antigen and the antibody.
- the antigen binding site suitable for internalization can be estimated from the molecular structure of the target, and it can be easily determined from the binding strength and physical properties of the antibody. It is difficult to guess an antibody with high chemical capacity. For this reason, obtaining an antibody having high internalization ability against a target antigen is an important issue in developing highly effective ADCs (Non-patent Document 9).
- An object of the present invention is to provide an antibody that specifically binds to GPR20-positive tumor cells such as GIST, to provide an antibody-drug conjugate containing the antibody, and to have a therapeutic effect on a tumor using the antibody-drug conjugate It is to provide a pharmaceutical, a method for treating a tumor using the pharmaceutical, the antibody, a method for producing the antibody-drug conjugate, and the like.
- the present inventors have intensively studied to achieve the above-mentioned problems, and consider that GPR20 is one of the molecules characterizing GIST and can be a therapeutic target specific to GIST, and the internalization activity of anti-human GPR20 antibody And the binding mode was examined, and an anti-GPR20 antibody having high internalization activity in an antibody exhibiting a specific binding mode was found. Further, an anti-GPR20 antibody-drug conjugate in which a drug that exhibits intracellular toxicity is bound to the anti-GPR20 antibody via a linker having a specific structure is used for GPR20-positive malignant tumors such as GIST expressing GPR20. Thus, the present invention has been completed by finding that it exhibits an antitumor effect. That is, the present invention includes the following inventions.
- the present invention (1) An antibody or a functional fragment of the antibody having the characteristics described in the following (a) and (b); (A) specifically binds to GPR20, and (B) has an internalization ability to be taken up into cells after binding to GPR20. (2) The antibody or the functional fragment of the antibody according to (1), wherein GPR20 is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 1.
- the antibody or the functional fragment of the antibody according to any one of (1) to (3), (5) Any one of (1) to (4) that specifically binds to a higher order structure consisting of the amino acid sequence of amino acid numbers 1 to 48 and the amino acid sequence of amino acid numbers 108 to 125 in SEQ ID NO: 1.
- a functional fragment of the antibody, (6) Specific to at least one amino acid residue selected from the amino acid sequences set forth in amino acid numbers 1 to 48 in SEQ ID NO: 1 and at least one amino acid residue selected from the amino acid sequences set forth in amino acid numbers 108 to 125
- CDRL1, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 92, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 11,
- C CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 4,
- CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 5,
- CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 6, and amino acid sequence set forth in SEQ ID NO: 9.
- CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 14
- CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 15
- CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 16, and consisting of the amino acid sequence set forth in SEQ ID NO: 19.
- CDRL1, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 20, CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 21, and (E) CDRH1 consisting of the amino acid sequence set forth in SEQ ID NO: 24, CDRH2 consisting of the amino acid sequence set forth in SEQ ID NO: 25, CDRH3 consisting of the amino acid sequence set forth in SEQ ID NO: 26, and amino acid sequence set forth in SEQ ID NO: 29 CDRL1, CDRL2 consisting of the amino acid sequence set forth in SEQ ID NO: 30, and CDRL3 consisting of the amino acid sequence set forth in SEQ ID NO: 31, (11)
- the variable region of the heavy chain according to any one of the following selected from the group consisting of the following (a) to (c), and any one of the selected from (d) to (h)
- the antibody or the functional fragment of the antibody according to any one of (1) to (14), which is humanized (16) The variable region of the heavy chain consisting of the amino acid sequence according to any one of the following (a) to (h), and the group consisting of (i) to (o) The antibody or the functional fragment of the antibody according to (15) having a light chain variable region comprising the amino acid sequence of any one of the above: (A) the amino acid sequence represented by amino acid numbers 20 to 142 in SEQ ID NO: 48, (B) the amino acid sequence of amino acid numbers 20 to 142 in SEQ ID NO: 50, (C) the amino acid sequence of amino acid numbers 20 to 142 in SEQ ID NO: 52, (D) the amino acid sequence of amino acid numbers 20 to 142 in SEQ ID NO: 54, (E) the amino acid sequence of amino acid numbers 20 to 142 in SEQ ID NO: 56, (F) the amino acid sequence of amino acid numbers 20 to 142 in SEQ ID NO: 44, (G) an amino acid sequence having at least 95% homology
- a method for producing the antibody or functional fragment of the antibody (27) An antibody obtained by the production method according to (26) or a functional fragment of the antibody, (28) The functional fragment of the antibody according to (27), wherein the functional fragment is selected from the group consisting of Fab, F (ab) 2, Fab ′ and Fv, (29) Addition of sugar chain to N-bond, addition of sugar chain to O-bond, N-terminal processing, C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation, methionine residue at N-terminal (1) to (19), comprising addition of a group, amidation of a proline residue and one or more modifications selected from the group consisting of heavy chains in which one or two amino acids are deleted at the carboxyl terminus 27) and the antibody according to any one of (28) or a functional fragment of the antibody, (30) The antibody according to (29), wherein one or two amino acids are deleted at the carboxyl terminus of the heavy chain, (31) The antibody according to (30), wherein one
- the antitumor compound has a-(CH 2 ) n 2- It binds to the carbonyl group of the C ( ⁇ O)-moiety, where GGFG represents an amino acid sequence linked by a peptide bond consisting of glycine-glycine-phenylalanine-glycine.
- GGFG represents an amino acid sequence linked by a peptide bond consisting of glycine-glycine-phenylalanine-glycine.
- the antibody is an antibody comprising a heavy chain and a light chain according to any one selected from the group consisting of the following (a) to (x) or a functional fragment of the antibody: (41) to (43)
- the antibody-drug conjugate according to any one of the items: (A) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 48, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 58; (B) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 48, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 60; (C) a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 48, and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 62
- Method of treatment (60) The method for treating a tumor according to (59), wherein the antitumor drug is a tyrosine kinase inhibitor, (61) The method of treating a tumor according to (60), wherein the tyrosine kinase inhibitor is at least one selected from sunitinib, imatinib, and regorafenib.
- the tumor treatment method according to any one of (56) to (61), wherein the tumor is a gastrointestinal stromal tumor that is resistant to a tyrosine kinase inhibitor, (63)
- a method for producing an antibody-drug conjugate comprising a step of reacting an antibody or a functional fragment of the antibody with a drug-linker intermediate compound, Consists of.
- the anti-GPR20 antibody of the present invention recognizes a higher order structure consisting of two extracellular regions of the amino acid sequence 1 to 48 from the N-terminus of GPR20 and the amino acid sequence 108 to 125, and has internalization activity It is characterized by.
- the anti-GPR20 antibody-drug conjugate obtained by binding a drug exhibiting intracellular toxicity to the anti-GPR20 antibody of the present invention via a linker having a specific structure is administered to a patient having a cancer cell expressing GPR20. Therefore, it can be expected to achieve an excellent antitumor effect and safety. That is, the anti-GPR20 antibody-drug conjugate of the present invention is useful as an antitumor agent.
- SEQ ID NO: 2 shows the heavy chain amino acid sequence (SEQ ID NO: 2) of rat anti-GPR20 antibody 04-046 and the nucleotide sequence of the cDNA encoding the heavy chain (SEQ ID NO: 32).
- 1 shows the amino acid sequence of the light chain (SEQ ID NO: 7) of rat anti-GPR20 antibody 04-046 and the nucleotide sequence of the cDNA encoding the light chain (SEQ ID NO: 34).
- 1 shows the amino acid sequence of the heavy chain (SEQ ID NO: 12) of rat anti-GPR20 antibody 04-079 and the nucleotide sequence of the cDNA encoding the heavy chain (SEQ ID NO: 36).
- 1 shows the amino acid sequence of the light chain (SEQ ID NO: 17) of rat anti-GPR20 antibody 04-079 and the nucleotide sequence of the cDNA encoding the light chain (SEQ ID NO: 38).
- 2 shows the amino acid sequence (SEQ ID NO: 22) of the heavy chain of rat anti-GPR20 antibody 04-126 and the nucleotide sequence (SEQ ID NO: 40) of the cDNA encoding the heavy chain.
- 2 shows the amino acid sequence (SEQ ID NO: 27) of the light chain of rat anti-GPR20 antibody 04-126 and the nucleotide sequence (SEQ ID NO: 42) of the cDNA encoding the light chain.
- SEQ ID NO: 44 shows the amino acid sequence of the heavy chain of human chimerized anti-GPR20 antibody 04-046Ch and the nucleotide sequence of the cDNA encoding the heavy chain (SEQ ID NO: 46).
- 1 shows the amino acid sequence of the light chain of human chimerized anti-GPR20 antibody 04-046Ch (SEQ ID NO: 45) and the nucleotide sequence of cDNA encoding the light chain (SEQ ID NO: 47).
- the amino acid sequence of the humanized h046-H4b type heavy chain (SEQ ID NO: 48) is shown.
- the amino acid sequence of the humanized h046-H4e type heavy chain (SEQ ID NO: 50) is shown.
- the amino acid sequence of the humanized h046-H5b type heavy chain (SEQ ID NO: 52) is shown.
- the amino acid sequence of the humanized h046-H8 type heavy chain (SEQ ID NO: 54) is shown.
- the amino acid sequence of the humanized h046-H10 type heavy chain (SEQ ID NO: 56) is shown.
- 1 shows the amino acid sequence of humanized h046-L1 type light chain (SEQ ID NO: 58).
- 1 shows the amino acid sequence of humanized h046-L2 type light chain (SEQ ID NO: 60).
- 2 shows the amino acid sequence of humanized h046-L6 type light chain (SEQ ID NO: 62).
- FIG. 1 shows the amino acid sequence (SEQ ID NO: 64) of a humanized h046-L7 type light chain.
- the flow cytometry analysis result at the time of making the culture supernatant of the hybridoma which produces a human GPR20 transient expression cell strain and anti-human GPR20 antibody react is shown.
- FIG. 5 shows concentration-dependent binding of a rat anti-human GPR20 antibody to a human GPR20-expressing cell line in flow cytometry analysis.
- FIG. 5 shows concentration-dependent binding of a rat anti-human GPR20 antibody to a human GPR20-expressing cell line in flow cytometry analysis.
- FIG. 5 shows concentration-dependent binding of a rat anti-human GPR20 antibody to a human GPR20-expressing cell line in flow cytometry analysis.
- Fig. 6 shows the internalization ability of rat anti-GPR20 antibody.
- the vertical axis shows the survival rate when a rat anti-GPR20 antibody and a saporin-labeled anti-rat IgG antibody are added to a human GPR20-expressing cell line (relative rate when the cell survival rate when no antibody is added is 100%).
- the control figure of the amino acid sequence of human GPR20 and mouse GPR20 is shown.
- the amino acid sequences indicated by EC1, EC2, EC3, and EC4 indicate the extracellular region, and the numbers in parentheses indicate the region portion by the amino acid number of human GPR20.
- FIG. 3 shows human GPR20 specific binding of human chimerized anti-GPR20 antibody 04-046Ch.
- Figure 3 shows human GPR20 specific binding of human chimerized anti-GPR20 antibody 04-046Ch.
- Fig. 5 shows GPR20 binding of humanized anti-GPR20 antibody.
- Figure 5 shows GPR20 binding of humanized anti-GPR20 antibody and antibody-drug conjugate.
- FIG. 2 shows the in vitro cell growth inhibitory activity of antibody-drug conjugate (1) against GPR20-expressing cells.
- 2 shows the in vitro cell growth inhibitory activity of antibody-drug conjugates (7), (8), and (9) against GPR20-expressing cells.
- the antitumor effect of antibody-drug conjugates (1) and (2) in a nude mouse model transplanted subcutaneously with GIST-T1 / GPR20 cells is shown.
- FIG. 6 shows the antitumor effect of antibody-drug conjugates (1) and (2) in a nude mouse model of GIST430 cell subcutaneous transplantation
- the antitumor effect of antibody-drug conjugates (6) and (9) in a nude mouse model transplanted subcutaneously with GIST-T1 / GPR20 cells is shown.
- FIG. 6 shows the antitumor effect of the antibody-drug conjugate (13) in a nude mouse model transplanted subcutaneously with GIST-T1 / GPR20 cells.
- the anti-tumor effect of antibody-drug conjugates (4), (13) in a GIST020 subcutaneously transplanted nude mouse model is shown.
- FIG. 5 shows the antitumor effect of antibody-drug conjugate (13) in a nude mouse model of GIST1 subcutaneous transplantation.
- FIG. 6 shows the antitumor effect of an antibody-drug conjugate (14) and an antibody-drug conjugate targeting HER2 in a nude mouse model of gastric cancer cell line NCI-N87 cells subcutaneously transplanted.
- FIG. 6 shows the antitumor effect of the antibody-drug conjugate (14) in a nude mouse model of a tumor GIST074 subcutaneously transplanted from a gastrointestinal stromal tumor patient who became unresponsive to Regorafenib treatment.
- the combination effect of antibody-drug conjugate (3) and Sunitinib in a human gastrointestinal stromal tumor cell line GIST430 / 654 subcutaneously transplanted nude mouse model having an Imatinib resistance mutation is shown.
- cancer and “tumor” are used interchangeably.
- the term “gene” includes not only DNA but also mRNA, cDNA and cRNA thereof.
- the term “polynucleotide” is used in the same meaning as nucleic acid, Also included are DNA, RNA, probes, oligonucleotides, and primers.
- polypeptide and “protein” are used without distinction.
- cell includes cells in an individual animal and cultured cells.
- GPR20 is used in the same meaning as the GPR20 protein.
- cytotoxic activity refers to causing pathological changes in cells in some form, not just direct trauma, but also DNA cleavage, base dimer formation, chromosomes. It causes structural and functional damages such as cell breakage, damage to cell division apparatus, and reduction of various enzyme activities.
- demonstrating toxicity in a cell means to exhibit toxicity in a cell in some form, and not just a direct trauma, but also DNA cleavage and formation of a base dimer. It means that it affects the structure and function of all cells and metabolism, such as chromosome breakage, damage to cell division apparatus, reduction of various enzyme activities, and suppression of cell growth factor action.
- epitope means a partial peptide or partial three-dimensional structure of GPR20 to which a specific anti-GPR20 antibody binds.
- the epitope that is a partial peptide of GPR20 can be determined by methods well known to those skilled in the art such as immunoassay.
- various partial structures of the antigen are prepared.
- a known oligonucleotide synthesis technique can be used. For example, after preparing a series of polypeptides sequentially shortened by an appropriate length from the C-terminal or N-terminal of GPR20 using a gene recombination technique well known to those skilled in the art, the reactivity of the antibody against them is examined.
- epitopes After determining the recognition site, epitopes can be determined by synthesizing shorter peptides and examining their reactivity with those peptides.
- an antibody that binds to a membrane protein composed of a plurality of extracellular domains uses a three-dimensional structure composed of a plurality of domains as an epitope, the conformation is modified by modifying the amino acid sequence of a specific extracellular domain. It is possible to determine which domain to bind to.
- An epitope that is a partial three-dimensional structure of an antigen to which a specific antibody binds can also be determined by specifying an amino acid residue of an antigen adjacent to the previous antibody by X-ray structural analysis.
- binding to the same epitope means an antibody that binds to a common epitope. If the second antibody binds to the partial peptide or the three-dimensional structure to which the first antibody binds, it can be determined that the first antibody and the second antibody bind to the same epitope. In addition, by confirming that the second antibody competes for the binding of the first antibody to the antigen (that is, the second antibody prevents the binding of the first antibody to the antigen), the specific epitope sequence Alternatively, even if the structure is not determined, it can be determined that it binds to the same epitope of the first antibody and the second antibody.
- the second antibody when the first antibody and the second antibody bind to the same epitope and the first antibody has a special effect such as antitumor activity or internalization activity, the second antibody also has the same activity. You can expect to have. Therefore, for the anti-GPR20 antibody, by confirming that the second anti-GPR20 antibody competes with the partial peptide to which the anti-GPR20 antibody of the first antibody binds, the first antibody and the second antibody are of GPR20. It can be determined that the antibody binds to the same epitope.
- CDR means a complementarity determining region (CDR). It is known that there are three CDRs in each of the heavy and light chains of an antibody molecule. CDRs, also called hypervariable regions, are sites in the variable regions of antibody heavy and light chains that have particularly high primary structure variability, and heavy and light chain polypeptide chains. In the primary structure of each, it is separated into three locations.
- CDRH1, CDRH2, CDRH3 from the amino terminal side of the heavy chain amino acid sequence
- CDRL1 from the amino terminal side of the light chain amino acid sequence.
- CDRL2 and CDRL3 CDRL1 from the amino terminal side of the light chain amino acid sequence.
- hybridize under stringent conditions means that hybridization is performed at 68 ° C. in a commercially available hybridization solution ExpressHyb Hybridization Solution (Clontech) or using a filter on which DNA is immobilized. After hybridization at 68 ° C. in the presence of 7-1.0 M NaCl, a 0.1-2 fold concentration SSC solution (1 fold concentration SSC consists of 150 mM NaCl, 15 mM sodium citrate) Hybridization under conditions that can be identified by washing at 68 ° C. or equivalent conditions.
- GPR20 is a seven-transmembrane protein consisting of 358 amino acids belonging to class A of the G protein-coupled receptor (GPCR) family, and has an N-terminal side extracellular and a C-terminal side intracellular.
- GPCR G protein-coupled receptor
- the GPR20 protein used in the present invention can be used by directly purifying from a GPR20-expressing cell of a human or non-human mammal (rat, mouse, etc.), or by preparing a cell membrane fraction of the cell, It can also be obtained by synthesizing GPR20 in vitro or by producing it in a host cell by genetic manipulation. Specifically, in genetic manipulation, GPR20 cDNA is incorporated into an expressible vector and then synthesized in a solution containing enzymes, substrates and energy substances necessary for transcription and translation, or other prokaryotic organisms or eukaryotics. The protein can be obtained by expressing GPR20 by transforming a host cell of an organism.
- GPR20-expressing cell or a cell line expressing GPR20 by the above-described genetic manipulation as a GPR20 protein.
- an expression vector incorporating GPR20 cDNA can be directly administered to an immunized animal to express GPR20 in the immunized animal.
- the DNA sequence and amino acid sequence of human GPR20 are published on public databases and can be referred to by accession numbers such as NM_005293 and NP_005284 (NCBI).
- the GPR20 includes proteins having a biological activity equivalent to that of the GPR20 consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, and / or added.
- the amino acid sequence of the human GPR20 protein is described in SEQ ID NO: 1 in the sequence listing, and the extracellular region is an extracellular domain (EC1) consisting of amino acid numbers 1 to 48 of SEQ ID NO: 1 in the sequence listing, amino acid numbers 108 to It consists of an extracellular domain consisting of 125 (EC2), an extracellular domain consisting of amino acids 190 to 196 (EC3), and an extracellular domain consisting of amino acids 260 to 275 (EC4).
- EC1 extracellular domain consisting of amino acid numbers 1 to 48 of SEQ ID NO: 1 in the sequence listing
- amino acid numbers 108 to It consists of an extracellular domain consisting of 125 (EC2), an extracellular domain consisting of amino acids 190 to 196 (EC3), and an extracellular domain consisting of amino acids 260 to 275 (EC4).
- anti-GPR20 antibody As an example of the anti-GPR20 antibody of the present invention, two extracellular regions of the amino acid sequence 1 to 48 from the N-terminus of GPR20 and the amino acid sequence 108 to 125 shown in SEQ ID NO: 1 in the sequence listing And an anti-GPR20 antibody that recognizes a higher-order structure consisting of and has internalization activity.
- anti-GPR20 antibody of the present invention a high amino acid sequence consisting of two extracellular regions of the amino acid sequence 1 to 48 from the N-terminus of GPR20 and the amino acid sequence 108 to 125 shown in SEQ ID NO: 1 in the sequence listing.
- examples thereof include an anti-GPR20 antibody that recognizes the following structure, binds to at least the 113th tyrosine, and has internalization activity.
- the anti-GPR20 antibody of the present invention specifically binds to a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, and the amino acid of amino acid number 113 in SEQ ID NO: 1 in the sequence listing is changed from tyrosine to other
- An anti-GPR20 antibody having internalization activity without binding to a polypeptide substituted with an amino acid for example, phenylalanine
- the anti-GPR20 antibody of the present invention may be derived from any species, but preferred examples include human, rat, mouse and rabbit. When derived from species other than human, it is preferable to chimerize or humanize using well-known techniques.
- the antibody of the present invention may be a polyclonal antibody or a monoclonal antibody, but a monoclonal antibody is preferred.
- the anti-GPR20 antibody of the present invention is an antibody that can target tumor cells, that is, has the property of recognizing tumor cells, the property of binding to tumor cells, the property of being taken up and internalized in tumor cells, and the like. Therefore, the anti-GPR20 antibody of the present invention and a compound having antitumor activity can be bound via a linker to form an antibody-drug conjugate.
- the binding property of the antibody to tumor cells can be confirmed using flow cytometry. Incorporation of antibodies into tumor cells is as follows: (1) An assay that visualizes antibodies taken into cells using a secondary antibody (fluorescent label) that binds to therapeutic antibodies with a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) Assay for measuring the amount of fluorescence taken into cells using a secondary antibody (fluorescent label) that binds to the therapeutic antibody (Molecular Biology of the Cell Vol. 15, 5268-5282). , December 2004) or (3) Mab-ZAP assay (Bio Technologies 28: 162-165) that uses toxins that bind to therapeutic antibodies to release toxins and inhibit cell growth when taken up into cells. J nuary 2000) can be confirmed using. As the immunotoxin, a recombinant complex protein protein of the catalytic domain of diphtheria toxin and protein G can also be used.
- high internalization ability refers to the survival rate of GPR20-expressing cells to which the antibody and saporin-labeled anti-rat IgG antibody are administered (relative rate with the cell survival rate when no antibody is added as 100%). Is preferably 70% or less, more preferably 60% or less.
- the antitumor antibody-drug conjugate of the present invention is bound with a compound that exhibits an antitumor effect, it is preferable but not essential that the antibody itself has an antitumor effect.
- the antibody For the purpose of specifically and selectively exerting the cytotoxicity of the antitumor compound in tumor cells, it is important and preferable that the antibody has a property of internalizing and transferring into the tumor cells.
- An anti-GPR20 antibody can be obtained by immunizing an animal with a polypeptide serving as an antigen and collecting and purifying the antibody produced in the living body using a method commonly practiced in this field. Since it is a seven-transmembrane protein, it is preferable to use a GPCR retaining a three-dimensional structure as an antigen.
- An example of such a method is a DNA immunization method.
- the origin of the antigen is not limited to humans, and animals can be immunized with antigens derived from animals other than humans such as mice and rats.
- an antibody applicable to a human disease can be selected by examining the cross-reactivity between an antibody that binds to the obtained heterologous antigen and a human antigen.
- Hybridomas can be established by fusing antibody-producing cells that produce antibodies against the antigen and myeloma cells to obtain monoclonal antibodies.
- An antigen can be obtained by causing a host cell to produce a gene encoding an antigen protein by genetic manipulation. Specifically, a vector capable of expressing an antigen gene may be prepared, introduced into a host cell to express the gene, and the expressed antigen may be purified. An antibody can also be obtained by using a method for immunizing an animal with an antigen-expressing cell or a cell line expressing the antigen by genetic manipulation as described above.
- the antigen protein cDNA is incorporated into an expression vector and administered to the immunized animal, and the antigen protein is expressed in the body of the immunized animal to produce an antibody against the antigen protein. Can be acquired.
- the anti-GPR20 antibody used in the present invention is not particularly limited.
- an antibody specified by the amino acid sequence shown in the sequence listing of the present application can be preferably used. it can.
- the anti-GPR20 antibody used in the present invention preferably has the following characteristics.
- an antibody having the following characteristics (A) specifically binds to GPR20 (b) has an activity of internalizing in GPR20-expressing cells by binding to GPR20 (2) the antibody or the antibody according to (1) above, wherein GPR20 is human GPR20, (3) Recognizes a higher-order structure consisting of two extracellular regions of the amino acid sequence 1 to 48 from the N-terminus of human GPR20 and the amino acid sequence 108 to 125, and has internalization activity.
- the method for obtaining an antibody against GPR20 of the present invention is not particularly limited as long as an anti-GPR20 antibody can be obtained. Since GPR20 is a transmembrane protein, GPR20 having a higher-order structure may be used as an antigen. preferable.
- the DNA immunization method is a technique for inducing immunity to an antigen by introducing an antigen expression plasmid into an individual animal such as a mouse or a rat and expressing the antigen in the individual.
- Gene transfer methods include direct injection of plasmids into muscle, intravenous injection of liposomes and polyethyleneimine and other introduction reagents, viral vector methods, and gold particles with plasmids attached to them by Gene Gun.
- the amount of expression is further improved by treating the muscle with hyaluronidase before intramuscular injection of the plasmid (McMahon JM1, Signori E, Wells KE, Fazio VM, Wells DJ. Gene Ther. 2001 Aug; 8 (16) : 1264-70).
- the hybridoma can also be produced by a known method, for example, using a Hybridune Hybridoma Production System (Cyto Pulse Sciences).
- GPR20 cDNA is incorporated into an expression vector (for example, pcDNA3.1: Thermo Fisher Scientific), and the vector is directly administered to an immunized animal (for example, rat or mouse) by a method such as electroporation or gene gun.
- an immune response can be induced by expressing GPR20 in the animal body.
- Administration of the vector by electroporation or the like may be performed once or multiple times, preferably multiple times, if necessary to increase the antibody titer.
- a tissue for example, lymph node
- a tissue for example, lymph node
- myeloma eg, mouse myeloma SP2 / 0-ag14 cells
- D cell fusion between antibody-producing cells and myeloma
- E selection of a hybridoma group producing the target antibody
- F Division into single cell clones (cloning)
- G In some cases, culturing hybridomas for producing a large amount of monoclonal antibodies, or raising animals transplanted with hybridomas, (H) Physiological activity (internalization activity) of the monoclonal antibody thus produced, and examination of its binding specificity, or assay of properties as a labeling reagent. Examples thereof include, but are not limited to, flow cytometry or Cell-ELISA.
- hybridoma strains thus established include anti-GPR20 antibody-producing hybridomas 04-046, 04-079 and 04-126.
- the antibody produced by the anti-GPR20 antibody-producing hybridoma 04-046 is referred to as “04-046 antibody” or simply “04-046”, and the antibody produced by the hybridoma 04-079 is “04-079 antibody” or simply “04-079” is described, and the antibody produced by hybridoma 04-126 is described as “04-126 antibody” or simply “04-0126”.
- the heavy chain of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of amino acid residues 143 to 475 is a constant region.
- variable region is CDRH1 consisting of an amino acid sequence consisting of 45th to 54th amino acid residues, CDRH2 consisting of an amino acid sequence consisting of 69th to 78th amino acid residues, and 118th to 131st It has CDRH3 consisting of an amino acid sequence consisting of amino acid residues.
- the heavy chain variable region of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
- CDRH1 of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing
- the amino acid sequence of CDRH3 is It has the amino acid sequence shown in SEQ ID NO: 6.
- the heavy chain sequence of the 04-046 antibody is shown in FIG.
- the light chain of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence
- the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region.
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- variable region is represented by SEQ ID NO: 7 in the sequence listing, CDRL1 consisting of the amino acid sequence consisting of the 43rd to 53rd amino acid residues, CDRL2 consisting of the amino acid sequence consisting of the 69th to 75th amino acid residues, and 108th to 116th. It has CDRL3 consisting of an amino acid sequence consisting of amino acid residues.
- the light chain variable region of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing.
- the CDRL1 of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing
- the amino acid sequence of CDRL3 is It has the amino acid sequence shown in SEQ ID NO: 11.
- the sequence of the light chain of the 04-046 antibody is shown in FIG.
- the heavy chain of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of amino acid residues 143 to 475 is a constant region.
- the variable region is represented by CDRH1 consisting of an amino acid sequence consisting of 45th to 54th amino acid residues, CDRH2 consisting of an amino acid sequence consisting of 69th to 78th amino acid residues, and 118th to 131st in SEQ ID NO: 12 of the Sequence Listing.
- CDRH3 consisting of an amino acid sequence consisting of amino acid residues.
- the heavy chain variable region of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 13 in the Sequence Listing.
- CDR-01 of antibody 04-079 has the amino acid sequence shown in SEQ ID NO: 14 of the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 15 of the sequence listing
- the amino acid sequence of CDRH3 is It has the amino acid sequence shown in SEQ ID NO: 16.
- the heavy chain sequence of the 04-079 antibody is shown in FIG.
- the light chain of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 17 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence
- the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region.
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- variable region is represented by SEQ ID NO: 17 in the sequence listing, CDRL1 consisting of the amino acid sequence consisting of the 43rd to 53rd amino acid residues, CDRL2 consisting of the amino acid sequence consisting of the 69th to 75th amino acid residues, and 108th to 116th. It has CDRL3 consisting of an amino acid sequence consisting of amino acid residues.
- the light chain variable region of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing.
- the CDRL1 of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 19 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing
- the amino acid sequence of CDRL3 is It has the amino acid sequence shown in SEQ ID NO: 21.
- the sequence of the light chain of the 04-079 antibody is shown in FIG.
- the heavy chain of antibody 04-126 has the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of amino acid residues 143 to 475 is a constant region.
- the variable region is represented by CDRH1 consisting of an amino acid sequence consisting of 45th to 54th amino acid residues, CDRH2 consisting of an amino acid sequence consisting of 69th to 78th amino acid residues, and 118th to 131st in SEQ ID NO: 22 of the Sequence Listing.
- the heavy chain variable region of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 23 of the Sequence Listing.
- CDRH1 of antibody 04-126 has the amino acid sequence shown in SEQ ID NO: 24 of the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing
- the amino acid sequence of CDRH3 is It has the amino acid sequence shown in SEQ ID NO: 26.
- the heavy chain sequence of the 04-126 antibody is shown in FIG.
- the light chain of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 27 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence
- the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region.
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- variable region is represented by SEQ ID NO: 27 in the sequence listing, CDRL1 consisting of the amino acid sequence consisting of the 43rd to 53rd amino acid residues, CDRL2 consisting of the amino acid sequence consisting of the 69th to 75th amino acid residues, and 108th to 116th. It has CDRL3 consisting of an amino acid sequence consisting of amino acid residues.
- the light chain variable region of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 28 of the Sequence Listing.
- the CDRL1 of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 29 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence shown in SEQ ID NO: 30 in the sequence listing
- the amino acid sequence of CDRL3 is It has the amino acid sequence shown in SEQ ID NO: 31.
- the sequence of the light chain of the 04-126 antibody is shown in FIG.
- the heavy chain amino acid sequence of the 04-046 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 32 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 32 of the Sequence Listing is a signal sequence, and the nucleotide sequence consisting of nucleotides 58 to 426 encodes the heavy chain variable region of the 04-046 antibody.
- the nucleotide sequence consisting of nucleotides 427 to 1425 encodes the heavy chain constant region of the 04-046 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 133 to 162 encoding CDRH1 in SEQ ID NO: 32, and the nucleotide sequence described in nucleotide numbers 205 to 234 encoding CDRH2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 352 to 393 encoding CDRH3.
- the nucleotide sequence of the heavy chain variable region of the 04-046 antibody is also shown in SEQ ID NO: 33 in the Sequence Listing.
- the sequence of SEQ ID NO: 32 is shown in FIG.
- the light chain nucleotide sequence of the 04-046 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 34 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 1 to 60 of the nucleotide sequence shown in SEQ ID NO: 34 of the Sequence Listing is a signal sequence, and the nucleotide sequence consisting of nucleotides 61 to 384 encodes the light chain variable region of the 04-046 antibody.
- the nucleotide sequence consisting of nucleotides 385 to 699 encodes the light chain constant region of the 04-046 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 34, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 348 encoding CDRL3.
- the nucleotide sequence of the light chain variable region of the 04-046 antibody is also shown in SEQ ID NO: 35 in the Sequence Listing.
- the sequence of SEQ ID NO: 34 is shown in FIG.
- the heavy chain nucleotide sequence of the 04-079 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 36 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 36 of the Sequence Listing is a signal sequence, and the nucleotide sequence consisting of nucleotides 58 to 426 encodes the heavy chain variable region of the 04-079 antibody.
- the nucleotide sequence consisting of nucleotides 427 to 1425 encodes the heavy chain constant region of the 04-079 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 133 to 162 encoding CDRH1 in SEQ ID NO: 36, and the nucleotide sequence described in nucleotide numbers 205 to 234 encoding CDRH2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 352 to 393 encoding CDRH3.
- the nucleotide sequence of the heavy chain variable region of the 04-079 antibody is also shown in SEQ ID NO: 37 in the Sequence Listing.
- the sequence of SEQ ID NO: 36 is shown in FIG.
- the light chain nucleotide sequence of the 04-079 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 38 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 1 to 60 of the nucleotide sequence shown in SEQ ID NO: 38 of the Sequence Listing is a signal sequence, and the nucleotide sequence consisting of nucleotides 61 to 384 encodes the light chain variable region of the 04-079 antibody.
- the nucleotide sequence consisting of nucleotides 385 to 699 encodes the light chain constant region of the 04-079 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 38, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 348 encoding CDRL3.
- the nucleotide sequence of the light chain variable region of the 04-079 antibody is also shown in SEQ ID NO: 39 in the Sequence Listing.
- the sequence of SEQ ID NO: 38 is described in FIG.
- the heavy chain nucleotide sequence of the 04-126 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 40 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 1 to 57 of the nucleotide sequence shown in SEQ ID NO: 40 of the Sequence Listing is a signal sequence, and the nucleotide sequence consisting of nucleotides 58 to 426 encodes the heavy chain variable region of the 04-126 antibody.
- the nucleotide sequence consisting of nucleotides 427 to 1425 encodes the heavy chain constant region of the 04-126 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 133 to 162 encoding CDRH1 in SEQ ID NO: 40, and the nucleotide sequence described in nucleotide numbers 205 to 234 encoding CDRH2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 352 to 393 encoding CDRH3.
- the nucleotide sequence of the heavy chain variable region of the 04-126 antibody is also shown in SEQ ID NO: 41 in the Sequence Listing.
- the sequence of SEQ ID NO: 40 is shown in FIG.
- the light chain nucleotide sequence of the 04-126 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 42 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 1 to 60 of the nucleotide sequence shown in SEQ ID NO: 42 of the Sequence Listing is a signal sequence, and the nucleotide sequence consisting of nucleotides 61 to 384 encodes the light chain variable region of the 04-126 antibody.
- the nucleotide sequence consisting of nucleotides 385 to 699 encodes the light chain constant region of the antibody 04-126.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 42, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 348 encoding CDRL3.
- the nucleotide sequence of the light chain variable region of the 04-126 antibody is also shown in SEQ ID NO: 43 of the Sequence Listing. The sequence of SEQ ID NO: 42 is described in FIG.
- the monoclonal antibody binds to the partial peptide or the three-dimensional structure to which the 04-046 antibody, 04-079 antibody or 04-126 antibody binds, the monoclonal antibody becomes the 04-046 antibody, 04-079 antibody. Alternatively, it can be determined that it binds to the same epitope as the 04-126 antibody. In addition, the monoclonal antibody competes with the binding of the 04-046 antibody, 04-079 antibody or 04-126 antibody to GPR20 (ie, the monoclonal antibody is the 04-046 antibody, 04-079 antibody or 04-126).
- the monoclonal antibody binds to the same epitope as the anti-GPR20 antibody even if the sequence or structure of the specific epitope has not been determined.
- the monoclonal antibody has the same antigen binding ability, biological activity and / or internalization activity as the 04-046 antibody, 04-079 antibody or 04-126 antibody. It is highly expected.
- the antibodies of the present invention include genetically engineered antibodies that have been artificially modified for the purpose of reducing the heterologous antigenicity against humans, such as chimera (Chimeric ) Antibodies, humanized antibodies, human antibodies and the like. These antibodies can be produced using known methods.
- chimeric antibody examples include antibodies in which the variable region and the constant region of the antibody are different from each other, for example, a chimeric antibody in which the variable region of a mouse or rat-derived antibody is joined to a human-derived constant region (Proc. Natl. Acad). Sci.U.S.A., 81, 6851-6855, (1984)).
- a chimeric antibody derived from rat anti-human GPR20 antibody 04-046 antibody comprises a heavy chain comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 3 and a light chain comprising the light chain variable region represented by SEQ ID NO: 8. It is an antibody and may have a constant region derived from any human.
- a chimeric antibody derived from the rat anti-human GPR20 antibody 04-079 antibody comprises a heavy chain comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 13 and a light chain comprising the light chain variable region represented by SEQ ID NO: 18. It is an antibody and may have a constant region derived from any human.
- a chimeric antibody derived from the rat anti-human GPR20 antibody 04-126 antibody comprises a heavy chain comprising a heavy chain variable region comprising the amino acid sequence represented by SEQ ID NO: 23 and a light chain comprising the light chain variable region represented by SEQ ID NO: 28 It is an antibody and may have a constant region derived from any human.
- a chimeric antibody 04-046Ch derived from the rat anti-human GPR20 antibody 04-046 antibody (hereinafter also referred to as “04-046Ch”).
- the amino acid sequence of the 04-046Ch antibody is a heavy chain having an amino acid sequence consisting of amino acid residues 20 to 472 in SEQ ID NO: 44 in the sequence listing and a light chain having an amino acid sequence consisting of 21 to 233 in SEQ ID NO: 45 in the sequence listing. Mention may be made of antibodies consisting of chains.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of residues 143 to 472 is a constant region.
- the sequence of SEQ ID NO: 44 is described in FIG.
- the amino acid sequence consisting of the 1st to 20th amino acid residues is a signal sequence
- the amino acid sequence consisting of the 21st to 128th amino acid residues is the variable region
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- the sequence of SEQ ID NO: 45 is described in FIG.
- the heavy chain amino acid sequence of the 04-046Ch antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 46 in the Sequence Listing.
- the nucleotide sequence consisting of the 1st to 57th nucleotides of the nucleotide sequence shown in SEQ ID NO: 46 of the Sequence Listing encodes the signal sequence of the 04-046Ch antibody, and the nucleotide sequence of 58-0 of the nucleotide sequence shown in SEQ ID NO: 46 of the Sequence Listing.
- the nucleotide sequence consisting of the 426th nucleotide encodes the heavy chain variable region sequence of the 04-046Ch antibody
- the nucleotide sequence consisting of the 427th to 1416th nucleotides of the nucleotide sequence shown in SEQ ID NO: 46 of the Sequence Listing is 04- It encodes the heavy chain constant region of the 046Ch antibody.
- the sequence of SEQ ID NO: 46 is set forth in FIG.
- the light chain amino acid sequence of the 04-046Ch antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 47 of the Sequence Listing.
- the nucleotide sequence consisting of the 1st to 60th nucleotides of the nucleotide sequence shown in SEQ ID NO: 47 of the sequence listing encodes the signal sequence of the 04-046Ch antibody, and 61 to 60 of the nucleotide sequence shown in SEQ ID NO: 47 of the sequence listing.
- the nucleotide sequence consisting of the 384th nucleotide encodes the light chain variable region sequence of the 04-046Ch antibody
- the nucleotide sequence consisting of the 385th to 699th nucleotides of the nucleotide sequence shown in SEQ ID NO: 47 of the Sequence Listing is 04- It encodes the light chain constant region of the 046Ch antibody.
- the sequence of SEQ ID NO: 47 is set forth in FIG.
- a humanized antibody an antibody in which only a complementarity determining region (CDR; complementarity determining region) is incorporated into a human-derived antibody (see Nature (1986) 321, p.522-525), a CDR sequence is obtained by CDR grafting.
- CDR complementarity determining region
- antibodies in which amino acid residues of some frameworks are transplanted into human antibodies International Publication No. 90/07861
- antibodies in which the amino acid sequences of some CDRs are modified while maintaining the ability to bind to antigens Can be mentioned.
- the humanized antibody derived from the 04-046, 04-079, or 04-126 antibody retains all six CDR sequences of the 04-046 antibody, 04-079 antibody, or 04-126 antibody. As long as it has internalization activity, it is not limited to a specific humanized antibody, and it is not limited to a specific humanized antibody as long as it has internalization activity while modifying the amino acid sequence of some CDRs.
- humanized antibodies of rat antibody 04-046 include (1) an amino acid sequence consisting of amino acid residues 20 to 142 of SEQ ID NO: 48, 50, 52, 54 or 56 in the sequence listing, (2) above ( 1) an amino acid sequence having at least 95% homology to the amino acid sequence, and (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above.
- a heavy chain comprising any one of the heavy chain variable regions, and (4) an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 58, 60, 62 or 64, (5) of (4) above
- Any combination of light chain comprising a light chain variable region of any one of the amino acid sequence can be mentioned.
- an antibody in which one of the heavy chain or the light chain is humanized and the other is a rat antibody or a chimeric antibody light chain or heavy chain can also be used.
- antibodies include (1) an amino acid sequence consisting of amino acid residues 20 to 472 of SEQ ID NO: 44 in the sequence listing, and (2) at least 95% or more of the amino acid sequence of (1) above.
- An amino acid sequence having homology and (3) a heavy chain comprising any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above, and (4)
- Any light chain comprising a light chain variable region consisting of any one of amino acid sequences wherein one or several amino acids are deleted, substituted or added in the amino acid sequence of (4) above Mention may be made of the combined.
- antibodies include (1) an amino acid sequence consisting of amino acid residues 20 to 142 of SEQ ID NO: 48, 50, 52, 54 or 56 in the sequence listing, (2) the above (1) Any one of an amino acid sequence having at least 95% homology to the amino acid sequence, and (3) an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (1) above A heavy chain comprising a heavy chain variable region consisting of two, and (4) an amino acid sequence consisting of amino acid residues 21 to 233 of SEQ ID NO: 45 of the Sequence Listing, (5) against the amino acid sequence of (4) above Amino acid sequence having at least 95% homology, and (6) any one of amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (4) above It may include any combination of the light chain comprising a Ranaru light chain variable region.
- Another example includes an antibody comprising a combination of any heavy chain and light chain selected from the following (7) to (19): (7) amino acid number 20 in SEQ ID NO: 44 A light chain consisting of the amino acid sequence described in amino acid numbers 21 to 234 in SEQ ID NO: 58, and (8) an amino acid sequence described in amino acid numbers 20 to 472 in SEQ ID NO: 44.
- SEQ ID NO: 48 the sequence consisting of amino acid residues 45 to 54 (GYTFTSYYIS) is CDRH1
- the sequence consisting of amino acid residues 69 to 78 is CDRH2
- the sequence consisting of amino acids 118 to 131 indicates CDRH3.
- SEQ ID NO: 50 a sequence consisting of amino acid residues 45 to 54 (GYTFTSYIS) is CDRH1
- a sequence consisting of amino acid residues 69 to 78 (FINPGSGTN) is a sequence consisting of CDRH2
- amino acid residues 118 to 131 indicates CDRH3.
- SEQ ID NO: 52 a sequence consisting of amino acid residues 45 to 54 (GYTFTSYIS) is CDRH1, a sequence consisting of amino acid residues 69 to 78 (FINPGSGTN) is a sequence consisting of CDRH2, and amino acid residues 118 to 131. (GAGGFLRIITKFDY) indicates CDRH3.
- GYTFTSYIS GYTFTSYIS
- FINPGSGTN amino acid residues 118 to 131.
- GGGFLRIITKFDY indicates CDRH3.
- SEQ ID NO: 56 the sequence consisting of amino acid residues 45 to 54 (GYTFTSYYIS) is CDRH1
- the sequence consisting of amino acid residues 69 to 78 is CDRH2
- the sequence consisting of amino acid residues 118 to 131 indicates CDRH3.
- the sequence consisting of the 44th to 54th amino acid residues (RASKSVSTYIH) is CDRL1
- the sequence consisting of the 70th to 76th amino acid residues (SASNLES) is CDRL2
- the amino acid numbers 109 to 117 are the amino acid residues.
- the resulting sequence represents CDRL3.
- the sequence consisting of amino acid residues 44 to 54 (RASKSVSTYIH) is CDRL1
- the sequence consisting of amino acid residues 70 to 76 (SASNLES) is CDRL2
- the amino acid residues 109 to 117 are amino acid residues.
- the resulting sequence (QQINELPYT) represents CDRL3.
- SEQ ID NO: 62 the sequence consisting of amino acid residues 44 to 54 (RASKSVSTYIH) is CDRL1, the sequence consisting of amino acid residues 70 to 76 (SASDRES) is CDRL2, and the amino acid residues 109 to 117 are amino acid residues.
- the resulting sequence represents CDRL3.
- SEQ ID NO: 64 the sequence consisting of amino acid residues 44 to 54 (RASKSVSTYIH) is CDRL1, the sequence consisting of amino acid residues 70 to 76 (SAGNLES) is CDRL2, and the amino acid residues 109 to 117 are amino acid residues.
- the resulting sequence (QQINELPYT) represents CDRL3.
- “several” means 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 Means 3 or 1 or 2;
- amino acid substitution in the present specification, conservative amino acid substitution is preferable.
- Conservative amino acid substitutions are those that take place within a group of amino acids that are related to an amino acid side chain.
- Such amino acid substitution is preferably performed within a range that does not deteriorate the properties of the substance having the original amino acid sequence.
- the heavy chain having a variable region consisting of the amino acid sequence described in amino acid numbers 20 to 142 in SEQ ID NO: 48 and the amino acid numbers 21 to 129 in SEQ ID NO: 64 An antibody comprising a light chain having a variable region comprising an amino acid sequence, a heavy chain having a variable region comprising an amino acid sequence represented by amino acid numbers 20 to 142 in SEQ ID NO: 52, and an amino acid represented by amino acid numbers 21 to 129 in SEQ ID NO: 60
- An antibody consisting of a light chain having a variable region consisting of a sequence, a heavy chain having a variable region consisting of an amino acid sequence described in amino acid numbers 20 to 142 in SEQ ID NO: 54, and an amino acid sequence described in amino acid numbers 21 to 129 in SEQ ID NO: 58 An antibody consisting of a light chain having a variable region consisting of An antibody comprising a heavy chain having a variable region consisting of the amino acid sequence described in amino acid numbers 20
- an antibody consisting of a heavy chain consisting of the amino acid sequence described in amino acid numbers 20 to 472 in SEQ ID NO: 48 and a light chain consisting of the amino acid sequence described in amino acid numbers 21 to 234 in SEQ ID NO: 64 an antibody consisting of a heavy chain consisting of the amino acid sequence set forth in amino acid numbers 20 to 472 in SEQ ID NO: 52 and a light chain consisting of the amino acid sequence set forth in amino acid numbers 21 to 234 in SEQ ID NO: 60
- amino acid numbers 20 to 472 in SEQ ID NO: 54 An antibody consisting of a heavy chain consisting of the amino acid sequence of No. 5 and a light chain consisting of the amino acid sequence of SEQ ID No.
- an antibody having a biological activity equivalent to that of each of the above antibodies by combining a sequence having high homology with the above heavy chain amino acid sequence and light chain amino acid sequence. Such homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, most preferably 99% or more. Homology.
- An antibody having biological activity equivalent to that of each of the above antibodies can also be selected by combining an amino acid sequence in which one to several amino acid residues are substituted, deleted, or added to the heavy chain or light chain amino acid sequence. Is possible.
- Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Jhangbund, ZhangD. (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402).
- Blast algorithm can be found on the Internet at www. ncbi. nlm. nih. It can also be used by accessing gov / blast.
- the amino acid sequence consisting of the 1st to 19th amino acid residues is a signal sequence, and the 20th to 142nd amino acids.
- the amino acid sequence consisting of residues is a variable region, and the amino acid sequence consisting of amino acid residues 143 to 472 is a constant region.
- the sequence of SEQ ID NO: 48 is shown in FIG. 9, the sequence of SEQ ID NO: 50 in FIG. 10, the sequence of SEQ ID NO: 52 in FIG. 11, the sequence of SEQ ID NO: 54 in FIG. 12, and the sequence of SEQ ID NO: 56 in FIG. Are listed.
- the amino acid sequence consisting of the 1st to 20th amino acid residues is a signal sequence, and the 21st to 129th amino acid residues
- the amino acid sequence consisting of is the variable region, and the amino acid sequence consisting of the 130th to 234th amino acid residues is the constant region.
- the sequence of SEQ ID NO: 58 is shown in FIG. 14, the sequence of SEQ ID NO: 60 is shown in FIG. 15, the sequence of SEQ ID NO: 62 is shown in FIG. 16, and the sequence of SEQ ID NO: 64 is shown in FIG.
- Examples of the antibody of the present invention further include a human antibody that binds to GPR20.
- the anti-GPR20 human antibody means a human antibody having only the gene sequence of an antibody derived from a human chromosome.
- the anti-GPR20 human antibody is a method using a human antibody-producing mouse having a human chromosome fragment containing the heavy and light chain genes of a human antibody (Tomizuka, K. et al., Nature Genetics (1997) 16, p. 133. -143, Kuroiwa, Y. et.al., Nucl.Acids Res. (1998) 26, p.3447-3448; Yoshida, H. et.al., Animal Cell Technology: Basic and Applied Aspects.10.
- the endogenous immunoglobulin heavy chain and light chain loci are disrupted, and instead, a human immunoglobulin is produced via a yeast artificial chromosome (YAC) vector or the like.
- YAC yeast artificial chromosome
- genetically modified animals into which heavy and light chain loci have been introduced they can be produced by creating knockout animals and transgenic animals and crossing these animals together.
- eukaryotic cells are transformed with cDNA encoding each of the heavy and light chains of such a human antibody, preferably a vector containing the cDNA, to produce a gene recombinant human monoclonal antibody.
- This antibody can also be obtained from the culture supernatant by culturing the transformed cells.
- eukaryotic cells preferably CHO cells
- mammalian cells such as lymphocytes and myeloma can be used.
- a method for obtaining a human antibody derived from phage display selected from a human antibody library (Wormstone, IM et al, Investigative Ophthalmology & Visual Science. (2002) 43 (7), p. 2301-2308; Mé, S. et.al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p.189-203; Siriwardena, D. et., Ophthalmology (2002) 109 (3), p. 427-431 etc.) are also known.
- a phage display method (Nature Biotechnology (2005), 23, (9), p. 1105) in which a variable region of a human antibody is expressed on a phage surface as a single chain antibody (scFv) and a phage that binds to an antigen is selected. ⁇ 1116) can be used.
- the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.
- a human antibody can be obtained by preparing an expression vector having the sequence, introducing it into a suitable host and expressing it (WO 92/01047). No. 92/20791, 93/06213, 93/11236, 93/19172, 95/01438, 95/15388, Annu. Rev. Immunol (1994) 12, p. 433-455, Nature Biotechnology (2005) 23 (9), pp. 1105-1116).
- the human antibody is converted to the 04-046 antibody, 04-079 antibody, or 04-126. It can be determined that it binds to the same epitope as the antibody.
- the human antibody competes for binding of the 04-046 antibody, 04-079 antibody or 04-126 antibody to GPR20 (ie, the human antibody is the 04-046 antibody, 04-079 antibody or 04-126).
- the human antibody can be treated with the 04-046 antibody, 04-079 antibody, or 04-126 antibody even if the specific epitope sequence or structure has not been determined. It can be determined that it binds to the same epitope. If the epitopes are confirmed to be identical, it is strongly expected that the human antibody has a biological activity equivalent to that of the 04-046 antibody, 04-079 antibody, or 04-126 antibody.
- the chimeric antibody, humanized antibody, or human antibody obtained by the above method can be evaluated for binding to an antigen by a known method or the like, and a suitable antibody can be selected.
- An example of another index for comparing the properties of antibodies is antibody stability.
- DSC Differential scanning calorimetry
- Tm thermal denaturation midpoint
- the difference in thermal stability can be compared by measuring the Tm value using DSC and comparing the values.
- Tm thermal denaturation midpoint
- a suitable antibody can be selected using stability as an index.
- Other indicators for selecting antibodies include high yields in suitable host cells and low aggregation in aqueous solutions. For example, since the antibody with the highest yield does not necessarily exhibit the highest thermal stability, it is necessary to select the most suitable antibody for human administration based on a comprehensive judgment based on the above-mentioned indicators. .
- the antibody of the present invention includes a modified antibody.
- the modified product means a product obtained by chemically or biologically modifying the antibody of the present invention.
- Chemical modifications include chemical modifications to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and the like.
- Biological modifications include post-translational modifications (eg, glycosylation to N- or O-links, N- or C-terminal processing, deamidation, aspartic acid isomerization, methionine oxidation) And those obtained by adding a methionine residue to the N-terminal by expression using a prokaryotic host cell.
- modified products of the antibody of the present invention for example, an enzyme label, a fluorescent label, and an affinity label are also included in the meaning of such a modified product.
- a modified product of the antibody of the present invention is useful for improving antibody stability and blood retention, reducing antigenicity, detecting or isolating an antibody or antigen, and the like.
- the antibody of the present invention includes an antibody whose sugar chain modification is regulated.
- an antibody gene When an antibody gene is once isolated and then introduced into an appropriate host to produce an antibody, a combination of an appropriate host and an expression vector can be used.
- Specific examples of the antibody gene include a combination of a gene encoding the heavy chain sequence of the antibody described herein and a gene encoding the light chain sequence.
- the heavy chain sequence gene and the light chain sequence gene When transforming a host cell, the heavy chain sequence gene and the light chain sequence gene can be inserted into the same expression vector, or can be inserted into separate expression vectors. is there.
- animal cells When eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.
- animal cells mammalian cells such as COS cells (Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650) which are monkey cells, mouse fibroblasts NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strain (Urlauub, G. and Chasin, LA Proc. Natl. Acad. Sci. US A. (1980) 77, p.4126-4220), FreeStyle 293F cells (Invitrogen).
- COS cells Gluzman, Y. Cell (1981) 23, p.175-182, ATCC CRL-1650
- mouse fibroblasts NIH3T3 ATCC No. CRL-1658
- an antibody can be obtained by introducing a desired antibody gene into these cells by transformation, and culturing the transformed cells in vitro.
- the yield may vary depending on the sequence of the antibody, and it is possible to select an antibody having an equivalent binding activity that can be easily produced as a drug using the yield as an index. Therefore, the antibody of the present invention includes a step of culturing the transformed host cell and a step of collecting the target antibody or a functional fragment of the antibody from the culture obtained in the step. An antibody obtained by the method for producing the antibody is also included.
- the antibody according to the present invention also includes the antibody subjected to the modification and the functional fragment of the antibody, a deletion in which one or two amino acids are deleted at the heavy chain carboxyl terminus, and amidated Such deletion forms (for example, heavy chain in which the proline residue at the carboxyl terminal site is amidated) and the like are also included.
- the carboxyl-terminal deletion of the heavy chain of the antibody according to the present invention is not limited to the above type.
- the two heavy chains constituting the antibody according to the present invention may be either one of the heavy chains selected from the group consisting of the full length and the above-mentioned deletion, or a combination of any two of them. It may be a thing.
- the amount ratio of each deletion can be influenced by the type and culture conditions of cultured mammalian cells producing the antibody according to the present invention, but the main component of the antibody according to the present invention is the carboxyl in both of the two heavy chains. A case where one terminal amino acid residue is deleted can be mentioned.
- Examples of the isotype of the antibody of the present invention include IgG (IgG1, IgG2, IgG3, IgG4) and the like, and preferably IgG1 or IgG2.
- the biological activity of an antibody generally includes an antigen-binding activity, an activity that is internalized in a cell that expresses the antigen by binding to the antigen, an activity that neutralizes the activity of the antigen, an activity that enhances the activity of the antigen, Examples include antibody-dependent cytotoxicity (ADCC) activity, complement-dependent cytotoxicity (CDC) activity, and antibody-dependent cell-mediated phagocytosis (ADCP).
- the function of the antibody according to the present invention is directed to GPR20. It is a binding activity, preferably an activity that is internalized in a GPR20-expressing cell by binding to GPR20.
- the antibody of the present invention may have ADCC activity, CDC activity and / or ADCP activity in addition to cell internalization activity.
- the obtained antibody can be purified to homogeneity.
- separation and purification methods used for ordinary proteins may be used.
- antibodies can be separated and purified by appropriately selecting and combining column chromatography, filter filtration, ultrafiltration, salting out, dialysis, preparative polyacrylamide gel electrophoresis, isoelectric focusing, etc. (Stratesies) for Protein Purification and Characterization: A Laboratory Course Manual, Daniel R.Marshak et al.eds, Cold Spring Harbor Laboratory Press (1996); Antibodies:. A Laboratory Manual.Ed Harlow and David Lane, Cold Spring Harbor Laboratory ( 988)) it is not intended to be limited thereto.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.
- chromatography can be performed using liquid chromatography such as HPLC or FPLC.
- columns used for affinity chromatography include protein A columns and protein G columns.
- protein A columns As a column using a protein A column, Hyper D, POROS, Sepharose F.R. F. (Pharmacia) and the like.
- Anti-GPR20 antibody-drug conjugate (1) Drug The anti-GPR20 antibody obtained in the above-mentioned "2. Production of anti-GPR20 antibody” binds the drug via a linker structure portion, thereby anti-GPR20 antibody-drug conjugate. It can be.
- the drug is not particularly limited as long as it has a substituent or a partial structure that can be bonded to the linker structure.
- the anti-GPR20 antibody-drug conjugate can be used for various applications depending on the drug to be bound.
- drugs examples include substances having anti-tumor activity, substances having an effect on blood diseases, substances having an effect on autoimmune diseases, anti-inflammatory substances, antibacterial substances, antifungal substances, antiparasitic substances, anti-parasitic substances, Examples include viral substances and anti-anesthetic substances.
- the antitumor compound is not particularly limited as long as it is a compound having an antitumor effect and has a substituent and a partial structure that can be bonded to a linker structure.
- a part or all of the linker is cleaved in the tumor cell to release the antitumor compound portion and to exhibit an antitumor effect.
- the linker is cleaved at the binding site with the drug, the antitumor compound is released in its original structure, and its original antitumor effect is exhibited.
- the anti-GPR20 antibody obtained in “2. Production of anti-GPR20 antibody” can be converted into an anti-GPR20 antibody-drug conjugate by binding an antitumor compound via a linker structure moiety.
- an antitumor compound used in the present invention is exetecan ((1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-, a camptothecin derivative. 4-methyl-1H, 12H-benzo [de] pyrano [3 ′, 4 ′: 6,7] indolidino [1,2-b] quinoline-10,13 (9H, 15H) -dione;
- Exatecan is a compound that exhibits an excellent antitumor effect even in such a state, although it may be released in tumor cells in a state where a part of the linker is bound.
- Exatecan has a camptothecin structure, so that in an acidic aqueous medium (for example, about pH 3), the equilibrium is biased to a structure in which a lactone ring is formed (ring-closed), whereas in a basic aqueous medium (for example, about pH 10), the lactone ring is It is known that the equilibrium is biased to the ring-opened structure (ring-opened body). Even drug conjugates into which exatecan residues corresponding to such a ring-closed structure and ring-opened structure are introduced are expected to have an equivalent antitumor effect, and any of them are included in the scope of the present invention. Nor.
- antitumor compounds examples include antitumor compounds described in the literature (Pharmacological Reviews, 68, p3-19, 2016). Examples thereof include doxorubicin, calchemamicin, and dolastatin. ) 10, oristatins (Auristatins) such as monomethyl oristatin E (MMAE), monomethyl oristatin F (MMAF), maytansinoids (maytansinoids) such as DM1 and DM4, SG2000 (pyrolobenzodiazepine) dimer SJG-136), SN-38, which is a derivative of camptothecin, CC-1065, etc.
- oristatins such as monomethyl oristatin E (MMAE), monomethyl oristatin F (MMAF)
- maytansinoids maytansinoids
- DM1 and DM4 SG2000 (pyrolobenzodiazepine) dimer SJG-136
- SN-38 which is a derivative of camptothecin,
- Karumaishin compound (Duocarmycins), amanitins (Amanitin), mention may be made of daunorubicin, mitomycin C, bleomycin, Shikuroshichijin, vincristine, vinblastine, methotrexate, platinum-based antitumor agents (cisplatin or derivatives thereof), taxol or derivatives thereof.
- the number of drugs bound to one antibody molecule is an important factor affecting the effectiveness and safety of the antibody-drug conjugate.
- Antibody-drug conjugates are manufactured by specifying reaction conditions such as the amount of raw materials and reagents to be reacted so that the number of drug bonds is constant. What is a chemical reaction of low molecular weight compounds? Unlike, it is usually obtained as a mixture of different numbers of drugs combined.
- the number of drugs bound to one antibody molecule is specified and expressed as an average value, that is, the average number of drug bonds.
- the number of drug bindings is shown unless an antibody-drug conjugate having a specific drug binding number contained in an antibody-drug conjugate mixture having a different drug binding number is indicated. Mean value.
- the number of Exatecan binding to the antibody molecule is controllable, and about 1 to 10 Exatecan can be bound as the average number of drugs bound per antibody, preferably 2 to 8, more preferably Is from 5 to 8, more preferably from 7 to 8, even more preferably 8.
- a person skilled in the art can design a reaction for binding the required number of drugs to the antibody from the description of the examples of the present application, and can obtain an antibody-drug conjugate in which the number of bindings of exatecan is controlled. .
- Linker structure A linker structure for binding a drug to the anti-GPR20 antibody in the anti-GPR20 antibody-drug conjugate of the present invention will be described.
- the linker structure for binding the anti-GPR20 antibody and the drug is not particularly limited as long as it can be used as the antibody-drug conjugate, and should be appropriately selected and used according to the purpose of use. Can do.
- linker structure known document (Pharmacol Rev 68: 3 - 19 , January 2016, Protein Cell DOI 10.1007 / s13238-016-0323-0 etc.) can be mentioned linkers described in more detail Examples include VC (valine-citrulline), MC (maleinamide caproyl), SMCC (succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate: succinimidyl 4- (N- maleimidotyl) cyclohexane-1-carboxylate), SPP (N-succinimidyl 4- (2-pyridyldithio) pentanoic acid: N-su cinimidyl 4- (2-pyridyldithio) pentanoate, SS (disulfide), SPDB (N-succimidyl 4- (2-pyridyldithio) butyrate) N-succinimidyl 4- (2-
- linker structure for example, a linker structure described in US Patent Publication US2016 / 0297890 (for example, those described in paragraphs [0260] to [0289]) can be mentioned.
- the left end of the structure shown below is a binding site with an antibody, and the right end is a binding site with a drug.
- GGFG in the following linker structure represents an amino acid sequence connected by a peptide bond composed of glycine-glycine-phenylalanine-glycine.
- the antitumor compound is bonded to the carbonyl group of CH 2 —O—CH 2 —C ( ⁇ O) — at the terminal opposite to (-Succinimid-3-yl-N) (in the above example, the right terminal).
- “-(Succinimid-3-yl-N)-” is the following formula:
- the 3rd position in this partial structure is a binding site to the anti-GPR20 antibody.
- This binding to the antibody at the 3-position is characterized by binding by forming a thioether.
- the nitrogen atom at position 1 of this structural moiety is bound to the methylene carbon atom present in the linker containing the structure.
- a drug-linker structure portion having the following structure is preferably bound to the antibody.
- These drug-linker structure moieties may be bound to 1 to 10 as the average number of bonds per antibody, preferably 2 to 8, more preferably 5 to 8, and still more preferably 7 To 8, more preferably 8.
- An antibody that can be used in the antibody-drug conjugate of the present invention has an internalization activity described in the above-mentioned section “2. Production of anti-GPR20 antibody” and Examples. There is no particular limitation as long as it is an anti-GPR20 antibody and a functional fragment of the antibody.
- AB represents an antibody having a sulfhydryl group.
- L 1 is It is represented by the structure of-(Succinimid-3-yl-N)-.
- L 1 ′ represents a maleimidyl group represented by the following formula.
- the compound of the formula (1) is described as a structure in which one structural part from the drug to the linker end is bound to one antibody. This is for convenience of explanation. In practice, there are many cases where a plurality of the structural parts are actually bound to one antibody molecule. This situation is the same in the following description of the manufacturing method.
- the compound (2) which can be obtained by a known method (for example, available by the method described in US2016 / 297890 published patent publication (for example, the method described in paragraphs [0336] to [0374])) and a sulfhydryl group
- the antibody-drug conjugate (1) can be produced by reacting an antibody (3a) having
- An antibody (3a) having a sulfhydryl group can be obtained by a method well known to those skilled in the art (Hermanson, GT, Bioconjugate Technologies, pp. 56-136, pp. 456-493, Academic Press (1996)).
- Traut's reagent is allowed to act on the amino group of the antibody;
- N-succinimidyl S-acetylthioalkanoates are allowed to act on the amino group of the antibody, followed by hydroxylamine;
- N-succinimidyl 3- (pyridyldithio) ) Propionate is allowed to act, and then a reducing agent is allowed to act;
- a reducing agent such as dithiothreitol, 2-mercaptoethanol, tris (2-carboxyethyl) phosphine hydrochloride (TCEP) is allowed to act on the antibody, and the intra-antibody chain
- TCEP (2-carboxyethyl) phos
- TCEP as a reducing agent is used in an amount of 0.3 to 3 molar equivalents per inter-chain disulfide in the antibody, and reacted with the antibody in a buffer containing a chelating agent, whereby the antibody inner chain Antibodies in which the interdisulfide is partially or completely reduced can be obtained.
- the chelating agent include ethylenediaminetetraacetic acid (EDTA) and diethylenetriaminepentaacetic acid (DTPA). These may be used at a concentration of 1 mM to 20 mM.
- EDTA ethylenediaminetetraacetic acid
- DTPA diethylenetriaminepentaacetic acid
- the buffer solution sodium phosphate, sodium borate, sodium acetate solution and the like can be used.
- the antibody (3a) having a partially or completely reduced sulfhydryl group can be obtained by reacting the antibody with TCEP at 4 to 37 ° C. for 1 to 4 hours.
- a drug-linker moiety can be bound by a thioether bond by carrying out a reaction for adding a sulfhydryl group to the drug-linker moiety.
- antibody-drug conjugate (1 ) can be manufactured.
- a solution in which the compound (2) is dissolved may be added to a buffer solution containing the antibody (3a) having a sulfhydryl group and reacted.
- a buffer solution a sodium acetate solution, sodium phosphate, sodium borate, or the like may be used.
- the pH during the reaction is 5 to 9, and more preferably, the reaction may be performed in the vicinity of pH 7.
- an organic solvent such as dimethylsulfoxide (DMSO), dimethylformamide (DMF), dimethylacetamide (DMA), N-methyl-2-pyridone (NMP) can be used.
- the organic solvent solution in which the compound (2) is dissolved may be reacted by adding 1 to 20% v / v to a buffer solution containing the antibody (3a) having a sulfhydryl group.
- the reaction temperature is 0 to 37 ° C., more preferably 10 to 25 ° C., and the reaction time is 0.5 to 2 hours.
- the reaction can be terminated by deactivating the reactivity of the unreacted compound (2) with a thiol-containing reagent.
- a thiol-containing reagent is, for example, cysteine or N-acetyl-L-cysteine (NAC). More specifically, the reaction can be completed by adding 1 to 2 molar equivalents of NAC to the compound (2) used and incubating at room temperature for 10 to 30 minutes.
- the produced antibody-drug conjugate (for example, antibody-drug conjugate (1)) is concentrated, buffer exchanged, purified, antibody concentration and per antibody molecule by the following common operations.
- the antibody-drug conjugate (1) can be identified.
- the gel filtration purification operation in which this fractionated fraction is again applied to the NAP-25 column and eluted with a buffer solution, is repeated 2 to 3 times in total, thereby allowing unbound drug linkers and low molecular weight compounds (tris (2-carboxyethyl) phosphine) Antibody-drug conjugates without the hydrochloride (TCEP), N-acetyl-L-cysteine (NAC), dimethyl sulfoxide) were obtained.
- TCEP hydrochloride
- NAC N-acetyl-L-cysteine
- dimethyl sulfoxide dimethyl sulfoxide
- the total absorbance at a certain wavelength is equal to the sum of the absorbances of all absorbing species present in the system [addition of absorbance], so the molar extinction coefficient of the antibody and drug changes before and after conjugation of the antibody and drug. Assuming that there is no antibody, the antibody concentration and the drug concentration in the antibody-drug conjugate are expressed by the following relational expression.
- a 280 represents the absorbance of the antibody-drug conjugate aqueous solution at 280 nm
- a 370 represents the absorbance of the antibody-drug conjugate aqueous solution at 370 nm
- a A, 280 represents the absorbance of the antibody at 280 nm
- a A , 370 indicates the absorbance of the antibody at 370 nm
- AD, 280 indicates the absorbance of the conjugate precursor at 280 nm
- AD, 370 indicates the absorbance of the conjugate precursor at 370 nm
- ⁇ A, 280 is at 280 nm.
- ⁇ A, 370 shows the molar extinction coefficient of the antibody at 370 nm
- ⁇ D, 280 shows the molar extinction coefficient of the conjugate precursor at 280 nm
- ⁇ D, 370 shows the conjugate at 370 nm.
- C A antibody - indicates antibody concentration in drug conjugates
- C D antibody - shows the drug concentration in the drug conjugate.
- ⁇ A, 280, ⁇ A, 370, ⁇ D, 280, ⁇ D, 370 values prepared in advance (calculated estimated values or actual values obtained from UV measurement of compounds) are used.
- ⁇ A, 280 can be estimated from the amino acid sequence of an antibody by a known calculation method (Protein Science, 1995, vol. 4, 2411-2423).
- ⁇ A, 370 is usually zero.
- ⁇ D, 280 and ⁇ D, 370 are obtained by measuring the absorbance of a solution in which the conjugate precursor to be used is dissolved at a certain molar concentration. Long).
- C A and C D can be obtained by measuring A 280 and A 370 of the antibody-drug conjugate aqueous solution and substituting these values into equations (1) and (2) to solve the simultaneous equations. Furthermore C D can be drug average binding per antibody determined by the dividing in C A.
- HPLC analysis HPLC analysis is performed under the following measurement conditions.
- HPLC system Agilent 1290 HPLC system (Agilent Technologies) Detector: UV absorbance meter (measurement wavelength: 280 nm)
- Mobile phase A aqueous solution containing 0.10% trifluoroacetic acid (TFA), 15% 2-propanol
- Mobile phase B acetonitrile solution containing 0.075% TFA, 15% 2-propanol
- Gradient program 14% -36% (0 min-15 min), 36% -80% (15 min-17 min), 80% -14% (17 min-17.01 min), 14% (17.01 min-25 min)
- HPLC system Agilent 1290 HPLC system (Agilent Technologies)
- Detector UV absorbance meter (measurement wavelength: 280 nm)
- F-3 Data Analysis F-3-1
- the light chain of an antibody is expressed as L i and the heavy chain as H i according to the number of drugs bound (where i represents the number of drugs bound. That is, in the present invention).
- the number of drug bonds is expressed as L 0 , L 1 , H 0 , H 1 , H 2 , H 3 .
- a light chain (L 0 ) and a heavy chain (H 0 ) of an antibody to which no drug is bound a light chain with one drug bound: L 1 and a heavy chain with one drug bound: H 1
- the heavy chain with two drugs bonded: H 2 and the heavy chain with three drugs bonded: H 3 increase in hydrophobicity and increase in retention time in proportion to the number of drugs bonded, for example, L 0 , L 1 , H 0 , H 1 , H 2 , H 3 in this order.
- the detection peak can be assigned to any one of L 0 , L 1 , H 0 , H 1 , H 2 , and H 3 by comparing the holding times with L 0 and H 0 .
- the peak area value is corrected according to the following formula using the molar extinction coefficient of the light chain, heavy chain, and drug linker according to the number of bonds of the drug linker.
- the molar extinction coefficient (280 nm) of the light and heavy chains in each antibody is determined by the known calculation method (Protein Science, 1995, vol. 4, 2411-2423). Values deduced from the sequence can be used. In the case of h046-H4e / L7, according to the amino acid sequence, 26210 was used as the molar extinction coefficient of the light chain and 68990 was used as the estimated value as the molar extinction coefficient of the heavy chain.
- the molar extinction coefficient (280 nm) of the drug linker is the measured molar extinction coefficient (280 nm) of the compound obtained by reacting each drug linker with mercaptoethanol or N-acetylcysteine and converting the maleimide group to succinimide thioether. It was.
- the wavelength at which the absorbance is measured can be appropriately set by those skilled in the art, but is preferably a wavelength at which the peak of the antibody can be measured, and more preferably 280 nm.
- F-3-3 Calculate the peak area ratio (%) of each chain with respect to the total peak area correction value according to the following formula.
- a Li, A Hi: L i , H i each peak area correction value F-3-4 antibody - drug average bond number per antibody per molecule in drug conjugates is calculated according to the following equation.
- Drug average number of bonds (L 0 peak area ratio x0 + L 0 peak area ratio x1 + H 0 peak area ratio x0 + H 1 peak area ratio x1 + H 2 peak area ratio x2 + H 3 peak area ratio x3) / 100 ⁇ 2
- Drug average number of bonds (L 0 peak area ratio x0 + L 0 peak area ratio x1 + H 0 peak area ratio x0 + H 1 peak area ratio x1 + H 2 peak area ratio x2 + H 3 peak area ratio x3) / 100 ⁇ 2
- conjugates for example, about ⁇ 1 having the same average number of drugs prepared under the same conditions into a new lot. it can. In that case, the average number of drugs falls within the average number of drugs before mixing.
- an antibody-drug conjugate used in the present invention includes the formula
- A represents the binding position with the antibody
- an anti-GPR20 antibody disclosed in the present specification and a drug-linker structure moiety represented by the above-mentioned antibody-drug conjugates.
- a partial structure comprising a linker and a drug in an antibody-drug conjugate is also referred to as “drug-linker structure”, “drug-linker structure part”, or “drug linker”.
- This drug linker is a thiol group (in other words, a sulfur atom of a cysteine residue) generated at a disulfide bond site between antibody chains (between two heavy chains and heavy chains and between two heavy chains and light chains). Is bound to.
- AB represents the anti-GPR20 antibody disclosed in the present specification, and is bound to a drug linker via a sulfhydryl group derived from the antibody.
- n is synonymous with so-called DAR (Drug-to-Antibody Ratio) and indicates a drug antibody ratio per antibody. That is, it shows the number of drugs bound to one molecule of antibody, which is a numerical value specified and expressed as an average value, that is, an average drug binding number.
- n may be 2 to 8, preferably 5 to 8, more preferably 7 to 7, as measured by common operation F. 8 is even more preferable.
- the antibody represented by AB is any one selected from the group consisting of the following (a) to (y):
- Examples include antibody-drug conjugates or pharmacologically acceptable salts thereof, comprising the heavy chain and light chain antibodies or functional fragments thereof according to item 1: from the following (a) to (y):
- anti-GPR20 antibody and the functional fragment of the antibody of the present invention have internalization activity, they can be used as antibodies used in antibody-drug conjugates.
- Anti-GPR20 antibody-drug conjugate a drug having antitumor activity such as cytotoxic activity is used as a drug.
- a conjugate of an anti-GPR20 antibody having internalization activity and / or a functional fragment of the antibody and a drug having antitumor activity such as cytotoxic activity is used for cancer cells expressing GPR20. Since it exhibits antitumor activity, it can be used as a pharmaceutical, particularly as a therapeutic and / or prophylactic agent for cancer.
- the anti-GPR20 antibody-drug conjugate of the present invention becomes a hydrate by absorbing moisture or adhering adsorbed water by being left in the atmosphere, or by recrystallization or purification operation. In some cases, such water-containing compounds or pharmacologically acceptable salts are also included in the present invention.
- a pharmacologically acceptable acid addition salt can be formed as desired.
- acid addition salts include hydrohalides such as hydrofluoride, hydrochloride, hydrobromide and hydroiodide; nitrates, perchlorates, sulfates and phosphates.
- Inorganic acid salts such as methane sulfonate, trifluoromethane sulfonate, lower alkane sulfonate such as ethane sulfonate; aryl sulfonate such as benzene sulfonate and p-toluene sulfonate; formate , Acetate, trifluoroacetate, malate, fumarate, succinate, citrate, tartrate, oxalate, maleate and other organic acid salts; ornithate, glutamate, asparagine Examples thereof include amino acid salts such as acid salts.
- a pharmacologically acceptable base addition salt can be formed as desired.
- base addition salts include alkali metal salts such as sodium salts, potassium salts and lithium salts; alkaline earth metal salts such as calcium salts and magnesium salts; inorganic salts such as ammonium salts; dibenzylamine salts and morpholine.
- phenylglycine alkyl ester salt ethylenediamine salt, N-methylglucamine salt, diethylamine salt, triethylamine salt, cyclohexylamine salt, dicyclohexylamine salt, N, N'-dibenzylethylenediamine salt, diethanolamine salt, N-benzyl-N
- organic amine salts such as-(2-phenylethoxy) amine salt, piperazine salt, tetramethylammonium salt, and tris (hydroxymethyl) aminomethane salt.
- the present invention can also include anti-GPR20 antibody-drug conjugates in which one or more of the atoms making up the antibody-drug conjugate is replaced with an isotope of that atom.
- isotopes There are two types of isotopes, radioactive isotopes and stable isotopes. Examples of isotopes include hydrogen isotopes ( 2 H and 3 H), carbon isotopes ( 11 C, 13 C and 14 C), nitrogen isotopes ( 13 N and 15 N), oxygen isotopes ( 15 O, 17 O and 18 O), fluorine isotopes ( 18 F) and the like.
- compositions comprising isotope-labeled antibody-drug conjugates are useful, for example, as therapeutic agents, prophylactic agents, research reagents, assay reagents, diagnostic agents, in vivo diagnostic imaging agents, and the like.
- Isotope-labeled antibody-drug conjugates and any ratio mixtures of isotope-labeled antibody-drug conjugates are all encompassed by the present invention.
- the isotope-labeled antibody-drug conjugate is produced by a method known in the art, for example, by using an isotope-labeled raw material instead of the raw material in the production method of the present invention described later. Can do.
- In vitro cell killing activity can be measured, for example, by cell growth inhibitory activity.
- a cancer cell line overexpressing GPR20 can be cultured, anti-GPR20 antibody-drug conjugates can be added to the culture system at various concentrations, and the focus activity, colony formation and spheroid growth inhibitory activity can be measured. it can.
- a cancer cell line derived from gastrointestinal stromal tumor GIST
- cell growth inhibitory activity against gastrointestinal stromal tumor can be examined.
- the therapeutic effect on cancer using an in vivo experimental animal is, for example, that an anti-GPR20 antibody-drug conjugate is administered to a nude mouse transplanted with a tumor cell line that highly expresses GPR20, and changes in cancer cells are observed.
- an animal model in which cells derived from gastrointestinal stromal tumors are transplanted into immunodeficient mice, the therapeutic effect on gastrointestinal stromal tumors can be measured.
- the type of cancer to which the anti-GPR20 antibody-drug conjugate of the present invention is applied is not particularly limited as long as it expresses GPR20 in a cancer cell to be treated, but for example, esophagus, stomach, Although digestive organ cancers, such as a small intestine and a large intestine, can be mentioned, as long as GPR20 is expressed, it is not restrict
- a more preferable example of cancer is gastrointestinal stromal tumor (GIST).
- GIST gastrointestinal stromal tumor
- the anti-GPR20 antibody-drug conjugate of the present invention can be suitably administered to a mammal, but is more preferably a human.
- the substance used in the pharmaceutical composition containing the anti-GPR20 antibody-drug conjugate of the present invention is appropriately selected and applied from the dosage additive and other dosage agents ordinarily used in this field at the dosage and concentration. Can do.
- the anti-GPR20 antibody-drug conjugate of the present invention can be administered as a pharmaceutical composition comprising one or more pharmaceutically compatible ingredients.
- the pharmaceutical composition typically includes one or more pharmaceutical carriers (eg, sterile liquids (eg, water and oils (petroleum, animal, vegetable, or synthetic origin oils (eg, peanut oil)). Water) is a more typical carrier when the pharmaceutical composition is administered intravenously, saline solution, and aqueous dextrose and glycerol solutions. Can also be used as a liquid carrier, in particular for injectable solutions Suitable pharmaceutical excipients are known in the art. Alternatively, emulsifiers or pH buffering agents can be included Examples of suitable pharmaceutical carriers are “Remington's Pharmaceutical Sciences” by EW Martin. Described s ". As a formulation corresponds to the mode of administration.
- Various delivery systems are known and can be used to administer the anti-GPR20 antibody-drug conjugates of the invention.
- Introduction methods include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, and subcutaneous routes. Administration can be, for example, by infusion or bolus injection. In certain preferred embodiments, administration of the antibody-drug conjugate is by infusion. Parenteral administration is the preferred route of administration.
- the pharmaceutical composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans.
- compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
- the medicament may also include a solubilizing agent and a local anesthetic (eg, lignocaine) to relieve pain at the site of injection.
- a local anesthetic eg, lignocaine
- the ingredients are combined separately or in unit dosage form (eg, as a dry lyophilized powder or anhydrous concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent).
- the medicament can be dispensed, for example, in an infusion bottle containing sterile pharmaceutical grade water or saline. If administered by injection, an ampoule of sterile water for injection or saline can be provided, eg, so that the ingredients can be mixed prior to administration.
- the pharmaceutical composition of the present invention may be a pharmaceutical composition containing only the anti-GPR20 antibody-drug conjugate of the present application, or may contain an anti-GPR20 antibody-drug conjugate and at least one other cancer therapeutic agent. It may be a pharmaceutical composition.
- the anti-GPR20 antibody-drug conjugate of the present invention can be administered together with other cancer therapeutic agents, thereby enhancing the anticancer effect.
- Other anticancer agents used for such purposes may be administered to the individual simultaneously or separately with the antibody-drug conjugate, or at different administration intervals. Also good.
- cancer therapeutic agents include tyrosine kinase inhibitors including Imatinib, Sunitinib, Regofenib and the like, CDK4 / 6 inhibitors including Palociclib and the like, HSP90 inhibitors including TAS-116 and the like, MEK inhibitors including MEK162 and the like, And immune checkpoint inhibitors including nivolumab, pembrolizumab, ipilimumab and the like, but are not limited as long as they have antitumor activity.
- tyrosine kinase inhibitors including Imatinib, Sunitinib, Regofenib and the like
- CDK4 / 6 inhibitors including Palociclib and the like
- HSP90 inhibitors including TAS-116 and the like
- MEK inhibitors including MEK162 and the like
- immune checkpoint inhibitors including nivolumab, pembrolizumab, ipilimumab and the like, but are not limited as long
- Such a pharmaceutical composition may be formulated as a freeze-dried preparation or a liquid preparation as a preparation having the selected composition and the required purity.
- a pharmaceutical composition When formulated as a lyophilized formulation, it may be a formulation containing appropriate formulation additives used in this field.
- liquid preparations can be formulated as liquid preparations containing various preparation additives used in this field.
- the anti-GPR20 antibody-drug conjugate contained in the pharmaceutical composition of the present invention has the affinity of the antibody-drug conjugate for the antigen, that is, dissociation for the antigen.
- Kd value the higher the affinity (the lower the Kd value), the more effective the drug can be exerted even with a small dose. Therefore, in determining the dose of the antibody-drug conjugate, the dose can be set based on the affinity state between the antibody-drug conjugate and the antigen.
- the antibody-drug conjugate of the present invention is administered to humans, for example, about 0.001 to 100 mg / kg may be administered once or multiple times at intervals of 1 to 180 days. Preferably, 0.1 to 50 mg / kg, more preferably 1 to 15 mg / kg may be administered multiple times at intervals of once every 2 to 3 weeks.
- Example 1 Acquisition of rat anti-human GPR20 antibody having internalization activity 1
- a cDNA encoding human GPR20 protein (NP_005284) is obtained from human brain according to a method known to those skilled in the art.
- a human GPR20 expression vector pcDNA3.1-hGPR20 was prepared by amplification by PCR using a template and incorporation into a mammalian expression vector.
- the amino acid sequence of human GPR20 is shown in SEQ ID NO: 1 in the sequence listing. Large-scale preparation of pcDNA3.1-hGPR20 plasmid DNA was performed using EndoFree Plasmid Giga Kit (QIAGEN).
- Hybridoma preparation After lymph node cells and mouse myeloma SP2 / 0-ag14 cells were electro-fused using a Hybridum Hybridoma Production System (Cyto Pulse Sciences), ClonCell-HY SelemDelC Suspended at 37 ° C., diluted and cultured under conditions of 37 ° C. and 5% CO 2 . Each appearing hybridoma colony was recovered as a monoclone, suspended in ClonCell-HY Selection Medium E (StemCell Technologies), and cultured under conditions of 37 ° C. and 5% CO 2 . After moderate cell growth, a frozen stock of each hybridoma cell was prepared and the culture supernatant was collected and used for screening of hybridomas producing anti-GPR20 antibodies.
- a Hybridoma preparation After lymph node cells and mouse myeloma SP2 / 0-ag14 cells were electro-fused using a Hybridum Hybridoma Production System (Cyto Pulse Sciences),
- OPD coloring solution 0.05 M trisodium citrate, 0.1 M disodium hydrogenphosphate.12 water, pH 4. 5
- OPD coloring solution 0.05 M trisodium citrate, 0.1 M disodium hydrogenphosphate.12 water, pH 4. 5
- o-phenylenediamine dihydrochloride o-phenylenediamine dihydrochloride (Wako Pure Chemical Industries, Ltd.) and H 2 O 2 dissolved in 0.4 mg / mL and 0.6% (v / v), respectively, were added at 100 ⁇ L / well. did.
- the color development reaction was carried out with occasional stirring, 1M HCl was added at 100 ⁇ L / well to stop the color development reaction, and then the absorbance at 490 nm was measured with a plate reader (ENVISION: PerkinElmer).
- the hybridoma producing a culture supernatant showing higher absorbance in the pcDNA3.1-hGPR20 expression vector-introduced 293 ⁇ cells is specific for human GPR20 expressed on the cell membrane surface was selected as a hybridoma producing a binding antibody.
- Example 1 The suspension of transiently expressed 293T cells prepared in -5-1 was centrifuged, and the supernatant was removed. Then, the hybridoma culture supernatant was added to each suspension, and suspended at 4 ° C. for 1 hour. Left to stand. After washing twice with 5% FBS-containing PBS, Anti-Rat IgG FITC conjugate (SIGMA) diluted 500-fold with 5% FBS-containing PBS was added and suspended, and left at 4 ° C. for 1 hour.
- SIGMA Anti-Rat IgG FITC conjugate
- the hybridoma producing an antibody in which the histogram of pcDNA3.1-hGPR20-introduced 293T cells is shifted to the strong fluorescence intensity side with respect to the fluorescence intensity histogram of pcDNA3.1-introducing 293T cells, which is a control, is used as an antibody-producing hybridoma that binds to human GPR20.
- 178 clones were selected.
- FIG. 18 shows clones 04-002, 04-006, 04-013, 04-014, 04-020, 04-021, 04-037, 04-046, 04 as examples of antibodies specifically bound to human GPR20.
- Rat anti-human GPR20 monoclonal antibody was purified from the hybridoma culture supernatant.
- a rat anti-GPR20 monoclonal antibody-producing hybridoma was grown to a sufficient amount with ClonCell-HY Selection Medium E (StemCell Technologies), and then Ultra Low IgG FBS (Life Technologies Inc., LifSolMe, with the addition of 20% of the selenium-rich selenium) The medium was changed to 4) and cultured for 4-5 days. The main culture supernatant was collected, passed through a 0.8 ⁇ m filter, and then passed through a 0.2 ⁇ m filter to remove insoluble matters.
- the antibody was purified from the above hybridoma supernatant in a one-step process by Protein G affinity chromatography (under 4 to 6 ° C.).
- the buffer substitution step after protein G affinity chromatography purification was performed at 4 to 6 ° C.
- the culture supernatant of the hybridoma was applied to a column packed with Protein G (GE Healthcare Bioscience) equilibrated with PBS. After all of the culture supernatant liquid entered the column, the column was washed with PBS twice the column volume. Next, elution was performed with 0.1 M glycine / hydrochloric acid aqueous solution (pH 2.7), and fractions containing the antibody were collected.
- the collected fraction was immediately adjusted to pH 7.0 to 7.5 by adding 1 M Tris-HCl (pH 9.0), and then the Centrifugal UF Filter Device VIVASPIN 20 (fractionated molecular weight UF30K, Sartorius, 4 to 6 ° C.) ) was used to replace the buffer with PBS and concentrated to an antibody concentration of 0.2 mg / mL or more. Finally, it was filtered through a Minisart-Plus filter (Sartorius) to obtain a purified antibody sample.
- VIVASPIN 20 fractionated molecular weight UF30K, Sartorius, 4 to 6 ° C.
- Example 2 In vitro evaluation of rat anti-GPR20 antibody 2) -1 Evaluation of binding ability of rat anti-GPR20 antibody by flow cytometry In order to evaluate the binding ability to GPR20, 1) pcDNA3 prepared by the method shown in -5-1 .1-hGPR20-introduced 293T cell suspension was centrifuged and the supernatant was removed.
- Rat anti-human GPR20 monoclonal antibody having internalization activity prepared in 8) -8 (clone number was 04-002, 04-006, 04-013, 04-014, 04-021, 04-037, 04-046, 04-047, 04-067, 04-068, 04-079, 04-114, 04-115, 04- 125, 04-126, 04-127, 04-133, 04-139 and 04-163) and rat anti-human GP
- Two R20 monoclonal antibodies (13-024, 13-048) or rat IgG control (R & D Systems) were added and suspended, and the mixture was allowed to stand at 4 ° C. for 1 hour.
- the internalization activity of the purified rat anti-GPR20 antibody was the anti-rat IgG antibody reagent Rat bound with a toxin (saporin) that inhibits protein synthesis as in 1) -6. -Evaluated using ZAP (ADVANCED TARGETING SYSTEMS). That is, HEK293 cells stably expressing GPR20-EGFP protein with EGFP linked to the C-terminal side of human GPR20 were seeded at 2.5 ⁇ 10 3 cells / well and cultured overnight at 37 ° C. and 5% CO 2.
- Rat anti-GPR20 antibody final concentration: 0.012 to 1 ⁇ g / mL
- Rat-ZAP final concentration: 0.5 ⁇ g / mL
- the cell growth inhibitory effect by the addition of the anti-GPR20 antibody was expressed as a relative activity with the number of viable cells being 100% when no antibody was added.
- Each anti-GPR20 antibody most suppressed cell proliferation when added at a final concentration of 0.11 or 0.33 ⁇ g / mL.
- Rat IgG2a and IgG2b isotype control antibodies were used as negative control antibodies that recognize antigens unrelated to GPR20.
- Example 3 Determination of nucleotide sequence of cDNA encoding variable region of rat anti-GPR20 antibody Twenty-one rat anti-GPR20 antibodies (04-002, 04-006, 04-013, whose internalization activity was evaluated in Example 2) 04-014, 04-021, 04-037, 04-046, 04-047, 04-067, 04-068, 04-079, 04-114, 04-115, 04-125, 04-126, 04- 127, 04-133, 04-139, 04-163, 13-024, 13-048) The nucleotide sequence of cDNA encoding the variable region was determined by the following method.
- RNA washing solution A (10 mM Tris-HCl (pH 7.5), 0.15 M LiCl, 1 mM EDTA, 0.1% LiDS, 0.1% Triton X-100) and a solution for cDNA synthesis (50 mM Tris X-100).
- rat immunoglobulin heavy and light chain variable region gene fragments Wash the magnetic beads with TE solution (10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.1% Triton X-100). Later, rat immunoglobulin heavy chain and light chain genes were amplified using 5'-RACE PCR. Specifically, the magnetic beads were transferred to a PCR reaction solution (0.2 ⁇ M primer, 0.2 mM dNTP, 0.25 unit PrimeSTAR HS DNA Polymerase (TAKARA)), and a reaction at 94 ° C. for 30 seconds to 68 ° C. for 90 seconds was performed for 35 cycles. .
- the primer sets used are as follows. In the primer sequence, D represents a mixed base composed of A, G, and T, and N represents a mixed base composed of A, C, G, and T.
- PCR primer set (for heavy chain) 5′-GCTAGCGCTACCGCGACTCAGATCCCCCCCCCCCCCCDN-3 ′ (Nhe-polyCS) (SEQ ID NO: 66) 5′-TCACTGAGCTGGGTGAGAGTGTAGAGCCCC-3 ′ (rIg ⁇ -AS1) (SEQ ID NO: 67) 5′-TCACCGAGCTGCTGAGGGGTGTAGAGCCCC-3 ′ (rIg ⁇ -AS2) (SEQ ID NO: 68) PCR primer set (for light chain) 5′-GCTAGCGCTACCGCGACTCAGATCCCCCCCCCCCCCCDN-3 ′ (Nhe-polyCS) (SEQ ID NO: 66) (same as for heavy chain) 5′-TCAGTAACACTGTCCAGGACACCATTCTC-3 ′ (rIg ⁇ -AS) (SEQ ID NO: 69)
- a sequence analysis of the nucleotide sequence was performed on the fragment amplified by the PCR reaction.
- 5′-CTGGCTCAGGGAAAATAGCCC-3 ′ (rIg ⁇ -seq) (SEQ ID NO: 70) as a heavy chain sequence primer
- 5′-TCCGTTGCTAACTGTTTCC-3 ′ (rIg ⁇ -seq) (SEQ ID NO: 71) as a light chain sequence primer
- Each of the oligonucleotides having Sequence analysis was performed using a gene sequence analyzer (“ABI PRISM 3700 DNA Analyzer; Applied Biosystems” or “Applied Biosystems 3730xl Analyzer; applied Cysystem”). Technologies) and GeneAmp 9700 (Applied Biosystems).
- base sequence and amino acid sequence of the constant region of rat heavy chain IgG2b reference was made to the base sequence and amino acid sequence of AABR03048905 (IGHG2B * 01) published in IMGT, the international ImM GeneTics information system (registered trademark). Further, the base sequence and amino acid sequence of the constant region of rat light chain IgK were referred to the base sequence and amino acid sequence of V01241 (IGKC * 01) published in the same system.
- the heavy chain of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of amino acid residues 143 to 475 is a constant region.
- the variable region has CDRH1 consisting of the amino acid sequence described in 45 to 54, CDRH2 consisting of the amino acid sequence described in 69 to 78, and CDRH3 consisting of the amino acid sequence described in 118 to 131 in SEQ ID NO: 2 in the sequence listing. .
- the heavy chain variable region of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing.
- CDRH1 of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing
- the amino acid sequence of CDRH3 is It has the amino acid sequence shown in SEQ ID NO: 6.
- the heavy chain sequence of the 04-046 antibody is shown in FIG.
- the light chain of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 7 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 21 is a signal sequence
- the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region.
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- the variable region has a CDRL1 consisting of the amino acid sequence described in 43 to 53, a CDRL2 consisting of the amino acid sequence described in 69 to 75, and a CDRL3 consisting of the amino acid sequence described in 108 to 116 in SEQ ID NO: 7 of the Sequence Listing.
- the light chain variable region of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing.
- the CDRL1 of the 04-046 antibody has the amino acid sequence shown in SEQ ID NO: 9 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence shown in SEQ ID NO: 10 in the sequence listing
- the amino acid sequence of CDRL3 is It has the amino acid sequence shown in SEQ ID NO: 11.
- the sequence of the light chain of the 04-046 antibody is shown in FIG.
- the heavy chain of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 12 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of amino acid residues 143 to 475 is a constant region.
- the variable region has CDRH1 consisting of the amino acid sequence described in 45 to 54, CDRH2 consisting of the amino acid sequence described in 69 to 78, and CDRH3 consisting of the amino acid sequence described in 118 to 131 in SEQ ID NO: 12 of the Sequence Listing. .
- the heavy chain variable region of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 13 in the Sequence Listing.
- CDR-01 of antibody 04-079 has the amino acid sequence shown in SEQ ID NO: 14 of the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 15 of the sequence listing
- the amino acid sequence of CDRH3 is It has the amino acid sequence shown in SEQ ID NO: 16.
- the heavy chain sequence of the 04-079 antibody is shown in FIG.
- the light chain of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 17 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence
- the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region.
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- the variable region has a CDRL1 consisting of the amino acid sequence described in 43 to 53, a CDRL2 consisting of the amino acid sequence described in 69 to 75, and a CDRL3 consisting of the amino acid sequence described in 108 to 116 in SEQ ID NO: 17 of the Sequence Listing. .
- the light chain variable region of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing.
- the CDRL1 of the 04-079 antibody has the amino acid sequence shown in SEQ ID NO: 19 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence shown in SEQ ID NO: 20 in the sequence listing
- the amino acid sequence of CDRL3 is It has the amino acid sequence shown in SEQ ID NO: 21.
- the sequence of the light chain of the 04-079 antibody is shown in FIG.
- the heavy chain of antibody 04-126 has the amino acid sequence shown in SEQ ID NO: 22 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 19 is a signal sequence
- the amino acid sequence consisting of amino acid residues 20 to 142 is a variable region.
- the amino acid sequence consisting of amino acid residues 143 to 475 is a constant region.
- the variable region has CDRH1 consisting of the amino acid sequence described in 45 to 54, CDRH2 consisting of the amino acid sequence described in 69 to 78, and CDRH3 consisting of the amino acid sequence described in 118 to 131 in SEQ ID NO: 22 of the Sequence Listing. .
- the heavy chain variable region of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 23 of the Sequence Listing.
- CDRH1 of antibody 04-126 has the amino acid sequence shown in SEQ ID NO: 24 of the sequence listing
- the amino acid sequence of CDRH2 has the amino acid sequence shown in SEQ ID NO: 25 of the sequence listing
- the amino acid sequence of CDRH3 is It has the amino acid sequence shown in SEQ ID NO: 26.
- the heavy chain sequence of the 04-126 antibody is shown in FIG.
- the light chain of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 27 in the sequence listing.
- the amino acid sequence consisting of amino acid residues 1 to 20 is a signal sequence
- the amino acid sequence consisting of amino acid residues 21 to 128 is a variable region.
- the amino acid sequence consisting of the 129th to 233rd amino acid residues is a constant region.
- the variable region has, in SEQ ID NO: 27 of the Sequence Listing, CDRL1 consisting of the amino acid sequence described in 43 to 53, CDRL2 consisting of the amino acid sequence described in 69 to 75, and CDRL3 consisting of the amino acid sequence described in 108 to 116. .
- the light chain variable region of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 28 of the Sequence Listing.
- the CDRL1 of the 04-126 antibody has the amino acid sequence shown in SEQ ID NO: 29 in the sequence listing
- the amino acid sequence of CDRL2 has the amino acid sequence shown in SEQ ID NO: 30 in the sequence listing
- the amino acid sequence of CDRL3 is It has the amino acid sequence shown in SEQ ID NO: 31.
- the sequence of the light chain of the 04-126 antibody is shown in FIG.
- the heavy chain amino acid sequence of the 04-046 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 32 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 58 to 426 of the nucleotide sequence shown in SEQ ID NO: 32 in the sequence listing encodes the heavy chain variable region of antibody 04-046, and the nucleotide sequence consisting of nucleotides 427 to 1425 Encodes the heavy chain constant region of the 04-046 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 133 to 162 encoding CDRH1 in SEQ ID NO: 32, and the nucleotide sequence described in nucleotide numbers 205 to 234 encoding CDRH2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 352 to 393 encoding CDRH3.
- the nucleotide sequence of the heavy chain variable region of the 04-046 antibody is also shown in SEQ ID NO: 33 in the Sequence Listing.
- the sequence of SEQ ID NO: 32 is shown in FIG.
- the light chain amino acid sequence of the 04-046 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 34 in the sequence listing.
- the nucleotide sequence consisting of nucleotides 61 to 384 of the nucleotide sequence shown in SEQ ID NO: 34 of the sequence listing encodes the light chain variable region of antibody 04-046, and the nucleotide sequence consisting of nucleotides 385 to 699 Encodes the light chain constant region of the 04-046 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 34, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 348 encoding CDRL3.
- the nucleotide sequence of the light chain variable region of the 04-046 antibody is also shown in SEQ ID NO: 35 in the Sequence Listing.
- the sequence of SEQ ID NO: 34 is shown in FIG.
- the heavy chain amino acid sequence of the 04-079 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 36 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 58 to 426 of the nucleotide sequence shown in SEQ ID NO: 36 in the sequence listing encodes the heavy chain variable region of antibody 04-079 and nucleotide sequence consisting of nucleotides 427 to 1425 Encodes the heavy chain constant region of the 04-079 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 133 to 162 encoding CDRH1 in SEQ ID NO: 36, and the nucleotide sequence described in nucleotide numbers 205 to 234 encoding CDRH2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 352 to 393 encoding CDRH3.
- the nucleotide sequence of the heavy chain variable region of the 04-079 antibody is also shown in SEQ ID NO: 37 in the Sequence Listing. The sequence of SEQ ID NO: 37 is described in FIG.
- the light chain amino acid sequence of the 04-079 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 38 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 61 to 384 of the nucleotide sequence shown in SEQ ID NO: 38 of the sequence listing encodes the light chain variable region of antibody 04-079, and the nucleotide sequence consisting of nucleotides 385 to 699 Encodes the light chain constant region of the 04-079 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 38, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 348 encoding CDRL3.
- the nucleotide sequence of the light chain variable region of the 04-079 antibody is also shown in SEQ ID NO: 39 in the Sequence Listing.
- the sequence of SEQ ID NO: 38 is described in FIG.
- the heavy chain amino acid sequence of the 04-126 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 40 in the Sequence Listing.
- the nucleotide sequence consisting of nucleotides 58 to 426 of the nucleotide sequence shown in SEQ ID NO: 40 of the Sequence Listing encodes the heavy chain variable region of antibody 04-126, and the nucleotide sequence consisting of nucleotides 427 to 1425 Encodes the heavy chain constant region of the 04-126 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 133 to 162 encoding CDRH1 in SEQ ID NO: 40, and the nucleotide sequence described in nucleotide numbers 205 to 234 encoding CDRH2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 352 to 393 encoding CDRH3.
- the nucleotide sequence of the heavy chain variable region of the 04-126 antibody is also shown in SEQ ID NO: 41 in the Sequence Listing.
- the sequence of SEQ ID NO: 40 is shown in FIG.
- the light chain amino acid sequence of the 04-126 antibody is encoded by the nucleotide sequence shown in SEQ ID NO: 42 in the sequence listing.
- the nucleotide sequence consisting of nucleotides 61 to 384 of the nucleotide sequence shown in SEQ ID NO: 42 of the sequence listing encodes the light chain variable region of antibody 04-126, and the nucleotide sequence consisting of nucleotides 385 to 699 Encodes the light chain constant region of the 04-126 antibody.
- the nucleotide sequence encoding the variable region is the polynucleotide consisting of the nucleotide sequence described in nucleotide numbers 127 to 159 encoding CDRL1 in SEQ ID NO: 42, and the nucleotide sequence described in nucleotide numbers 205 to 225 encoding CDRL2.
- the polynucleotide has a polynucleotide consisting of the nucleotide sequence of nucleotide numbers 322 to 348 encoding CDRL3.
- the nucleotide sequence of the light chain variable region of the 04-1216 antibody is also shown in SEQ ID NO: 43 of the Sequence Listing. The sequence of SEQ ID NO: 42 is described in FIG.
- Example 4 Analysis of GPR20 binding site of anti-GPR20 monoclonal antibody
- the binding site of the anti-human GPR20 antibody was classified by measuring the binding ability to the human / mouse chimeric GPR20 substituted with the above sequence by Cell-ELISA method.
- FIG. 21 is a control diagram of amino acid sequences of human GPR20 (NP_005284) and mouse GPR20 (NP_775541).
- Four extracellular regions EC1 to EC4 are represented by the amino acid sequence numbers of human GPR20, and EC1 is 1 to 48, EC2 indicates 108-125, EC3 indicates 190-196, and EC4 indicates 260-275.
- cDNA is artificially synthesized based on the Ref Seq sequence NM_173365 of mouse GPR20 and cloned into a pcDNA-DEST40 expression vector (Invitrogen). An expression vector pcDNA-mGPR20 was constructed.
- coli TOP10 (Invitrogen), a human GPR20 with a FLAG tag added to the N-terminus and a human GPR20 expression vector in which EC2 or EC3 or EC4 is replaced with a mouse GPR20-derived sequence are constructed. did.
- the primer sets used for each PCR are as follows.
- PCR primer set (for human GPR20 with FLAG tag at the N-terminus) 5′-GACTACAAAGACGATGGAGACAAGCCCTCTGTGTCTCCAGC-3 ′ (NFLAG-1; SEQ ID NO: 72) 5'-CTTGTCGTCATCGTCTTTGTAGTCCATGGTGGAGCCTGC-3 '(NFLAG-2; SEQ ID NO: 73) PCR primer set (for human GPR20 with EC2 replaced with mouse GPR20-derived sequence) 5′-ACCGCGCTTCGCTGTGTTCTACGGCGCCAG-3 ′ (mEC2-1; SEQ ID NO: 74) 5′-CTGGCGCCGTAGAACACAGCGAAGCGGT-3 ′ (mEC2-2; SEQ ID NO: 75) PCR primer set (for human GPR20 with EC3 replaced with mouse GPR20-derived sequence) 5′-TGTCCGGTGCTGGGCGGAGATCCGGGTGGACGATCATGCTGCCGTGTTCTT-3 ′ (mEC3-1; SEQ ID NO
- PCR primer set (for human GPR20 with EC1 replaced by mouse GPR20-derived sequence) 5′-GCCGCTGATGGGCGTGCACGGAGCCATCT-3 ′ (mEC1-1; SEQ ID NO: 80) 5′-AGAGGGCATGGTGGAGCTCTGCTTT-3 ′ (mEC1-2; SEQ ID NO: 81)
- a PCR reaction was similarly carried out using the following primer set for 20 cycles to amplify a DNA fragment encoding the EC1 region of mouse GPR20. .
- PCR primer set (for human GPR20 with EC1 replaced with mouse GPR20-derived sequence) 5′-AGGCTCCCACCATGCCCCTGCCGTTGTCTATGA-3 ′ (mEC1-3; SEQ ID NO: 82) 5′-CACCGCCATCAGCCGCTTGCCACAGGCTGGGGAAGGTGGCTTGCA-3 ′ (mEC1-4; SEQ ID NO: 83)
- the two kinds of DNA fragments were subjected to agarose gel electrophoresis according to a conventional method, and DNA fragments of the desired size were isolated using QIAquick Gel Extraction Kit (Promega). These two DNA fragments were ligated by the reaction of In-Fusion HD enzyme (Clontech) and transformed into E. coli TOP10 (Invitrogen) to construct a human GPR20 expression vector in which EC1 was replaced with a mouse GPR20-derived sequence. .
- human GPR20 is a cell expressing human full-length GPR20
- FLAG-huGPR20 is a cell expressing human GPR20 with a FLAG tag added to the N-terminus
- mouse GPR20 is a cell expressing mouse GPR20
- hGPR20_mECD1 is a cell in which chimeric GPR20 is expressed by replacing ECD1 of human GPR20 with ECD1 in mouse GPR20
- hGPR20_mECD2 is a cell in which chimeric GPR20 is expressed by replacing ECD2 in human GPR20 with ECD2 in mouse GPR20
- hGPR20_mECD3 is in human GPR20 Cells expressing chimeric GPR20 in which ECD3 is replaced with ECD3 of mouse GPR20
- hGPR20_mECD4 is EPR4 of human GPR20 and GPR of mouse 0 shows a cell chimeras GPR20 was expressed was replaced with ECD
- Group A 04-046, 04-079 and 04-126 do not bind to mouse GPR20 and human GPR20 EC1 (ECD1 When the EC2 (also referred to as ECD2) or GPR20 derived from mouse was substituted, the binding was lost. This indicates that 04-046, 04-079 and 04-126 recognize the higher order structure of EC1 and EC2 of GPR20.
- Group B 04-021 showed weak binding to mouse GPR20, and when EC1 of human GPR20 was replaced with mouse-derived GPR20, the binding was attenuated to the same extent as binding to mouse GPR20. Moreover, since it did not show the binding property to human GPR20 with a FLAG tag added to the N-terminus, it was shown that the vicinity of the N-terminus of EC1 of GPR20 was recognized.
- Group C 13-024 and 13-048 do not bind to mouse GPR20, and the binding of EC1 of human GPR20 to mouse-derived GPR20 significantly decreased, indicating that it binds to EC1. It was done.
- Table 1 shows the correspondence relationship between the extracellular region of GPR20 recognized by each antibody from the above experiments and the internalization activity of the antibody shown in FIG. 20 (the lower the cell viability, the stronger the internalization activity).
- EC1 corresponds to 1-48
- EC2 corresponds to 108-125
- EC3 corresponds to 190-196
- EC4 corresponds to 260-275.
- the cell survival rate is considered to have a high internalization activity of less than 70%. It was revealed that all of these antibodies were from Group A. That is, it was shown that antibodies showing high internalization activity are concentrated on the antibodies of group A that recognize the higher order structure consisting of EC1 and EC2 of human GPR20.
- Example 5 Preparation of human chimerized anti-GPR20 antibody 04-046Ch 5) -1 Construction of expression vector of human chimerized anti-GPR20 antibody 5) Construction of 1-1 chimera and humanized antibody light chain expression vector pCMA-LK Plasmid pcDNA3 .3-TOPO / LacZ (Invitrogen) digested with restriction enzymes XbaI and PmeI, a fragment of about 5.4 kb, a DNA encoding the human ⁇ chain secretion signal and the human ⁇ chain constant region shown in SEQ ID NO: 84 The DNA fragment containing the sequence was ligated using In-Fusion Advantage PCR cloning kit (Clontech) to prepare pcDNA3.3 / LK.
- In-Fusion Advantage PCR cloning kit (Clontech)
- PCR was carried out with the following primer set, and the obtained 3.8 kb fragment was phosphorylated and self-ligated to downstream of the CMV promoter to establish a signal sequence, cloning site, and human kappa chain.
- Chimeric and humanized antibody light chain expression vectors pCMA-LK with normal regions were constructed.
- Primer set 5'-TATACCGTCGCACCTCTAGCTAGAGCTTGGC-3 '(3.3-F1; SEQ ID NO: 85) 5′-GCTATGGCAGGGCCTGCCGCCCCCGACGTTG-3 ′ (3.3-R1; SEQ ID NO: 86)
- chimeric and humanized antibody IgG1-type heavy chain expression vector pCMA-G1 A DNA fragment obtained by digesting pCMA-LK with XbaI and PmeI to remove the ⁇ chain secretion signal and the human ⁇ chain constant region; A DNA fragment comprising a human heavy chain signal sequence shown in SEQ ID NO: 87 and a DNA sequence encoding the amino acid of the human IgG1 constant region was ligated using an In-Fusion Advantage PCR cloning kit (Clontech), and downstream of the CMV promoter. A chimeric and humanized antibody IgG1-type heavy chain expression vector pCMA-G1 having a signal sequence, cloning site, human IgG1 heavy chain constant region was constructed.
- a DNA fragment containing the DNA sequence encoding the variable region of the heavy chain of the human chimerized anti-GPR20 antibody is amplified with KOD-Plus- (TOYOBO) and the following primer set, and chimeric and humanized Heavy chain expression vector of human chimerized anti-GPR20 antibody 04-046Ch by inserting the antibody IgG1-type heavy chain expression vector pCMA-G1 into the site cut with restriction enzyme BlpI using In-Fusion HD PCR cloning kit (Clontech) Built.
- the obtained expression vector was designated as "pCMA / 04-046Ch-H".
- amino acid sequence of the heavy chain of human chimerized anti-GPR20 antibody 04-046Ch is shown in SEQ ID NO: 44.
- the nucleotide sequence of SEQ ID NO: 46 and the amino acid sequence of SEQ ID NO: 44 are also shown in FIG. Primer set 5′-AGCTCCCAGATGGGTGCTGAGC-3 ′ (EG-Inf-F; SEQ ID NO: 88) 5′-GGGGCCCTTGGGGAGGCTGAGC-3 ′ (EG1-Inf-R; SEQ ID NO: 89)
- a DNA fragment containing the DNA sequence encoding the variable region of the light chain of the human chimerized anti-GPR20 antibody is amplified with KOD-Plus- (TOYOBO) and the following primer set, and chimeric and humanized Construction of human chimerized anti-GPR20 antibody 04-046Ch light chain expression vector by inserting the antibody light chain expression vector pCMA-LK into the site cleaved with the restriction enzyme BsiWI using In-Fusion HD PCR cloning kit (Clontech) did.
- the obtained expression vector was designated as “pCMA / 04-046Ch-L”.
- amino acid sequence of the light chain of human chimerized anti-GPR20 antibody 04-046Ch is shown in SEQ ID NO: 45.
- the nucleotide sequence of SEQ ID NO: 47 and the amino acid sequence of SEQ ID NO: 45 are also shown in FIG.
- Heavy chain expression vector (0.24 mg) and light chain expression prepared by dissolving 1.8 mg of Polyethyleneimine (Polyscience # 24765) in 20 mL of Opti-Pro SFM medium (Invitrogen) and then using NucleoBond Xtra (TaKaRa) Vector (0.36 mg) was added to 20 mL Opti-Pro SFM medium (Invitrogen). 20 mL of the expression vector / Opti-Pro SFM mixture was added to 20 mL of the polyethylenemine / Opti-Pro SFM mixture, gently stirred, allowed to stand for another 5 minutes, and then added to the FreeStyle 293F cells.
- the human chimerized anti-GPR20 antibody obtained by a combination of pCMA / 04-046Ch-H and pCMA / 04-046Ch-L was named “04-046Ch”.
- Example 5 pcDNA3.1-hGPR20 or pcDNA3.1 was transiently introduced into 293T cells using Lipofectamine 2000 in the same manner as in Example 5-5-1, under conditions of 37 ° C. and 5% CO 2 .
- the cell suspension was prepared after overnight culture at Human chimerized anti-GPR20 antibody 04-046Ch or human IgG as a negative control was added to these cell suspensions, and the mixture was allowed to stand at 4 ° C.
- PE-labeled F (ab ′) 2 Fragment anti-human IgG, Fc ⁇ antibody JACKSON IMMUNORESEARC
- FC500 Beckman Coulter
- Example 6 Production of humanized anti-GPR20 antibody 6) -1 Humanized body design of anti-GPR20 antibody 04-046 6) -1-1 Molecular modeling of variable region of 04-046 Molecular modeling of variable region of 04-046 was performed by a method known as homology modeling (Methods in Enzymology, 203, 121-153, (1991)). Primary sequence of variable region of human immunoglobulin registered in Protein Data Bank (Nuc. Acid Res. 35, D301-D303 (2007)) (three-dimensional structure derived from X-ray crystal structure is available) ) was compared with the variable region of 04-046 determined in Example 3) -2.
- 1SY6 and 1LK3 were selected as the sequences with the highest sequence identity to the heavy chain and light chain variable regions of 04-046.
- the three-dimensional structure of the framework region was created by combining the coordinates of 1SY6 and 1LK3 corresponding to the heavy chain and light chain of 04-046 to obtain a “framework model”.
- a representative conformation for each CDR was then incorporated into the framework model.
- an energy calculation was performed to eliminate adverse interatomic contacts. The above procedure was performed using a commercially available protein tertiary structure analysis program Discovery Studio (Accelrys).
- the human gamma chain subgroup 1 consensus sequence for the heavy chain and the human kappa chain subgroup 1 consensus sequence for the light chain are selected as acceptors based on their high sequence identity in the framework region. It was. The amino acid residues in the framework region for the acceptor were aligned with the amino acid residues for 04-046 to identify the position where a different amino acid was used. The positions of these residues were analyzed using the 04-046 three-dimensional model constructed above, and the donor residues to be grafted on the acceptor are described by Queen et al. (Proc. Natl. Acad. Sci. USA 86, 1000029-10033 (1989)). The sequence of humanized h046 was constructed as described in the examples below by transferring several selected donor residues into the acceptor antibody.
- the amino acid sequence of the humanized h046-H4b type heavy chain is set forth in SEQ ID NO: 48 in the sequence listing.
- the sequence consisting of the 1st to 19th amino acid residues of the amino acid sequence shown in SEQ ID NO: 48 represents a signal sequence
- the sequence consisting of the 20th to 142nd amino acid residues represents a heavy chain variable region
- the 143th to 472nd sequences A sequence consisting of amino acid residues represents a heavy chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 48 is set forth in SEQ ID NO: 49 of the Sequence Listing.
- sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 49 represents a signal sequence
- sequence consisting of nucleotides 58 to 426 represents the heavy chain variable region sequence
- sequence consisting of nucleotides 427 to 1416 Indicates the sequence of the heavy chain constant region.
- amino acid sequence of SEQ ID NO: 48 is also shown in FIG.
- humanized h046-H4e type heavy chain (sometimes referred to as “h046-H4e”).
- the amino acid sequence of the humanized h046-H4e type heavy chain is set forth in SEQ ID NO: 50 of the Sequence Listing.
- the sequence consisting of amino acid residues 1 to 19 of the amino acid sequence of SEQ ID NO: 50 is a signal sequence
- the sequence consisting of amino acid residues 20 to 142 is a heavy chain variable region
- the sequence consisting of amino acid residues 143 to 472 Indicates the heavy chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 50 is set forth in SEQ ID NO: 51 of the Sequence Listing.
- sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 51 is a signal sequence
- sequence consisting of nucleotides 58 to 426 is the heavy chain variable region sequence
- sequence consisting of nucleotides 427 to 1416 is the heavy chain constant. Codes an array of regions.
- amino acid sequence of SEQ ID NO: 50 is also shown in FIG.
- the sequence consisting of amino acid residues 1 to 19 in the amino acid sequence of SEQ ID NO: 52 is a signal sequence, the sequence consisting of amino acid residues 20 to 142 is a heavy chain variable region, and the sequence consisting of amino acid residues 143 to 472 Indicates the heavy chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 52 is described in SEQ ID NO: 53 of the Sequence Listing.
- sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 53 is a signal sequence
- sequence consisting of nucleotides 58 to 426 is a heavy chain variable region sequence
- sequence consisting of nucleotides 427 to 1416 is a heavy chain constant region It encodes an array.
- amino acid sequence of SEQ ID NO: 52 is also described in FIG.
- sequence consisting of the 1st to 19th amino acid residues, the sequence consisting of the 20th to 142nd amino acid residues, and the sequence consisting of the 143th to 472th amino acid residues of the amino acid sequence of SEQ ID NO: 54 are respectively a signal sequence and a heavy chain It corresponds to the variable region and heavy chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 54 is set forth in SEQ ID NO: 55 of the Sequence Listing.
- sequence consisting of nucleotides 1 to 57, the sequence consisting of nucleotides 58 to 426, and the sequence consisting of nucleotides 427 to 1416 of the nucleotide sequence of SEQ ID NO: 55 are a signal sequence, a heavy chain variable region sequence, and a heavy chain constant, respectively.
- the normal region sequence is encoded.
- the amino acid sequence of SEQ ID NO: 54 is also shown in FIG.
- the amino acid sequence of the humanized h046-H10 type heavy chain is described in SEQ ID NO: 56 in the sequence listing.
- the sequence consisting of amino acid residues 1 to 19 of the amino acid sequence of SEQ ID NO: 56 is a signal sequence
- the sequence consisting of amino acid residues 20 to 142 is a heavy chain variable region
- the sequence consisting of amino acid residues 143 to 472 The heavy chain constant region is indicated.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 56 is set forth in SEQ ID NO: 57 of the Sequence Listing.
- sequence consisting of nucleotides 1 to 57 of the nucleotide sequence of SEQ ID NO: 57 is a signal sequence
- sequence consisting of nucleotides 58 to 426 is the heavy chain variable region sequence
- sequence consisting of nucleotides 427 to 1416 is the heavy chain constant region It encodes an array.
- amino acid sequence of SEQ ID NO: 56 is also shown in FIG.
- leucine to valine 36th glutamine to aspartic acid, 41st serine to threonine, 58th arginine to lysine, 59th serine to proline, 61st glutamine to lysine, 62st Of glutamine to alanine, 95th aspartic acid to serine, 96th proline to serine, 97th valine to leucine, 98th Glutamic acid to glutamine, No. 100) aspartic acid to glutamic acid, No. 102 isoleucine to phenylalanine, No. 104 asparagine to threonine, No. 119 alanine to glutamine, No. 123 leucine to valine, No.
- a humanized h046 light chain designed by replacing leucine with isoleucine, alanine 128 with threonine, and inserting serine between 29 and 30 is referred to as “humanized h046-L1 type light chain” (“ h046-L1) ”).
- the amino acid sequence of the humanized h046-L1 type light chain is set forth in SEQ ID NO: 58 in the Sequence Listing.
- the sequence consisting of amino acid residues 1 to 20 in the amino acid sequence of SEQ ID NO: 58 is a signal sequence
- the sequence consisting of amino acid residues 21 to 129 is the light chain variable region
- the sequence consisting of amino acid residues 130 to 234 Indicates the light chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 58 is set forth in SEQ ID NO: 59 of the Sequence Listing.
- the sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 58 is a signal sequence
- the sequence consisting of nucleotides 61 to 387 is the light chain variable region sequence
- the sequence consisting of nucleotides 388 to 702 is the light chain constant region It encodes an array.
- the amino acid sequence of SEQ ID NO: 58 is also shown in FIG.
- h046-L2 type light chain (h046-L2)
- the amino acid sequence of the humanized h046-L2 type light chain is set forth in SEQ ID NO: 60 in the sequence listing.
- the sequence consisting of amino acid residues 1 to 20 of the amino acid sequence of SEQ ID NO: 60 is a signal sequence, the sequence consisting of amino acid residues 21 to 129 is the light chain variable region, and the sequence consisting of amino acid residues 130 to 234 Corresponds to the light chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 60 is set forth in SEQ ID NO: 61 in the sequence listing.
- sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 61 is a signal sequence
- sequence consisting of nucleotides 61 to 387 is the light chain variable region sequence
- sequence consisting of nucleotides 388 to 702 is the light chain constant region It encodes an array.
- the amino acid sequence of SEQ ID NO: 60 is also shown in FIG.
- the amino acid sequence of the humanized h046-L6 type light chain is set forth in SEQ ID NO: 62 in the sequence listing.
- the sequence consisting of the 1st to 20th amino acid residues of the amino acid sequence of SEQ ID NO: 62 is a signal sequence
- the sequence consisting of the 21st to 129th amino acid residues is the light chain variable region
- the sequence consisting of the 130th to 234th amino acid residues Indicates the light chain constant region.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 62 is described in SEQ ID NO: 63 of the sequence listing.
- the sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 63 is a signal sequence
- the sequence consisting of nucleotides 61 to 387 is the light chain variable region sequence
- the sequence consisting of nucleotides 388 to 702 is the light chain constant region It encodes an array.
- the amino acid sequence (SASDRES) of CDRL2 is described in SEQ ID NO: 92 in the sequence listing.
- the amino acid sequence of SEQ ID NO: 62 is also shown in FIG. 6) -3-4 Humanized h046-L7 type light chain Of the light chain of human chimeric antibody 04-046Ch shown in SEQ ID NO: 45, variable region 23rd valine is glutamine and 29th alanine is serine.
- the humanized h046 light chain was named “humanized h046-L7 type light chain” (sometimes referred to as “h046-L7”).
- the amino acid sequence of the humanized h046-L7 type light chain is set forth in SEQ ID NO: 64 in the sequence listing.
- the sequence consisting of amino acid residues 1 to 20 in the amino acid sequence of SEQ ID NO: 64 is a signal sequence
- the sequence consisting of amino acid residues 21 to 129 is the light chain variable region
- the sequence consisting of amino acid residues 130 to 234 The light chain constant region is indicated.
- the nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 64 is set forth in SEQ ID NO: 65 of the Sequence Listing.
- sequence consisting of nucleotides 1 to 60 of the nucleotide sequence of SEQ ID NO: 65 is a signal sequence
- sequence consisting of nucleotides 61 to 387 is the light chain variable region sequence
- sequence consisting of nucleotides 388 to 702 is the light chain constant region It encodes an array.
- the amino acid sequence of CDRL2 (SAGNLES) is described in SEQ ID NO: 93 in the sequence listing.
- the amino acid sequence of SEQ ID NO: 64 is also shown in FIG.
- An antibody consisting of a humanized h046-H4e type heavy chain and a humanized h046-L6 type light chain was designed and named “humanized h046-H4e / L6” (sometimes referred to as “h046-H4e / L6”).
- An antibody consisting of a humanized h046-H4e type heavy chain and a humanized h046-L7 type light chain was designed and named “humanized h046-H4e / L7” (sometimes referred to as “h046-H4e / L7”).
- An antibody consisting of a humanized h046-H4b type heavy chain and a humanized h046-L1 type light chain was designed and named “humanized h046-H4b / L1” (sometimes referred to as “h046-H4b / L1”).
- An antibody consisting of a humanized h046-H4b type heavy chain and a humanized h046-L2 type light chain was designed and named “humanized h046-H4b / L2” (sometimes referred to as “h046-H4b / L2”).
- An antibody consisting of a humanized h046-H4b type heavy chain and a humanized h046-L6 type light chain was designed and named “humanized h046-H4b / L6” (sometimes referred to as “h046-H4b / L6”).
- An antibody consisting of a humanized h046-H4b type heavy chain and a humanized h046-L7 type light chain was designed and named “humanized h046-H4b / L7” (sometimes referred to as “h046-H4b / L7”).
- An antibody consisting of a humanized h046-H5b type heavy chain and a humanized h046-L1 type light chain was designed and named “humanized h046-H5b / L1” (sometimes referred to as “h046-H5b / L1”).
- An antibody consisting of a humanized h046-H5b type heavy chain and a humanized h046-L2 type light chain was designed and named “humanized h046-H5b / L2” (sometimes referred to as “h046-H5b / L2”).
- An antibody consisting of a humanized h046-H5b type heavy chain and a humanized h046-L6 type light chain was designed and named “humanized h046-H5b / L6” (sometimes referred to as “h046-H5b / L6”).
- An antibody consisting of a humanized h046-H5b type heavy chain and a humanized h046-L7 type light chain was designed and named “humanized h046-H5b / L7” (sometimes referred to as “h046-H5b / L7”).
- An antibody consisting of a humanized h046-H8 type heavy chain and a humanized h046-L1 type light chain was designed and named “humanized h046-H8 / L1” (sometimes referred to as “h046-H8 / L1”).
- An antibody consisting of a humanized h046-H8 type heavy chain and a humanized h046-L2 type light chain was designed and named “humanized h046-H8 / L2” (sometimes referred to as “h046-H8 / L2”).
- An antibody consisting of a humanized h046-H8 type heavy chain and a humanized h046-L6 type light chain was designed and named “humanized h046-H8 / L6” (sometimes referred to as “h046-H8 / L6”).
- An antibody consisting of a humanized h046-H8 type heavy chain and a humanized h046-L7 type light chain was designed and named “humanized h046-H8 / L7” (sometimes referred to as “h046-H8 / L7”).
- An antibody consisting of a humanized h046-H10 type heavy chain and a humanized h046-L1 type light chain was designed and named “humanized h046-H10 / L1” (sometimes referred to as “h046-H10 / L1”).
- An antibody consisting of a humanized h046-H10 type heavy chain and a humanized h046-L2 type light chain was designed and named “humanized h046-H10 / L2” (sometimes referred to as “h046-H10 / L2”).
- An antibody consisting of a humanized h046-H10 type heavy chain and a humanized h046-L6 type light chain was designed and named “humanized h046-H10 / L6” (sometimes referred to as “h046-H10 / L6”).
- An antibody consisting of a humanized h046-H10 type heavy chain and a humanized h046-L7 type light chain was designed and named “humanized h046-H10 / L7” (sometimes referred to as “h046-H10 / L7”).
- An antibody consisting of a human chimeric c046 heavy chain and a humanized h046-L1 type light chain was designed and named “human chimeric c046 / L1” (sometimes referred to as “h046-Hwt / L1”).
- An antibody consisting of a human chimeric c046 heavy chain and a humanized h046-L2 type light chain was designed and named “human chimeric c046 / L2” (sometimes referred to as “h046-Hwt / L2”).
- An antibody consisting of a human chimeric c046 heavy chain and a humanized h046-L6 type light chain was designed and named “human chimeric c046 / L6” (sometimes referred to as “h046-Hwt / L6”).
- An antibody consisting of a human chimeric c046 heavy chain and a humanized h046-L7 type light chain was designed and named “human chimeric c046 / L7” (sometimes referred to as “h046-Hwt / L7”).
- the antibody designed as described above can be prepared according to Example 6) -5 and Example 6) -7, and can be evaluated according to Example 6) -6, Example 8) and Example 9). .
- a humanized h046-H4b type heavy chain expression vector was constructed by introducing mutations using the following primer set and KOD-Plus- Mutagenesis Kit (TOYOBO).
- the constructed expression vector was designated as “pCMA / h046-H4b”.
- the nucleotide sequence of the humanized h046-H4b type heavy chain is shown in SEQ ID NO: 49, and the amino acid sequence is shown in SEQ ID NO: 48.
- a humanized h046-H5b type heavy chain expression vector was constructed by introducing a mutation in the same manner as 6) -5-1-1 using plasmid B as a template.
- the constructed expression vector was designated as “pCMA / h046-H5b”.
- the nucleotide sequence of the humanized h046-H5b type heavy chain is shown in SEQ ID NO: 53, and the amino acid sequence is shown in SEQ ID NO: 52.
- the nucleotide sequence of the humanized h046-H10 type heavy chain is shown in SEQ ID NO: 57, and the amino acid sequence is shown in SEQ ID NO: 56.
- 5′-GTGAGCTGCAAGGCCAGCGGCTACACCCTTCACC-3 ′ H10-F; SEQ ID NO: 100
- a DNA fragment containing a DNA sequence encoding the variable region of humanized h046-L1 was amplified with KOD-Plus- (TOYOBO) and the following primer set, and Example 5) -1-
- the humanized h046-L1 expression vector by inserting the chimeric and humanized antibody light chain expression vector pCMA-LK constructed in 1 into the site cleaved with the restriction enzyme BsiWI using the In-Fusion HD PCR cloning kit (Clontech). Built.
- the obtained expression vector was designated as “pCMA / h046-L1”.
- the amino acid sequence of humanized h046-L1 is shown in SEQ ID NO: 58.
- Primer set 5'-CTGTGGATCTCCGGCCGCGTACGCC-3 '(CM-LKF; SEQ ID NO: 90) 5′-GGAGGGGGCGCCACCCGTACG-3 ′ (KCL-Inf-R; SEQ ID NO: 102)
- Example 6 A humanized h046-L7 expression vector was constructed in the same manner as in 5-2-1. The obtained expression vector was designated as “pCMA / h046-L7”. The amino acid sequence of humanized h046-L7 is shown in SEQ ID NO: 64.
- Humanized h046-H4b / L7 was obtained by a combination of pCMA / h046-H4b and pCMA / h046-L7.
- Humanized h046-H5b / L2 was obtained by a combination of pCMA / h046-H5b and pCMA / h046-L2.
- Humanized h046-Hwt / L6 was obtained by a combination of pCMA / 04-046Ch-H and pCMA / h046-L6.
- pCMA / h046-H8 and pCMA / h046-L1 By combining pCMA / h046-H8 and pCMA / h046-L1 to obtain humanized h046-H8 / L1, pCMA / h046-H10 and pCMA / h046-L1 can be combined with humanized h046-H10 / L1. I got it. Humanized h046-H10 / L6 was obtained by a combination of pCMA / h046-H10 and pCMA / h046-L6.
- Example 6 Flow of GPR20 binding of humanized anti-GPR20 antibody prepared in 5-3 Evaluation was made by cytometry.
- Example 1) After pcDNA3.1-hGPR20 was transiently introduced into 293T cells using Lipofectamine 2000 in the same manner as in Example 5-5-1 and cultured overnight at 37 ° C. and 5% CO 2. A cell suspension was prepared. A humanized anti-GPR20 antibody was added to these cell suspensions and allowed to stand at 4 ° C. for 1 hour, then washed twice with 5% FBS-containing PBS and diluted 320-fold with 5% FBS-containing PBS.
- the horizontal axis represents the fluorescence intensity of PE representing the amount of antibody binding
- the vertical axis represents the number of cells.
- the shaded area is a negative control in which no anti-GPR20 antibody was reacted
- the white outline is that when each anti-GPR20 antibody was reacted, the fluorescence intensity was enhanced by binding of the antibody to GPR20 on the cell surface. Show.
- Example 6 The internalization activity of the humanized anti-GPR20 antibody prepared in 5-3 is the same as in 1) -6. Evaluation was performed using an anti-human IgG antibody reagent Hum-ZAP (ADVANCED TARGETING SYSTEMS) to which a toxin (saporin) to inhibit was bound. That is, HEK293 cells stably expressing GPR20-EGFP protein with EGFP linked to the C-terminal side of human GPR20 were seeded at 2.5 ⁇ 10 3 cells / well and cultured overnight at 37 ° C. and 5% CO 2.
- ADVANCED TARGETING SYSTEMS anti-human IgG antibody reagent Hum-ZAP
- humanized anti-GPR20 antibody final concentration: 0.015 to 1.27 ⁇ g / mL
- Hum-ZAP final concentration: 1 ⁇ g / mL
- h046_H4b type heavy chain expression vector was constructed according to the protocol of GS Gene Expression System (registered trademark) of Lonza.
- the constructed expression vector was designated as “GSV-h046_H4b”.
- humanized h046-L6 type light chain expression vector It is represented by nucleotide numbers 61 to 702 of the nucleotide sequence (2) of the humanized h046_L6 type light chain represented by SEQ ID NO: 110 in the Sequence Listing.
- a DNA fragment containing a sequence encoding a humanized h046_L6 type light chain was synthesized (GENEART artificial gene synthesis service).
- a humanized h046_L6 type light chain expression vector was constructed from the synthesized DNA fragment according to the protocol of GS Gene Expression System (registered trademark) of Lonza. The constructed expression vector was designated as “GSV-h046_L6”.
- Humanized h046_L7 represented by nucleotide numbers 61 to 702 of the nucleotide sequence of humanized h046_L7 type light chain represented by SEQ ID NO: 111 in the Sequence Listing
- a DNA fragment containing a sequence encoding a type light chain was synthesized (Geneart Artificial Gene Synthesis Service).
- a humanized h046_L7 type light chain expression vector was constructed from the synthesized DNA fragment according to the protocol of GS Gene Expression System (registered trademark) of Lonza. The constructed expression vector was named “GSV-h046_L7”.
- Example 6 The humanized h046-H4b / L7 expression vector constructed in 7-3-1, DGV-h046_H4bL7-GS, was transfected into CHOK1SV cells (Lonza), and the humanized h046-H4b / L7 production strain Built. The resulting production strain was named “GPR1-12”.
- Example 6 -7-4-2 Production of humanized h046-H5b / L2 producing cells
- the resulting production strain was named “GPR2-15”.
- the obtained culture broth was diluted with C36 medium so as to have a cell density of 30 ⁇ 10 4 cells / mL and charged into WAVE CELLBAG (GE Healthcare Bioscience), 37 ° C., 5% CO 2 , air supply amount 0.3 L / Min, rocking 18-24 rpm, angle 6-8 ° for 10 days.
- FM4Ae2 medium self-prepared was added daily for 6% of the charged amount per day.
- the obtained culture broth was roughly filtered with a depth filter Millistak MC0HC054H1 (Merck Millipore), and then filtered with a 0.22 ⁇ m filter (Sartorius) attached to Flexboy Bags. This filtrate was designated as “h046-H4b / L7 culture supernatant”.
- Example 6 -7-5-2 Humanized h046-H5b / L2 producing cell culture Humanized h046- produced in Example 6) -7-4-2 as in Example 6) -7-5-1
- the H5b / L2 production strain “GPR2-15” was cultured and expanded, and fed batch culture was performed using a culture apparatus Wave reactor (GE Healthcare Japan). The cells were diluted with C36 medium to a cell density of 30 ⁇ 10 4 cells / mL, charged into WAVE CELLBAG (GE Healthcare Bioscience), and cultured for 11 days. The obtained culture broth was filtered, and this filtrate was designated as “h046-H5b / L2 culture supernatant”.
- Example 6 Similar to Example 7-7-5-1, humanized h046- produced in Example 6) -7-4-3
- the Hwt / L6 production strain “GPR3-2” was cultured and expanded, and fed batch culture was performed using a culture apparatus Wave reactor (GE Healthcare Japan). The cells were diluted with C36 medium to a cell density of 30 ⁇ 10 4 cells / mL, charged into WAVE CELLBAG (GE Healthcare Bioscience), and cultured for 14 days. The obtained culture broth was filtered, and this filtrate was named “h046-Hwt / L6 culture supernatant”.
- Example 6 Similar to Example 7-7-5-1, humanized h046- produced in Example 6) -7-4-4 The H8 / L1 production strain “GPR4-1” was cultured and expanded, and fed-batch culture was performed using a culture apparatus Wave reactor (GE Healthcare Japan). The cells were diluted with C36 medium to a cell density of 30 ⁇ 10 4 cells / mL, charged into WAVE CELLBAG (GE Healthcare Bioscience), and cultured for 11 days. The obtained culture broth was filtered, and this filtrate was designated as “h046-H8 / L1 culture supernatant”.
- Example 6 -7-5-5 Culture of humanized h046-H10 / L1 producing cells
- Example 6 Similar to Example 7-7-5-1, humanized h046- produced in Example 6) -7-4-5
- the H10 / L1 production strain “GPR5-10” was cultured and expanded, and fed-batch culture was performed using a culture apparatus Wave reactor (GE Healthcare Japan). It diluted with C36 culture medium so that it might become a cell density of 30 * 10 ⁇ 4 > cells / mL, and it prepared for WAVE CELLBAG (GE Healthcare Bioscience), and cultured for 10 days. The obtained culture broth was filtered, and this filtrate was named “h046-H10 / L1 culture supernatant”.
- Example 6 -7-5-6 Culture of humanized h046-H10 / L6 producing cells Example 6) Similar to Example 7-7-5-1, humanized h046- prepared in Example 6) -7-4-6
- the H10 / L6 production strain “GPR6-7” was cultured and expanded, and fed-batch culture was performed using a culture apparatus Wave reactor (GE Healthcare Japan).
- the cells were diluted with C36 medium to a cell density of 30 ⁇ 10 4 cells / mL, charged into WAVE CELLBAG (GE Healthcare Bioscience), and cultured for 12 days.
- the obtained culture broth was filtered, and this filtrate was designated as “h046-H10 / L6 culture supernatant”.
- Example 6 -7-5-7 Culture of humanized h046-H4e / L7-producing cells Humanized h046- produced in Example 6) -7-4-7 as in Example 6) -7-5-1
- the H4e / L7 production strain “GPE-23” was cultured and expanded, and fed-batch culture was performed using a culture apparatus Wave reactor (GE Healthcare Japan). The cells were diluted with C36 medium to a cell density of 30 ⁇ 10 4 cells / mL, charged into WAVE CELLBAG (GE Healthcare Bioscience), and cultured for 11 days. The obtained culture broth was filtered, and this filtrate was named “h046-H4e / L7 culture supernatant”.
- Example 6 “h046-H4b / L7 obtained in 7-5-1
- the “culture supernatant” was purified by a three-step process of rProtein A affinity chromatography, anion exchange chromatography, and cation exchange chromatography. First, the culture supernatant was applied to rProtein A affinity chromatography resin equilibrated with PBS. After all of the culture solution entered the column, the column was washed with PBS, a buffer solution containing arginine, and PBS.
- the rProtein A purified pool was then applied to an anion exchange chromatographic resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool. The AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer.
- the recovered solution was filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain a CEX purification pool.
- the CEX purified pool was concentrated to an antibody concentration of 20 mg / mL with Pellicon 3 Cassette 30 kDa (Merck Millipore), and then replaced with a histidine buffer (25 mM histidine, 5% Sorbitol, pH 6.0). Finally, a 0.22 ⁇ m filter that was filtered through Stericup-GV (Merck Millipore) was used as a purified sample.
- the purified sample was named “h046_H4b / L7”.
- the recovered solution was neutralized with Tris buffer, roughly filtered with a glass fiber filter AP20 (Merck Millipore), and then filtered with Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an rProtein A purification pool. .
- the rProtein A purified pool was applied to an anion exchange chromatography resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool.
- Stericup-GV Merck Millipore
- the AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer. Next, elution was carried out using an acetate buffer containing a high concentration of NaCl, and an absorption peak at 280 nm was collected. The recovered solution was filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain a CEX purification pool.
- Stericup-GV Merck Millipore
- the CEX purified pool was concentrated to an antibody concentration of 40 mg / mL with Pellicon 3 Cassette 30 kDa (Merck Millipore), and then replaced with histidine buffer (25 mM histidine, 5% Sorbitol, pH 6.0). Finally, a 0.22 ⁇ m filter that was filtered through Stericup-GV (Merck Millipore) was used as a purified sample.
- the purified sample was named “h046_H5b / L2”.
- the rProtein A purified pool was then applied to an anion exchange chromatographic resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool. The AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer.
- the recovered solution was filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain a CEX purification pool.
- the CEX purified pool was concentrated to an antibody concentration of 40 mg / mL with Pellicon 3 Cassette 30 kDa (Merck Millipore), and then replaced with histidine buffer (25 mM histidine, 5% Sorbitol, pH 6.0). Finally, a 0.22 ⁇ m filter that was filtered through Stericup-GV (Merck Millipore) was used as a purified sample.
- the purified sample was named “h046-Hwt / L6”.
- the rProtein A purified pool was then applied to an anion exchange chromatographic resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool. The AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer.
- the recovered solution was filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain a CEX purification pool.
- the CEX purified pool was concentrated to an antibody concentration of 40 mg / mL with Pellicon 3 Cassette 30 kDa (Merck Millipore), and then replaced with histidine buffer (25 mM histidine, 5% Sorbitol, pH 6.0). Finally, a 0.22 ⁇ m filter that was filtered through Stericup-GV (Merck Millipore) was used as a purified sample.
- the purified sample was named “h046-H8 / L1”.
- the recovered solution was neutralized with Tris buffer, roughly filtered with a glass fiber filter AP20 (Merck Millipore), and then filtered with Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an rProtein A purification pool. .
- the rProtein A purified pool was then applied to an anion exchange chromatographic resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool. The AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer.
- the rProtein A purified pool was then applied to an anion exchange chromatographic resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool. The AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer.
- the recovered solution was filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain a CEX purification pool.
- the CEX purified pool was concentrated to an antibody concentration of 40 mg / mL with Pellicon 3 Cassette 30 kDa (Merck Millipore), and then replaced with histidine buffer (25 mM histidine, 5% Sorbitol, pH 6.0). Finally, a 0.22 ⁇ m filter that was filtered through Stericup-GV (Merck Millipore) was used as a purified sample.
- the purified sample was named “h046-H10 / L6”.
- the rProtein A purified pool was then applied to an anion exchange chromatographic resin equilibrated with PBS. After all of the Apply solution entered the column, PBS was passed through. An absorption peak at 280 nm when the flow-through fraction and PBS were passed was collected. The pH of the recovered liquid was adjusted with acetic acid, and filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain an AEX purification pool. The AEX purification pool was then applied to a cation exchange chromatographic resin equilibrated with acetate buffer. After all of the apply solution entered the column, the column was washed with an acetate buffer.
- the recovered solution was filtered through Stericup-GV (Merck Millipore), which is a 0.22 ⁇ m filter, to obtain a CEX purification pool.
- the CEX purified pool was concentrated to an antibody concentration of 40 mg / mL with Pellicon 3 Cassette 30 kDa (Merck Millipore), and then replaced with histidine buffer (25 mM histidine, 5% Sorbitol, pH 6.0). Finally, a 0.22 ⁇ m filter that was filtered through Stericup-GV (Merck Millipore) was used as a purified sample.
- the purified sample was named “h046-H4e / L7”.
- Step 1 Antibody-drug conjugate (1)
- Antibody reduction Using 04-046Ch prepared in Example 5) -2, using the common procedure B (using 1.47 mLmg ⁇ 1 cm ⁇ 1 as the 280 nm extinction coefficient) and C described in Production Method 1, Prepared to 10 mg / mL with PBS 6.0 / EDTA.
- a 9.4 mM TCEP Tokyo Chemical Industry Co., Ltd.
- aqueous solution (0.104 mL; 5.0 equivalents for one antibody molecule
- a 1 M aqueous dipotassium hydrogen phosphate solution Nacalai Tesque, Inc.
- 0.170 mL was added. After confirming that the pH of this solution was within 7.4 ⁇ 0.1, the disulfide bond at the interchain part of the antibody was reduced by incubating at 37 ° C. for 1 hour.
- Step 1 Antibody-drug conjugate (2)
- Antibody reduction Using 04-046Ch prepared in Example 5) -2, using the common procedure B (using 1.47 mLmg ⁇ 1 cm ⁇ 1 as the 280 nm extinction coefficient) and C described in Production Method 1, Prepared to 10 mg / mL with PBS 6.0 / EDTA.
- a 9.4 mM TCEP Tokyo Chemical Industry Co., Ltd.
- aqueous solution (0.104 mL; 5.0 equivalents for one antibody molecule
- a 1 M aqueous dipotassium hydrogen phosphate solution Nacalai Tesque, Inc.
- 0.0509 mL was added. After confirming that the pH of this solution was within 7.0 ⁇ 0.1, the disulfide bond at the interchain portion of the antibody was reduced by incubating at 37 ° C. for 1 hour.
- Step 1 Antibody-drug conjugate (3)
- -7-6-1 was used as the common procedure B described in production method 1 (using 1.31 mLmg -1 cm -1 as the 280 nm extinction coefficient)
- C were adjusted to 10 mg / mL with PBS 6.0 / EDTA.
- a 10 mM TCEP Tokyo Chemical Industry Co., Ltd.
- aqueous solution 0.237 mL; 5.5 equivalents per molecule of antibody
- a 1 M dipotassium hydrogen phosphate aqueous solution Nacalai Tesque, Inc .; 0 0.0940 mL
- Step 1 Antibody-drug conjugate (4) Antibody reduction: h046-H4b / L7 prepared in Example 6) -7-6-1 was used as the common procedure B described in production method 1 (using 1.31 mLmg -1 cm -1 as the 280 nm extinction coefficient) And using C, it was adjusted to 10 mg / mL with PBS 6.0 / EDTA.
- This solution (300 mL) was put into a 1000 mL Erlenmeyer flask made of polycarbonate, 1 M aqueous solution of dipotassium hydrogenphosphate (4.80 mL) was added at room temperature with stirring with a magnetic stirrer, and then 10 mM TCEP aqueous solution (11.3 mL; antibody 1 5.5 equivalents to molecule). After confirming that the pH of this solution was within 7.0 ⁇ 0.1, the stirring was stopped, and the disulfide bond at the interchain portion of the antibody was reduced by incubating at 37 ° C. for 2 hours.
- Ultrafiltration membrane Merck, Pellicon XL, Cassette, Ultracell 30KDa
- tube pump US Cole Palmer Master Flex Pump model 77521-40, pump head model 7518-00
- Ultrafiltration purification was performed using an ultrafiltration apparatus composed of US Coal Palmer Master Flex Tube L / S16). That is, by performing ultrafiltration purification while adding ABS (a total of 3.00 L) as a purification buffer to the reaction solution, unbound drug linkers and other low molecular weight reagents are removed and the buffer solution is replaced with ABS. And further concentrated.
- the obtained purified solution was subjected to microfiltration (0.22 ⁇ m, PVDF membrane, twice) to obtain 83.0 mL of a solution containing the title antibody-drug conjugate “h046-H4b / L7-ADC1”. .
- Antibody concentration 23.1 mg / mL, antibody yield: 2.94 g (98%), average number of drugs bound per antibody molecule measured in common operation E (n): 5.8; Measured average drug binding number per antibody molecule (n): 7.4.
- Step 1 Antibody-drug conjugate (5)
- Example 6 A method similar to Step 1 of Example 7-3 using h046-H5b / L2 prepared in -7-6-2 (using 1.31 mLmg ⁇ 1 cm ⁇ 1 as the extinction coefficient of 280 nm) Gave the title antibody-drug conjugate “h046-H5b / L2-ADC1”.
- Antibody concentration 2.04 mg / mL, antibody yield: 61.3 mg (98%), average number of drugs per antibody molecule measured in common operation E (n): 5.4; in common operation F Measured average drug binding number per antibody molecule (n): 7.9.
- Step 1 Antibody-drug conjugate (6)
- Example 6 The same method as in Step 1 of Example 7-3 using h046-Hwt / L6 (using 1.31 mLmg ⁇ 1 cm ⁇ 1 as the 280 nm extinction coefficient) prepared in -7-6-3 Gave the title antibody-drug conjugate “h046-Hwt / L6-ADC1”.
- Antibody concentration 2.03 mg / mL, antibody yield: 60.9 mg (95%), drug average binding number per antibody molecule measured in common operation E (n): 5.6; in common operation F Average number of drugs bound per molecule of antibody measured (n): 7.8.
- Step 1 Antibody-drug conjugate (7)
- -7-6-1 was used as the common procedure B described in production method 1 (using 1.31 mLmg -1 cm -1 as the 280 nm extinction coefficient) And C were adjusted to 10 mg / mL with PBS 6.0 / EDTA.
- a 10 mM TCEP Tokyo Chemical Industry Co., Ltd.
- aqueous solution 0.193 mL; 5.5 equivalents per molecule of antibody
- a 1 M dipotassium hydrogen phosphate aqueous solution Nacalai Tesque, Inc .; 0 0.052 mL
- Step 1 Antibody-drug conjugate (8)
- Step 1 Antibody-drug conjugate (9)
- Example 6) Using h046-Hwt / L6 (using 1.31 mLmg ⁇ 1 cm ⁇ 1 as the 280 nm extinction coefficient) prepared in 7-6-3, Example 7) -8 Same as Step 1 By the method, the title antibody-drug conjugate “h046-Hwt / L6-ADC2” was obtained.
- Antibody concentration 2.10 mg / mL, antibody yield: 62.9 mg (99%), average number of drugs per antibody molecule measured in common operation E (n): 6.1; common operation F Measured average drug binding number per antibody molecule (n): 7.4.
- Step 1 Antibody-drug conjugate (10) Example 6) Using h046-H8 / L1 prepared in 7-6-4 (using 1.31 mLmg ⁇ 1 cm ⁇ 1 as the 280 nm extinction coefficient), the same as Example 7) -3 step 1 By the method, the title antibody-drug conjugate “h046-H8 / L1-ADC1” was obtained.
- Antibody concentration 1.75 mg / mL, antibody yield: 52.6 mg (86%), average number of drugs per antibody molecule measured in common operation E (n): 5.6; in common operation F Measured average drug binding number per antibody molecule (n): 7.9.
- Step 1 Antibody-drug conjugate (11)
- Example 6) Example 7) -3 Same as Step 1 using h046-H10 / L1 (using 1.31 mLmg ⁇ 1 cm ⁇ 1 as the extinction coefficient of 280 nm) prepared in -7-6-5
- the title antibody-drug conjugate “h046-H10 / L1-ADC1” was obtained.
- Antibody concentration 1.85 mg / mL, antibody yield: 55.6 mg (90%), average number of drugs per antibody molecule measured in common operation E (n): 5.6; in common operation F Measured average drug binding number per antibody molecule (n): 7.9.
- Step 1 Antibody-drug conjugate (12)
- Example 6) Using h046-H10 / L6 prepared in 7-6-6 (using 1.31 mLmg ⁇ 1 cm ⁇ 1 as the extinction coefficient of 280 nm), the same as Example 7) -3 step 1
- the title antibody-drug conjugate “h046-H10 / L6-ADC1” was obtained.
- Antibody concentration 1.83 mg / mL, antibody yield: 55.0 mg (87%), average number of drugs per antibody molecule measured in common operation E (n): 5.5; in common operation F Measured average drug binding number per antibody molecule (n): 8.0.
- Step 1 Antibody-drug conjugate (13)
- a 10 mM TCEP Tokyo Chemical Industry Co., Ltd.
- aqueous solution 0.12 mL; 5.5 equivalents per antibody molecule
- a 1 M dipotassium hydrogen phosphate aqueous solution Nacalai Tesque, Inc .; 0 .165 mL
- Step 1 Antibody-drug conjugate (14)
- Example 6) Using h046-H4e / L7 prepared in -7-6-7 (using 1.31 mLmg -1 cm -1 as the 280 nm extinction coefficient), the same method as in Step 7 of Example 7-13 Gave the title antibody-drug conjugate “h046-H4e / L7-ADC1”.
- Antibody concentration 3.14 mg / mL, antibody yield: 108 mg (99%), drug average binding number per antibody molecule measured in common operation E (n): 5.6; measured in common operation F Average number of drugs bound per antibody molecule (n): 7.6.
- Example 8 Evaluation of in vitro activity of antibody-drug conjugate 8) -1-1 Binding evaluation of humanized anti-GPR20 antibody and antibody-drug conjugate Example 6) Humanized anti-GPR20 prepared in 7-6 The GPR20 binding properties of the antibody and the antibody-drug conjugate prepared in Example 7 were evaluated by flow cytometry.
- Example 6) The binding to 293T cells into which pcDNA3.1-hGPR20 was transiently introduced in the same manner as in -6-1 was analyzed with a flow cytometer (BD FACSCant II: BD Bioscience). Concentration-dependent binding was confirmed.
- FIG. 25 shows a typical reaction example. In FIG.
- the horizontal axis represents antibody concentration (nM), and the vertical axis represents the amount of antibody binding due to MFI (mean fluorescence intensity).
- humanized anti-GPR20 antibodies h046-H4b / L7 and h046-H4e / L7, and antibody-drug conjugates (4) and (13) are in concentrations of pcDNA3.1-hGPR20-introduced 293T cells. The amount of binding increased dependently.
- GIST-T1 / GPR20 stable expression cell line The GIST-T1 / GPR20 stable expression cell line infects GIST-T1 cells (available from Cosmo Bio) with a recombinant retrovirus for human GPR20 expression. This was produced.
- a human GPR20-expressing retroviral vector was prepared using In-Fusion HD Cloning Kit (CLONTECH). That is, a PCR reaction was performed using the following primer set using the human GPR20 expression vector pcDNA3.1-hGPR20 prepared in Example 1 as a template. For this reaction, KOD FX DNA polymerase (Toyobo Co., Ltd.) was used, and the reaction was carried out at 98 ° C. for 10 seconds, 58 ° C. for 30 seconds, 68 ° C. for 2 minutes, 30 cycles, and then the obtained GPR20 cDNA fragment was contained.
- KOD FX DNA polymerase Toyobo Co., Ltd.
- the PCR product was subjected to agarose gel electrophoresis, and DNA of the desired size was extracted with QIAquick Gel Extraction Kit (QIAGEN).
- This DNA fragment is mixed with the retroviral vectors pLPCX (CLONTECH) digested with restriction enzymes EcoRI and NotI, In-Fusion HD Enzyme precix, ligated and then transformed into E. coli TOP10 (Invitrogen).
- pLPCX CRISPR20
- the primer sets used for PCR are as follows.
- PCR primer set (for In-Fusion cloning of pLPCX and GPR20 cDNA fragment) 5′-CTCAAGCTTCGAATTCACATGCCCCTCTGTTCTCCA-3 ′ (LPCX-1; SEQ ID NO: 112) 5′-TTGGCCGAGGGCGCCCTCTAAGCCCTCGGGCCCATTAG-3 ′ (LPCX-1; SEQ ID NO: 113) 8) -2-2
- LPCX-1 5′-CTCAAGCTTCGAATTCACATGCCCCTCTGTTCTCCA-3 ′
- LPCX-1 5′-TTGGCCGAGGGCGCCCTCTAAGCCCTCGGGCCCATTAG-3 ′
- the culture supernatant containing the replacement retrovirus was collected and added to the GIST-T1 cell culture system to infect the cells.
- GIST-T1 cells Three days after infection, GIST-T1 cells are seeded in a 96-well plate at 1 cell / well and cultured for a long time at 37 ° C. and 5% CO 2 in a medium supplemented with 0.3 to 1 ⁇ g / mL Puromycin.
- a cell line GIST-T1 / GPR20 that stably expresses GPR20 was established.
- FIG. 26 shows the concentration of the antibody-drug conjugate (1) prepared from the human chimerized antibody
- FIG. 27 shows the concentration when the antibody-drug conjugates (7), (8) and (9) prepared from the humanized antibody were added.
- Dependent cytostatic activity is shown.
- HIgG-ADC2 in this experiment is an antibody-drug conjugate prepared from human IgG that recognizes an antigen unrelated to GPR20, and was used as a negative control.
- Example 9 In vivo anti-tumor effect of antibody-drug conjugate The anti-tumor effect of antibody-drug conjugate was evaluated using an animal model in which cells derived from human gastrointestinal stromal tumors were transplanted into immunodeficient mice. 5-6 week old female BALB / c nude mice (CAnN.Cg-Foxnlnu / CrlCrlj, Charles River Japan) were acclimated under SPF conditions for 4-7 days prior to experimental use. Mice were fed a sterilized chow (FR-2, Funabashi Farms Co., Ltd) and sterilized tap water (prepared by adding 5-15 ppm sodium hypochlorite solution).
- the major axis and minor axis of the transplanted tumor were measured once or twice a week with an electronic digital caliper (CD-15CX, Mitutoyo Corp.), and the tumor volume was calculated according to the following formula.
- Tumor volume (mm 3 ) 1/2 ⁇ major axis (mm) ⁇ [minor axis (mm)] 2
- All antibody-drug conjugates were diluted with ABS buffer (10 mM-Acetate Buffer, 5% Sorbitol, pH 5.5) (NACALAI) and administered at a volume of 10 mL / kg via the tail vein.
- ABS buffer 10 mM-Acetate Buffer, 5% Sorbitol, pH 5.5
- an ABS buffer was administered in the same manner as a control group (vehicle group). Five to six mice per group were used in the experiment.
- hIgG-ADC1 and hIgG-ADC2 are antibody-drug conjugates made from human IgG that recognize an antigen unrelated to GPR20 and were used as negative controls.
- Example 9) -5 Antitumor effect (5)
- GIST-T1 / GPR20 cells were subcutaneously transplanted into female nude mice (Day 0) and randomly grouped on Day 17.
- Antibody-drug conjugate (13) was administered to Day 17 via the tail vein at doses of 0.3, 1, 3 mg / kg.
- Administration of the antibody-drug conjugate (13) decreased the tumor volume in a dose-dependent manner, and exhibited the effect of regressing the tumor at a dose of 1 mg / kg or more (FIG. 32).
- GIST020 obtained from National Institute of Biomedical Innovation, Health and Nutrition subcultured by subcutaneously transplanting a tumor extracted from a gastrointestinal stromal tumor patient of the small intestine into the subcutaneous skin of an immunodeficient mouse subcutaneously in the right flank of a female nude mouse After transplantation (Day 0), grouping was performed randomly on Day 55.
- Antibody-drug conjugates (4) and (13) were administered into Day 55 and 75 at a dose of 10 mg / kg via the tail vein.
- Administration of antibody-drug conjugates (4) and (13) significantly reduced the tumor volume compared to the control group, and both exhibited a tumor growth inhibitory effect (FIG. 33).
- GIST1 obtained from University of Toyama maintained by passage transplantation of the tumor extracted from a patient with gastrointestinal stromal tumor of the esophagus subcutaneously into an immunodeficient mouse was transplanted subcutaneously into the right flank of a female nude mouse (Day 0) A random grouping was performed on Day 38.
- Antibody-drug conjugate (13) was administered to Day 38, 59 at a dose of 3, 10 mg / kg via the tail vein.
- Administration of the antibody-drug conjugate (13) significantly reduced the tumor volume compared to the control group, and all exhibited a tumor growth inhibitory effect (FIG. 34).
- GIST074 obtained from National Institute of Biomedical Innovation, Health and Nutrition maintained by passage transplantation of a tumor derived from a gastrointestinal stromal tumor patient who has become unresponsive to Regorafenib treatment into a subcutaneous block of an immunodeficient mouse Subcutaneous transplantation was performed on the right flank of female nude mice (Day 0), and grouping was performed randomly on Day 29.
- Antibody-drug conjugate (14) was administered intravenously at Day 29, 50 at a dose of 10 mg / kg. Tumor growth was completely suppressed by administration of antibody-drug conjugate (14) (FIG. 36).
- the antibody-drug conjugate (14) also exerted an antitumor effect against gastrointestinal stromal tumors that are resistant to three tyrosine kinase inhibitors that are standard therapeutic agents.
- Imatinib and Sunitinib were orally administered once a day on the day marked with a triangle at doses of 150 and 40 mg / kg, respectively.
- Imatinib did not suppress tumor growth at all, but suppression of tumor growth was observed in the group administered with antibody-drug conjugate (3) and Sunitinib.
- a stronger drug effect was observed than the single agent, and tumor regression was observed.
- an anti-GPR20 antibody having internalization activity and an antibody-drug conjugate containing the antibody are provided.
- the antibody-drug conjugate can be used as a therapeutic agent for gastrointestinal stromal tumors.
- SEQ ID NO: 44 04-046 Ch heavy chain amino acid sequence
- SEQ ID NO: 45 04-046 Ch light chain amino acid sequence
- SEQ ID NO: 46 04-046 Ch antibody heavy chain nucleotide sequence
- SEQ ID NO: 47 04-046 Ch light chain nucleotide sequence sequence No.
- SEQ ID NO: 49 Nucleotide sequence of h046-H4b (1)
- SEQ ID NO: 50 amino acid sequence of h046-H4e SEQ ID NO: 51: nucleotide sequence of h046-H4e SEQ ID NO: 52: amino acid sequence of h046-H5b SEQ ID NO: 53: nucleotide sequence of h046-H5b (1)
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Abstract
Description
(1)以下の(a)及び(b)に記載の特性を有することを特徴とする抗体又は当該抗体の機能性断片;
(a)GPR20に特異的に結合する、及び
(b)GPR20に結合後、細胞内に取り込まれる内在化能を有する、
(2)GPR20が配列番号1に記載のアミノ酸配列からなるポリペプチドである(1)に記載の抗体又は当該抗体の機能性断片、
(3)配列番号1に記載のアミノ酸配列からなるポリペプチドには特異的に結合し、アミノ酸番号113のアミノ酸をY(チロシン)から他のアミノ酸に置換したポリペプチドには特異的に結合しない、(1)又は(2)に記載の抗体又は当該抗体の機能性断片、
(4)配列番号1に記載のアミノ酸配列からなるポリペプチドには特異的に結合し、アミノ酸番号113のアミノ酸をY(チロシン)からF(フェニルアラニン)に置換したポリペプチドには特異的に結合しない、(1)乃至(3)のいずれか1項に記載の抗体又は当該抗体の機能性断片、
(5)配列番号1においてアミノ酸番号1乃至48に記載のアミノ酸配列及びアミノ酸番号108乃至125に記載のアミノ酸配列からなる高次構造に特異的に結合する(1)乃至(4)のいずれか1項に記載の抗体又は当該抗体の機能性断片、
(6)配列番号1においてアミノ酸番号1乃至48に記載のアミノ酸配列から選択される少なくとも1つのアミノ酸残基及びアミノ酸番号108乃至125に記載のアミノ酸配列から選択される少なくとも1つのアミノ酸残基に特異的に結合する、(1)乃至(5)のいずれか1項に記載の抗体又は当該抗体の機能性断片、
(7)特異的に結合するアミノ酸の少なくとも1つが配列番号1においてアミノ酸番号113のアミノ酸である、(1)乃至(6)のいずれか1項に記載の抗体又は当該抗体の機能性断片、
(8)GPR20への結合に対して、以下の(a)乃至(c)からなる群から選択されるいずれか1項に記載の抗体と競合阻害活性を有する(1)乃至(7)のいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号2のアミノ酸番号20乃至475に示されるアミノ酸配列からなる重鎖及び配列番号7のアミノ酸番号21乃至233に示されるアミノ酸配列からなる軽鎖を有する抗体、
(b)配列番号12のアミノ酸番号20乃至475に示されるアミノ酸配列からなる重鎖及び配列番号17のアミノ酸番号21乃至233に示されるアミノ酸配列からなる軽鎖を有する抗体、及び、
(c)配列番号22のアミノ酸番号20乃至475に示されるアミノ酸配列からなる重鎖及び配列番号27のアミノ酸番号21乃至233に示されるアミノ酸配列からなる軽鎖を有する抗体、
(9)以下の(a)乃至(c)からなる群から選択されるいずれか1項に記載のCDRH1、CDRH2及びCDRH3、並びに(d)乃至(h)から選択されるいずれか1項に記載のCDRL1、CDRL2及びCDRL3を含む(1)乃至(8)から選択されるいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、
(b)配列番号14に記載のアミノ酸配列からなるCDRH1、配列番号15に記載のアミノ酸配列からなるCDRH2及び配列番号16に記載のアミノ酸配列からなるCDRH3、及び、
(c)配列番号24に記載のアミノ酸配列からなるCDRH1、配列番号25に記載のアミノ酸配列からなるCDRH2及び配列番号26に記載のアミノ酸配列からなるCDRH3、
(d)配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号10に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(e)配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号92に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(f)配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号93に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(g)配列番号19に記載のアミノ酸配列からなるCDRL1、配列番号20に記載のアミノ酸配列からなるCDRL2及び配列番号21に記載のアミノ酸配列からなるCDRL3、及び
(h)配列番号29に記載のアミノ酸配列からなるCDRL1、配列番号30に記載のアミノ酸配列からなるCDRL2及び配列番号31に記載のアミノ酸配列からなるCDRL3、
(10)以下の(a)乃至(e)から選択されるいずれか1項に記載のCDRH1、CDRH2及びCDRH3、並びにCDRL1、CDRL2及びCDRL3を含む(1)乃至(9)から選択されるいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、及び配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号10に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(b)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、及び配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号92に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(c)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、及び配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号93に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3
(d)配列番号14に記載のアミノ酸配列からなるCDRH1、配列番号15に記載のアミノ酸配列からなるCDRH2及び配列番号16に記載のアミノ酸配列からなるCDRH3、及び配列番号19に記載のアミノ酸配列からなるCDRL1、配列番号20に記載のアミノ酸配列からなるCDRL2及び配列番号21に記載のアミノ酸配列からなるCDRL3、及び
(e)配列番号24に記載のアミノ酸配列からなるCDRH1、配列番号25に記載のアミノ酸配列からなるCDRH2及び配列番号26に記載のアミノ酸配列からなるCDRH3、及び配列番号29に記載のアミノ酸配列からなるCDRL1、配列番号30に記載のアミノ酸配列からなるCDRL2及び配列番号31に記載のアミノ酸配列からなるCDRL3、
(11)以下の(a)乃至(c)からなる群から選択されるいずれか1項に記載の重鎖の可変領域、及び(d)乃至(h)から選択されるいずれか1項に記載の軽鎖の可変領域を有する(1)乃至(10)から選択されるいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、
(b)配列番号13に記載のアミノ酸配列からなる重鎖の可変領域、及び、
(c)配列番号23に記載のアミノ酸配列からなる重鎖の可変領域、からなる群から選択される重鎖の可変領域、
(d)配列番号8に記載のアミノ酸配列からなる軽鎖の可変領域、
(e)配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(f)配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(g)配列番号18に記載のアミノ酸配列からなる軽鎖の可変領域、及び、
(h)配列番号28に記載のアミノ酸配列からなる軽鎖の可変領域、
(12)以下の(a)乃至(e)から選択されるいずれか1項に記載の重鎖可変領域及び軽鎖可変領域を含む(1)乃至(11)から選択されるいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号8に記載のアミノ酸配列からなる軽鎖の可変領域、
(b)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(c)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(d)配列番号13に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号18に記載のアミノ酸配列からなる軽鎖の可変領域、
(e)配列番号23に記載のアミノ酸配列からなる重鎖の可変領域、及び、配列番号28に記載のアミノ酸配列からなる軽鎖の可変領域、
(13)定常領域がヒト由来定常領域である(1)乃至(12)のいずれか1項に記載の抗体又は当該抗体の機能性断片、
(14)配列番号44に記載のアミノ酸配列からなる重鎖及び配列番号45に記載のアミノ酸配列からなる軽鎖を含む(13)に記載の抗体又は当該抗体の機能性断片。
(15)ヒト化されている(1)乃至(14)のいずれか1項に記載の抗体又は当該抗体の機能性断片、
(16)以下の(a)乃至(h)からなる群から選択されるいずれか1項に記載のアミノ酸配列からなる重鎖の可変領域、及び(i)乃至(o)からなる群から選択されるいずれか1項に記載のアミノ酸配列からなる軽鎖の可変領域を有する(15)に記載の抗体又は当該抗体の機能性断片:
(a)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(b)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(c)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(d)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(e)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(f)配列番号44においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(g)(a)乃至(f)の配列において各CDR 配列以外のフレームワーク領域の配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、
(h)(a)乃至(g)の配列における各CDR 配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、
(i)配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(j)配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(k)配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(l)配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(m)配列番号45においてアミノ酸番号21乃至128に記載のアミノ酸配列、
(n)(i)乃至(m)の配列において各CDR 配列以外のフレームワーク領域の配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、及び
(o) (i)乃至(n)の配列における各CDR 配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、
(17)以下の(a)乃至(t)からなる群から選択されるいずれか1項に記載の重鎖可変領域及び軽鎖可変領域を含む(16)に記載の抗体又は抗体の機能性断片:
(a)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域および配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変流域、
(b)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる、重鎖可変領域および配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(c)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域および配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(d)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域および配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域からなる軽鎖可変領域、
(e)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(f)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(g)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(h)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(i)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(j)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(k)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(l)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(m)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(n)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(o)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(p)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(q)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(r)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(s)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、及び、
(t)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(18)以下の(a)乃至(x)から選択されるいずれか1項に記載の重鎖及び軽鎖を含む(16)又は(17)に記載の抗体又は抗体の機能性断片:
(a)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(b)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる、重鎖および配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(c)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(d)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(e)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(f)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(g)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(h)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(i)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(j)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(k)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(l)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(m)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(n)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(o)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(p)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(q)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(r)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(s)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(t)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(u)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(v)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(w)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(x)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(19)機能性断片がFab、F(ab)2、Fab’及びFvからなる群から選択される(1)乃至(18)のいずれか1項に記載の抗体の機能性断片、
(20)(1)乃至(19)のいずれか1項に記載の抗体又は当該抗体の機能性断片をコードするポリヌクレオチド、
(21)以下の(a)乃至(e)からなる群から選択されるいずれか1項に記載のポリヌクレオチドを含む(20)に記載のポリヌクレオチド:
(a)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号10に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRLをコードするポリヌクレオチド、
(b)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号92に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(c)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号93に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド
(d)配列番号14に記載のアミノ酸配列からなるCDRH1、配列番号15に記載のアミノ酸配列からなるCDRH2及び配列番号16に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号19に記載のアミノ酸配列からなるCDRL1、配列番号20に記載のアミノ酸配列からなるCDRL2及び配列番号21に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、及び、
(e)配列番号24に記載のアミノ酸配列からなるCDRH1、配列番号25に記載のアミノ酸配列からなるCDRH2及び配列番号26に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号29に記載のアミノ酸配列からなるCDRL1、配列番号30に記載のアミノ酸配列からなるCDRL2及び配列番号31に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(22)以下の(a)乃至(e)からなる群から選択されるいずれか1項に記載のポリヌクレオチドを含む(20)又は(21)に記載のポリヌクレオチド:
(a)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号8に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(b)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(c)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(d)配列番号13に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号18に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、及び、
(e)配列番号23に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号28に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(23)(20)乃至(22)のいずれか1項に記載のポリヌクレオチドを含有する発現ベクター、
(24)(23)に記載の発現ベクターにより形質転換された宿主細胞、
(25)宿主細胞が真核細胞である(23)に記載の宿主細胞、
(26)(24)又は(25)に記載の宿主細胞を培養する工程、及び当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程を含むことを特徴とする当該抗体又は当該抗体の機能性断片の製造方法、
(27)(26)に記載の製造方法により得られることを特徴とする抗体又は当該抗体の機能性断片、
(28)機能性断片がFab、F(ab)2、Fab’及びFvからなる群から選択される(27)に記載の抗体の機能性断片、
(29)N-結合への糖鎖付加、O-結合への糖鎖付加、N末のプロセッシング、C末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、N末にメチオニン残基の付加、プロリン残基のアミド化及びカルボキシル末端において1つ又は2つのアミノ酸が欠失した重鎖からなる群より選択される1又は2以上の修飾を含む(1)乃至(19)、(27)及び(28)からのいずれか1項に記載の抗体又は当該抗体の機能性断片、
(30)重鎖のカルボキシル末端において1つ又は2つのアミノ酸が欠失している(29)に記載の抗体、
(31)2本の重鎖の双方でカルボキシル末端において1つのアミノ酸が欠失している(30)に記載の抗体、
(32)重鎖のカルボキシル末端のプロリン残基が更にアミド化されている(29)乃至(31)のいずれか1項に記載の抗体、
(33)抗体依存性細胞傷害活性を増強させるために糖鎖修飾が調節されている(1)乃至(19)、及び(26)乃至(31)からのいずれか1項に記載の抗体又は当該抗体の機能性断片、
(34)(1)乃至(19)、及び(27)乃至(33)のいずれか1項に記載の抗体又は当該抗体の機能性断片に薬物が結合している抗体-薬物コンジュゲート、
(35)薬物が抗腫瘍性化合物である、(34)に記載の抗体-薬物コンジュゲート、
(36)
抗腫瘍性化合物が次式
(a) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(b) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(c) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
(d) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
(e) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(f) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
で示されるいずれかの構造のリンカーを介して、結合している(35)又は(36)に記載の抗体-薬物コンジュゲート。
(ここで、抗体は、-(Succinimid-3-yl-N)の末端において結合する。抗腫瘍性化合物は、1位のアミノ基の窒素原子を結合部位として、-(CH2)n2-C(=O)-部分のカルボニル基に結合する。上記式中GGFGは、グリシン-グリシン-フェニルアラニン-グリシンからなるペプチド結合でつながっているアミノ酸配列を示す。
-(Succinimid-3-yl-N)-は次式:
(38)リンカーが以下の(a)乃至(c)から選択されるいずれかの式で示される(34)乃至(37)のいずれか1項に記載の抗体-薬物コンジュゲート: (a) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
(b) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
(c) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(b) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
(a) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
(41)
以下の式化3(式中、Aは抗体との結合位置を示す)
で示される薬物-リンカー構造と、抗体とがチオエーテル結合によって結合した、(34)乃至(40)のいずれか1項に記載の抗体-薬物コンジュゲート
で示される構造を有する、(34)乃至(40)のいずれか1項に記載の抗体-薬物コンジュゲート:
ここで、ABは抗体又は該抗体の機能性断片を示す。nは抗体と結合している薬物-リンカー構造の1抗体あたりの平均結合数を示す。抗体とリンカーは抗体由来のスルフヒドリル基を介して結合している、
以下の式化5:
で示される(34)乃至(39)のいずれか1項に記載の抗体-薬物コンジュゲート:
ここで、ABは抗体又は該抗体の機能性断片を示す。nは抗体と結合している薬物-リンカー構造の1抗体あたりの平均結合数を示す。抗体とリンカーは抗体由来のスルフヒドリル基を介して結合している、
(a)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(b)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(c)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(d)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(e)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(f)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(g)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(h)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(i)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(j)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(k)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(l)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(m)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(n)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(o)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(p)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(q)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(r)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(s)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(t)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(u)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(v)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(w)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(x)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(45)重鎖がN-結合への糖鎖付加、O-結合への糖鎖付加、N末のプロセッシング、C末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、N末にメチオニン残基の付加、プロリン残基のアミド化及びカルボキシル末端において1つ又は2つのアミノ酸が欠失した重鎖からなる群より選択される1又は2以上の修飾を含む抗体である、(44)に記載の抗体-薬物コンジュゲート。
(46)選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が1から10個の範囲である(34)乃至(45)のいずれか1項に記載の抗体-薬物コンジュゲート、
(47)選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が2から8個の範囲である(34)乃至(46)のいずれか1項に記載の抗体-薬物コンジュゲート、
(48)選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が3から8個の範囲である(34)乃至(47)のいずれか1項に記載の抗体-薬物コンジュゲート、
(49)選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が7から8個の範囲である(34)乃至(48)のいずれか1項に記載の抗体-薬物コンジュゲート、
(50)選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が8である(32)乃至(49)のいずれか1項に記載の抗体-薬物コンジュゲート、
(51)(1)乃至(19)、及び(27)乃至(33)に記載の抗体又は当該抗体の機能性断片、(34)乃至(50)に記載の抗体-薬物コンジュゲートから選択されるいずれか、その塩、又はそれらの水和物を含むことを特徴とする医薬組成物、
(52)抗腫瘍薬であることを特徴とする、(51)に記載の医薬組成物、
(53)腫瘍がGPR20が発現している腫瘍であることを特徴とする、(52)に記載の抗腫瘍薬、
(54)腫瘍が消化管間質腫瘍であることを特徴とする、(52)又は(53)に記載の抗腫瘍薬、
(55)更に、他の抗腫瘍薬を含むことからなる、(51)乃至(54)のいずれか1項に記載の医薬組成物又は抗腫瘍薬。
(56)(1)乃至(19)、及び(27)乃至(33)に記載の抗体又は当該抗体の機能性断片、(34)乃至(50)に記載の抗体-薬物コンジュゲート、その塩、又はそれらの水和物から選択されるいずれかを個体に投与することを特徴とする腫瘍の治療方法。
(57)腫瘍が、消化管間質腫瘍であることを特徴とする、(56)に記載の治療方法、
(58)腫瘍が、チロシンキナーゼ阻害剤に抵抗性を示す腫瘍であることを特徴とする、(56)又は(57)に記載の治療方法、
(59)(1)乃至(19)、及び(27)乃至(33)に記載の抗体又は当該抗体の機能性断片、(34)乃至(50)に記載の抗体-薬物コンジュゲート、その塩、又はそれらの水和物から選択される少なくとも一つ、を含むことからなる医薬組成物及び少なくとも一つの抗腫瘍薬を、同時に、別々に又は連続して個体に投与することを特徴とする腫瘍の治療方法、
(60)抗腫瘍薬が、チロシンキナーゼ阻害剤であることを特徴とする、(59)に記載の腫瘍の治療方法、
(61)チロシンキナーゼ阻害剤が、スニチニブ(Sunitinib)、イマチニブ(Imatinib)、レゴラフェニブ(Regorafenib)から選択される少なくとも1つである、(60)に記載の腫瘍の治療方法、
(62)腫瘍が、チロシンキナーゼ阻害剤に抵抗性を示す消化管間質腫瘍であることを特徴とする、(56)乃至(61)のいずれか1項に記載の腫瘍の治療方法、
(63)(24)又は(25)に記載の宿主細胞を培養する工程、当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程、及び当該工程で得られた抗体又は当該抗体の機能性断片と薬物-リンカー中間化合物を反応させる工程を含むことを特徴とする、抗体-薬物コンジュゲートの製造方法、
からなる。
本明細書中において、「ポリヌクレオチド」という語は核酸と同じ意味で用いており、DNA、RNA、プローブ、オリゴヌクレオチド、及びプライマーも含まれる。
GPR20は、Gタンパク質共役受容体(GPCR)ファミリーのクラスAに属する、358アミノ酸からなる7回膜貫通型のタンパク質であり、N末端側を細胞外、C末端側を細胞内に持つ。
本発明の抗GPR20抗体の一例として、配列表の配列番号1に示すGPR20のN末端より1から48番目のアミノ酸配列、及び108から125番目のアミノ酸配列の2つの細胞外領域からなる高次構造を認識し、かつ内在化活性を有する抗GPR20抗体を挙げることができる。
(1)抗原の調製
抗原は抗原タンパク質をコードする遺伝子を遺伝子操作によって宿主細胞に産生させることによって得ることができる。具体的には、抗原遺伝子を発現可能なベクターを作製し、これを宿主細胞に導入して該遺伝子を発現させ、発現した抗原を精製すればよい。上記の遺伝子操作による抗原発現細胞、あるいは抗原を発現している細胞株を動物に免疫する方法を用いることによっても抗体を取得できる。
本発明で使用される抗GPR20抗体は、特に制限はないが、例えば、本願の配列表で示されたアミノ酸配列で特定される抗体を好適に使用することができる。本発明において使用される抗GPR20抗体としては、以下の特性を有するものが望ましい。
(1)以下の特性を有することを特徴とする抗体;
(a)GPR20に特異的に結合する
(b)GPR20と結合することによってGPR20発現細胞に内在化する活性を有する
(2)GPR20がヒトGPR20である上記(1)に記載の抗体又は当該抗体、
(3)ヒトGPR20のN末端より1から48番目のアミノ酸配列、及び108から125番目のアミノ酸配列の2つの細胞外領域からなる高次構造を認識し、かつ内在化活性を有する。
(a)GPR20のcDNAを発現ベクター(例えば、pcDNA3.1:Thermo Fisher Scientific)に組み込み、エレクトロポレーションや遺伝子銃等の方法によって、そのベクターを直接被免疫動物(例えば、ラットやマウス)に投与することによって、動物体内においてGPR20を発現させることによって、免疫反応を誘起させることができる。エレクトロポレーション等によるベクターの投与は抗体価を上げるために必要であれば、1回でも複数回でもよく、好ましくは複数回である。
(b)免疫反応を誘起された上述の動物より、抗体産生細胞を含む組織(例えばリンパ節)を採取する。
(c)骨髄腫細胞(以下「ミエローマ」という)(例えば、マウスミエローマSP2/0-ag14細胞)の調製、
(d)抗体産生細胞とミエローマとの細胞融合、
(e)目的とする抗体を産生するハイブリドーマ群の選別、
(f)単一細胞クローンへの分割(クローニング)、
(g)場合によっては、モノクローナル抗体を大量に製造するためのハイブリドーマの培養、又はハイブリドーマを移植した動物の飼育、
(h)このようにして製造されたモノクローナル抗体の生理活性(内在化活性)、及びその結合特異性の検討、あるいは標識試薬としての特性の検定
ここで用いられる抗体価の測定法としては、例えば、フローサイトメトリー又はCell-ELISA法を挙げることができるがこれらの方法に制限されない。
(3) その他の抗体
本発明の抗体には、上記GPR20に対するモノクローナル抗体に加え、ヒトに対する異種抗原性を低下させること等を目的として人為的に改変した遺伝子組換え型抗体、例えば、キメラ(Chimeric)抗体、ヒト化(Humanized)抗体、ヒト抗体等も含まれる。これらの抗体は、既知の方法を用いて製造することができる。
また、重鎖又は軽鎖の一方をヒト化し、他方をラット抗体やキメラ抗体の軽鎖又は重鎖とした抗体も用いることができる。そのような抗体の例としては、(1)配列表の配列番号44の20乃至472番目のアミノ酸残基からなるアミノ酸配列、(2)上記(1)のアミノ酸配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、及び(3)上記(1)のアミノ酸配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる重鎖、並びに(4)配列番号58、60、62又は64の21乃至129番目のアミノ酸残基からなるアミノ酸配列、(5)上記(4)のアミノ酸配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、及び(6)上記(4)のアミノ酸配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる軽鎖可変領域を含む軽鎖の任意の組合せを挙げることができる。そのような抗体の他の例としては、(1)配列表の配列番号48、50、52、54又は56の20乃至142番目のアミノ酸残基からなるアミノ酸配列、(2)上記(1)のアミノ酸配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、及び(3)上記(1)のアミノ酸配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる重鎖可変領域を含む重鎖、並びに、(4)配列表の配列番号45の21乃至233番目のアミノ酸残基からなるアミノ酸配列、(5)上記(4)のアミノ酸配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、及び(6)上記(4)のアミノ酸配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列のいずれか一つからなる軽鎖可変領域を含む軽鎖の任意の組合せを挙げることができる。また、別の例としては、以下の(7)ないし(19)から選択されるいずれかの重鎖及び軽鎖の組み合わせからなる抗体を挙げることができる:(7)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、(8)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、(9)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、(10)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖。
(1)薬物
上記「2.抗GPR20抗体の製造」にて取得された抗GPR20抗体はリンカー構造部分を介して薬物を結合させることによって、抗GPR20抗体-薬物コンジュゲートとすることができる。薬物としては、リンカー構造に結合できる置換基、部分構造を有するものであれば特に制限はない。抗GPR20抗体-薬物コンジュゲートは結合する薬物に応じて種々の用途に用いることができる。そのような薬物の例としては、抗腫瘍活性を有する物質、血液疾患に対する効果を有する物質、自己免疫疾患に対する効果を有する物質、抗炎症物質、抗菌物質、抗真菌物質、抗寄生虫物質、抗ウイルス物質、抗麻酔物質等を挙げることができる。
本発明の抗GPR20抗体-薬物コンジュゲートに結合される化合物として抗腫瘍性化合物を用いる例について以下に述べる。抗腫瘍性化合物としては、抗腫瘍効果を有する化合物であって、リンカー構造に結合できる置換基、部分構造を有するものであれば特に制限はない。抗腫瘍性化合物は、リンカーの一部又は全部が腫瘍細胞内で切断されて抗腫瘍性化合物部分が遊離されて抗腫瘍効果が発現される。リンカーが薬物との結合部分で切断されれば抗腫瘍性化合物が本来の構造で遊離され、その本来の抗腫瘍効果が発揮される。
本発明の抗GPR20抗体-薬物コンジュゲートにおいて薬物を抗GPR20抗体に結合させるリンカー構造について述べる。
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
さらにより好ましくは、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
を挙げることができる。
抗体は-(Succinimid-3-yl-N)の末端、(例えば、『-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-』においては、(-CH2CH2CH2CH2CH2-)が結合するのとは反対側の末端(左末端))で結合し、抗腫瘍性化合物は-(Succinimid-3-yl-N)とは反対側の末端(上記例では右末端の、CH2-O-CH2-C(=O)-のカルボニル基で結合する。『-(Succinimid-3-yl-N)-』は、次式
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)。
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-(NH-DX)、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)。
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-(NH-DX)。
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-(NH-DX)
本発明の抗体ー薬物コンジュゲートに用いることができる抗体は、上記「2.抗GPR20抗体の製造」の項及び実施例に記載の内在化活性を有する抗GPR20抗体及び該抗体の機能性断片であれば特に制限がない。
下記式(1)で示される抗体-薬物コンジュゲートのうち、チオエーテルを介して抗GPR20抗体とリンカー構造が結合しているものは抗GPR20抗体を還元してジスルフィド結合をスルヒドリル基に変換した抗体に対して、既知の方法によって入手しうる化合物(2)(例えば、US2016/297890号公開特許公報に記載の方法(例えば段落[0336]乃至[0374]に記載の方法)で入手可能)を反応させることによって製造することができる。例えば下記の方法によって製造することができる。
ここで、L1は、
-(Succinimid-3-yl-N)-の構造で示される。
L1’は次式で示される、マレイミジル基を示す。]
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
-(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
-(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
。
製造した抗体-薬物コンジュゲート(例えば、抗体―薬物コンジュゲート(1))は、以下の共通操作によって濃縮、バッファー交換、精製、抗体濃度及び抗体一分子あたりの薬物平均結合数の測定を行い、抗体-薬物コンジュゲート(1)の同定を行うことができる。
Amicon Ultra(50,000 MWCO,Millipore Corporation)の容器内に抗体もしくは抗体-薬物コンジュゲート溶液を入れ、遠心機(Allegra X-15R,Beckman Coulter,Inc.)を用いた遠心操作(2000G乃至3800Gにて5乃至20分間遠心)にて、抗体もしくは抗体-薬物コンジュゲート溶液を濃縮した。
UV測定器(Nanodrop 1000,Thermo Fisher Scientific Inc.)を用いて、メーカー規定の方法に従い、抗体濃度の測定を行った。その際に、抗体ごとに異なる280nm吸光係数(1.3mLmg-1cm-1乃至1.8mLmg-1cm-1)を用いた。
Sephadex G-25担体を使用したNAP-25カラム(Cat.No.17-0852-02,GE Healthcare Japan Corporation)を、メーカー規定の方法に従い、塩化ナトリウム(50mM)及びEDTA(2mM)を含むリン酸緩衝液(50mM,pH6.0)(本明細書でPBS6.0/EDTAと称する。)にて平衡化させた。このNAP-25カラム一本につき、抗体水溶液2.5mLをのせたのち、PBS6.0/EDTA3.5mLで溶出させた画分(3.5mL)を分取した。この画分を共通操作Aによって濃縮し、共通操作Bを用いて抗体濃度の測定を行ったのちに、PBS6.0/EDTAを用いて20mg/mLに抗体濃度を調整した。
市販のSorbitol(5%)を含む酢酸緩衝液(10mM,pH5.5;本明細書でABSと称する。)のいずれかの緩衝液でNAP-25カラムを平衡化させた。このNAP-25カラムに、抗体-薬物コンジュゲート反応水溶液(約2.5mL)をのせ、メーカー規定の量の緩衝液で溶出させることで、抗体画分を分取した。この分取画分を再びNAP-25カラムにのせ緩衝液で溶出させるゲルろ過精製操作を計2乃至3回繰り返すことで、未結合の薬物リンカーや低分子化合物(トリス(2-カルボキシエチル)ホスフィン塩酸塩(TCEP),N-アセチル-L-システイン(NAC),ジメチルスルホキシド)を除いた抗体-薬物コンジュゲートを得た。
抗体-薬物コンジュゲートにおける結合薬物濃度は、抗体-薬物コンジュゲート水溶液の280nm及び370nmの二波長におけるUV吸光度を測定したのちに下記の計算を行うことで、算出することができる。
A370=AD,370+AA,370=εD,370CD+εA,370CA 式(2)
抗体-薬物コンジュゲートにおける抗体一分子あたりの薬物平均結合数は、前述の「(4)-5 共通操作E」に加え、以下の方法を用いる高速液体クロマトグラフィー(HPLC)分析によっても求めることができる。以下に、抗体と薬物リンカーがジスルフィド結合している場合のHPLCによる薬物平均結合数の測定方法を記載する。当業者は、この方法を参照して、抗体と薬物リンカーとの結合様式に依存して適宜、HPLCにより薬物平均結合数を測定し得る。
抗体-薬物コンジュゲート溶液(約1mg/mL、60μL)をジチオトレイトール(DTT)水溶液(100mM、15μL)と混合する。混合物を37℃で30分インキュベートすることで、抗体-薬物コンジュゲートの軽鎖及び重鎖間のジスルフィド結合を切断したサンプルを、HPLC分析に用いる。
HPLC分析を、下記の測定条件にて行う。
検出器:紫外吸光度計(測定波長:280nm)
カラム:ACQUITY UPLC BEH Phenyl(2.1×50mm、1.7μm、130Å;Waters、P/N 186002884)
カラム温度:80℃
移動相A:0.10%トリフルオロ酢酸(TFA)、15%2-プロパノールを含む水溶液
移動相B:0.075%TFA、15%2-プロパノールを含むアセトニトリル溶液
グラジエントプログラム:14%-36%(0分-15分)、36%-80%(15分-17分)、80%-14%(17分―17.01分)、14%(17.01分―25分)
サンプル注入量:10μL
もしくは、
HPLCシステム:Agilent 1290 HPLCシステム(Agilent Technologies)
検出器:紫外吸光度計(測定波長:280nm)
カラム:PLRP-S(2.1×50mm、8μm、1000Å;Agilent Technologies、P/N PL1912-1802)
カラム温度:80℃
移動相A:0.04%TFA水溶液
移動相B:0.04%TFAを含むアセトニトリル溶液
グラジエントプログラム:29%-36%(0分-12.5分)、36%-42%(12.5分-15分)、42%-29%(15分―15.1分)、29%-29%(15.1分―25分)
サンプル注入量:15μL
F-3-1 薬物の結合数に応じて抗体の軽鎖をLi、重鎖をHiと表記する(ここでiは結合している薬物の数を示す。すなわち、本発明において薬物結合数はL0、L1、H0、H1、H2、H3と表記する。
薬物の結合していない抗体の軽鎖(L0)及び重鎖(H0)に対して、薬物が一つ結合した軽鎖:L1及び、薬物が1個結合した重鎖:H1、薬物が二つ結合した重鎖:H2、薬物が三つ結合した重鎖:H3は、結合した薬物の数に比例して疎水性が増し保持時間が大きくなることから、例えば、L0、L1、H0、H1、H2、H3の順に溶出される。L0及びH0との保持時間比較により検出ピークをL0、L1、H0、H1、H2、H3のいずれかに割り当てることができる。
F-3-4 抗体-薬物コンジュゲートにおける抗体一分子あたりの薬物平均結合数を、下式に従って計算する。
製造方法1における式(2)で示される化合物であるが、次式のいずれかである。
で示される薬物-リンカー構造部分と、本明細書で開示される抗GPR20抗体とがチオエーテル結合によって結合した抗体-薬物コンジュゲートを挙げることができる。
本発明においては、抗体-薬物コンジュゲートのうち、リンカー及び薬物からなる部分構造を、「薬物-リンカー構造」、「薬物ーリンカー構造部分」又は「薬物リンカー」とも称する。この薬物リンカーは抗体の鎖間のジスルフィド結合部位(2箇所の重鎖-重鎖間、及び2箇所の重鎖-軽鎖間)において生じたチオール基(言い換えれば、システイン残基の硫黄原子)に結合している。
(a)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(b)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(c)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(d)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(e)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(f)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(g)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(h)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(i)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(j)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(k)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(l)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(m)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(n)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(o)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(p)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(q)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(r)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(s)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(t)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(u)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(v)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(w)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(x)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(y)重鎖又は軽鎖がN-結合への糖鎖付加、O-結合への糖鎖付加、N末のプロセッシング、C末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、N末にメチオニン残基の付加、プロリン残基のアミド化及びカルボキシル末端において1つ又は2つのアミノ酸欠失からなる群より選択される1又は2以上の修飾を含む、(a)乃至(x)からなる群から選択されるのいずれか1項に記載の抗体。
上記、「2.抗GPR20抗体の製造」の項及び実施例に記載された本発明の抗GPR20抗体及び当該抗体の機能性断片は、腫瘍細胞表面のGPR20に結合し、内在化活性を有することから、単独であるいは他の薬剤と組み合わせて、医薬として、特に消化管質腫瘍等のがんの治療剤として用いることができる。
in vivoでの実験動物を用いたがんに対する治療効果は、例えば、GPR20を高発現している腫瘍細胞株を移植したヌードマウスに抗GPR20抗体-薬物コンジュゲートを投与し、がん細胞の変化を測定することができる。ここで、消化管間質腫瘍由来の細胞を免疫不全マウスに移植した動物モデルを用いることで、消化管間質腫瘍に対する治療効果を測定することができる。
本発明の抗GPR20抗体-薬物コンジュゲートは、哺乳動物に対して好適に投与することができるが、より好ましくはヒトである。
1)-1 ヒトGPR20発現ベクターの構築
ヒトGPR20タンパク質(NP_005284)をコードするcDNAは、当業者に公知な方法に従いヒト脳由来cDNAを鋳型としたPCR法により増幅され、哺乳動物発現用ベクターに組み込むことによってヒトGPR20発現ベクターpcDNA3.1-hGPR20が作製された。ヒトGPR20のアミノ酸配列を配列表の配列番号1に示す。pcDNA3.1-hGPR20プラスミドDNAの大量調製は、EndoFree Plasmid Giga Kit(QIAGEN社)を用いて行った。
免疫には6週齢のWKY/Izmラットの雌(日本エスエルシー社)が使用された。まずラット両足下腿部をHyaluronidase(SIGMA-ALDRICH社)にて前処理後、同部位にヒトGPR20発現ベクターpcDNA3.1-hGPR20が筋肉内注射された。続けて、ECM830(BTX社)を使用し、2ニードル電極を用いて、同部位にインビボエレクトロポレーションを実施した。二週間に一度、同様のインビボエレクトロポーレーションを繰り返した後、79日目にラットのリンパ節を採取し、ハイブリドーマ作製に用いた。
リンパ節細胞とマウスミエローマSP2/0-ag14細胞とをHybrimune Hybridoma Production System(Cyto Pulse Sciences社)を用いて電気細胞融合した後、ClonaCell-HY Selection Medium D(StemCell Technologies社)に懸濁し、希釈して37℃、5% CO2の条件下で培養した。出現した各々のハイブリドーマコロニーは、モノクローンとして回収され、ClonaCell-HY Selection Medium E(StemCell Technologies社)に懸濁して37℃、5% CO2の条件下で培養した。適度に細胞が増殖した後、各々のハイブリドーマ細胞の凍結ストックを作製すると共に、培養上清が回収され、抗GPR20抗体を産生するハイブリドーマのスクリーニングに用いられた。
1)-4-1 Cell-ELISA用抗原遺伝子発現細胞の調製
293α細胞(インテグリンαv及びインテグリンβ3を発現するHEK293由来の安定発現細胞株)を10% FBS含有DMEM培地中5x105細胞/10mLになるよう調製した。Lipofectamine 2000(インビトロジェン社)を用いた形質移入手順に従って、この293α細胞に対して、pcDNA3.1-hGPR20もしくは陰性コントロールとしてpcDNA3.1のDNAを導入し、96-well plate(Corning社)に100μLずつ分注後、10% FBS含有DMEM培地中で37℃、5% CO2の条件下で24から27時間培養した。得られた形質移入細胞を接着状態のまま、Cell-ELISAに使用した。
実施例1)-4-1で調製した発現ベクター導入293α細胞の培養上清を除去後、pcDNA3.1-hGPR20又はpcDNA3.1導入293α細胞の各々に対しハイブリドーマ培養上清を添加し、4℃で1時間静置した。well中の細胞を5% FBS含有PBS(+)で1回洗浄後、5% FBS含有PBS(+)で500倍に希釈したAnti-Rat IgG-Peroxidase antibody produced in rabbit(SIGMA社)を加えて、4℃で1時間静置した。well中の細胞を5% FBS含有PBS(+)で3回洗浄した後、OPD発色液(OPD溶解液(0.05 M クエン酸3ナトリウム、0.1M リン酸水素2ナトリウム・12水 pH4.5)にo-フェニレンジアミン二塩酸塩(和光純薬社)、H2O2をそれぞれ0.4mg/mL、0.6%(v/v)になるように溶解)を100μL/wellで添加した。時々攪拌しながら発色反応を行い、1M HClを100μL/wellで添加して発色反応を停止させた後、プレートリーダー(ENVISION:PerkinElmer社)で490nmの吸光度を測定した。コントロールのpcDNA3.1導入293α細胞と比較し、pcDNA3.1-hGPR20発現ベクター導入293α細胞の方でより高い吸光度を示した培養上清を産生するハイブリドーマが、細胞膜表面上に発現するヒトGPR20に特異的に結合する抗体を産生するハイブリドーマとして選択された。
1)-5-1 フローサイトメトリー解析用抗原遺伝子発現細胞の調製
293T細胞を5×104細胞/cm2になるよう225cm2フラスコ(住友ベークライト社)に播種し、10% FBS含有DMEM培地中で37℃、5% CO2の条件下で一晩培養した。この293T細胞に、pcDNA3.1-hGPR20及び陰性コントロールとしてpcDNA3.1を各々Lipofectamine 2000を用いて導入し、37℃、5% CO2の条件下でさらに一晩培養した。各発現ベクターを導入した293T細胞をTrypLE Express(Life Technologies社)で処理し、10% FBS含有DMEMで細胞を洗浄した後、5% FBS含有PBSに懸濁した。得られた細胞懸濁液をフローサイトメトリー解析に使用した。
実施例1)-4-2のCell-ELISAで陽性と判定されたハイブリドーマが産生する抗体のヒトGPR20に対する結合特異性をフローサイトメトリー法によりさらに確認した。実施例1)-5-1で調製した一過性発現293T細胞の懸濁液を遠心し、上清を除去した後、各々に対しハイブリドーマ培養上清を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、5% FBS含有PBSで500倍に希釈したAnti-Rat IgG FITC conjugate(SIGMA社)を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、2μg/mL 7-aminoactinomycin D(Molecular Probes社)を含む5% FBS含有PBSに再懸濁し、フローサイトメーター(FC500:BeckmanCoulter社)で検出を行った。データ解析はFlowjo(TreeStar社)で行った。7-aminoactinomycin D陽性の死細胞をゲートで除外した後、生細胞のFITC蛍光強度のヒストグラムを作成した。コントロールであるpcDNA3.1導入293T細胞の蛍光強度ヒストグラムに対しpcDNA3.1-hGPR20導入293T細胞のヒストグラムが強蛍光強度側にシフトしている抗体を産生するハイブリドーマをヒトGPR20に結合する抗体産生ハイブリドーマとして178クローン選択した。図18にヒトGPR20に特異的に結合した抗体の例としてクローン番号04-002、04-006、04-013、04-014、04-020、04-021、04-037、04-046、04-047、04-060、04-067、04-068、04-079、04-084、04-114、04-115、04-117、04-125、04-126、04-127、04-133、04-139、04-143、04-145、04-151及び04-163、並びにコントロール(W/O 1stAb)の結果を示す。図18の横軸はクローン番号、縦軸はMFI(mean fluorescence intensity)による抗体結合量を示す。
抗GPR20抗体の内在化活性は、タンパク質合成を阻害する毒素(サポリン)を結合させた抗ラットIgG試薬Rat-ZAP(ADVANCED TARGETING SYSTEMS)を用いて評価した。すなわち、ヒトGPR20を一過性に発現させた293α細胞を3x103 cells/wellで96wellプレートに播種して37℃、5% CO2の条件下で一晩培養後、プレートを氷上で冷やした上で抗GPR20抗体産生ハイブリドーマの培養上清20μLを添加し、4℃で1時間静置した。培養上清を吸引して除去した後、500 ng/mLのRat-ZAPを含むDMEM-10%FBSを添加し、3日間37℃、5%CO2条件下で培養した。生存細胞数は、CellTiter-Glo(登録商標) Luminescent Cell Viability AssayによるATPの定量で測定した。このスクリーニングでは、ラット抗GPR20抗体の内在化活性に依存してRat-ZAPが細胞内に取り込まれ、タンパク合成を阻害するサポリンが細胞内に放出されることで、細胞増殖が抑制される。細胞増殖抑制率60%以上を指標に選定した結果、内在化活性を有する抗GPR20抗体を産生する19種のハイブリドーマ(クローン番号は、04-002、04-006、04-013、04-014、04-021、04-037、04-046、04-047、04-067、04-068、04-079、04-114、04-115、04-125、04-126、04-127、04-133、04-139及び04-163)が選定された。
ラット抗ヒトGPR20モノクローナル抗体の重鎖のサブクラス、軽鎖のタイプは、RAT MONOCLONAL ANTIBODY ISOTYPING TEST KIT(DSファーマバイオメディカル社)により決定された。その結果、クローン番号04-047及び04-068はIgG2a、κ鎖、クローン番号04-002、04-006、04-013、04-014、04-021、04-037、04-046、04-067、04-079、04-114、04-115、04-125、04-126、04-127、04-133、04-139及び04-163はIgG2b、κ鎖であることが確認された。
ラット抗ヒトGPR20モノクローナル抗体は、ハイブリドーマ培養上清から精製した。
2)-1 フローサイトメトリーによるラット抗GPR20抗体の結合能評価
GPR20に対する結合能を評価するため、1)-5-1で示す方法により作製したpcDNA3.1-hGPR20導入293T細胞懸濁液を遠心し、上清を除去した後、1)-8で調製した内在化活性を有するラット抗ヒトGPR20モノクローナル抗体19種(クローン番号は、04-002、04-006、04-013、04-014、04-021、04-037、04-046、04-047、04-067、04-068、04-079、04-114、04-115、04-125、04-126、04-127、04-133、04-139及び04-163)及びラット抗ヒトGPR20モノクローナル抗体2種(13-024、13-048)、又はラットIgGコントロール(R&D Systems社)を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、5% FBS含有PBSで320倍に希釈したGoat Anti-Rat IgG(H+L), PE conjugate(Beckman Coulter社)を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、フローサイトメーター(FC500:Beckman Coulter社)で検出を行った。結果を図19に示す。図19において横軸は抗体濃度(nM)、縦軸はMFI(mean fluorescence intensity)による抗体結合量を示す。図19に示す通り、ラット抗ヒトGPR20抗体はいずれも、pcDNA3.1-hGPR20導入293T細胞に対して濃度依存的に結合量が増加した。一方、ラットIgG2a及びIgG2bアイソタイプコントロール抗体は、GPR20結合性を示さなかった。
精製したラット抗GPR20抗体の内在化活性は、1)-6と同様にタンパク質合成を阻害する毒素(サポリン)を結合させた抗ラットIgG抗体試薬Rat-ZAP(ADVANCED TARGETING SYSTEMS)を用いて評価した。すなわち、ヒトGPR20のC末端側にEGFPをつないだGPR20-EGFPタンパク質を安定発現するHEK293細胞を2.5x103cells/wellで播種して37℃、5% CO2の条件下で一晩培養後、ラット抗GPR20抗体(終濃度:0.012から1μg/mL)とRat-ZAP(終濃度:0.5μg/mL)を添加し、5日間培養後に生存細胞数をCellTiter-Glo(登録商標) Luminescent Cell Viability AssayによるATPの定量で測定した。抗GPR20抗体添加による細胞増殖抑制作用は、抗体を加えなかった時の生存細胞数を100%とした相対活性で表記した。各抗GPR20抗体は終濃度0.11又は0.33μg/mLで添加した際に最も細胞増殖を抑制した。図20に各抗体の最も細胞増殖を抑制した濃度における細胞生存率を示す。本実験において内在化活性が強い抗体は低い細胞生存率を示すと考えられる。ラットIgG2a及びIgG2bアイソタイプコントロール抗体(R&D Systems社)を、GPR20とは無関係な抗原を認識する陰性コントロール抗体として使用した。
実施例2で内在化活性を評価した21種のラット抗GPR20抗体(04-002、04-006、04-013、04-014、04-021、04-037、04-046、04-047、04-067、04-068、04-079、04-114、04-115、04-125、04-126、04-127、04-133、04-139、04-163、13-024、13-048)の可変領域をコードするcDNAのヌクレオチド配列は、以下に方法により決定した。
抗GPR20抗体産生ハイブリドーマの細胞溶解液(50mM Tris-HCl(pH7.5)、250mM LiCl、5mM EDTA(pH8)、0.5%ドデシル硫酸Li(LiDS)、2.5mM dithiothreitol(DTT))を、オリゴdT25が結合した磁気ビーズ(Dynabeads mRNA DIRECT Kit、Life Technologies社)と混合し、mRNAを磁気ビーズに結合させた。次に磁気ビーズをmRNA洗浄溶液A(10mM Tris-HCl(pH7.5)、0.15M LiCl、1mM EDTA、0.1% LiDS、0.1% TritonX-100)とcDNA合成用溶液(50mM Tris-HCl(pH8.3)、75mM KCl、3mM MgCl2、5mM DTT、0.5mM dNTP、0.2% TritonX-100、1.2unit RNase inhibitor(Life Technologies社)で1回ずつ洗浄した後、12unit SuperScriptIII Reverse Transcriptase(Life Technologies社)を加えたcDNA合成用溶液でcDNA合成を行った。続いて3’テーリング反応溶液(50mM リン酸カリウム、4mM MgCl2、0.5mM dGTP、0.2% TritonX-100、1.2unit RNase inhibitor(Life Technologies社))で洗浄した後、48unit Terminal Transferase, recombinant(Roche社)を加えた反応溶液で3’テーリング反応を行った。
磁気ビーズをTE溶液(10mM Tris-HCl(pH7.5)、1mM EDTA、0.1% TritonX-100)にて洗浄後、5’-RACE PCR法を用いてラット免疫グロブリン重鎖及び軽鎖遺伝子の増幅を行った。即ち、磁気ビーズをPCR反応溶液(0.2μM プライマー、0.2mM dNTP、0.25unit PrimeSTAR HS DNA Polymerase(TAKARA社))に移し、94℃30秒-68℃90秒の反応を35サイクル行った。用いたプライマーセットは下記の通り。プライマー配列中におけるDはA、G、Tからなる混合塩基、NはA、C、G、Tの混合塩基であることを示している。
5’-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCDN-3’(Nhe-polyC-S)(配列番号66)
5’-TCACTGAGCTGGTGAGAGTGTAGAGCCC-3’(rIgγ-AS1)(配列番号67)
5’-TCACCGAGCTGCTGAGGGTGTAGAGCCC-3’(rIgγ-AS2)(配列番号68)
PCRプライマーセット(軽鎖用)
5’-GCTAGCGCTACCGGACTCAGATCCCCCCCCCCCCCDN-3’(Nhe-polyC-S)(配列番号66)(重鎖用と同じ)
5’-TCAGTAACACTGTCCAGGACACCATCTC-3’(rIgκ-AS)(配列番号69)
シークエンス解析は遺伝子配列解析装置(「ABI PRISM 3700 DNA Analyzer;Applied Biosystems」あるいは「Applied Biosystems 3730xl Analyzer;Applied Biosystems」)を用いて実施し、シークエンス反応は、Dye Terminator Cycle Sequencing System with AmpliTaq DNA polymerase(Life Technologies社)及びGeneAmp 9700(Applied Biosystems社)を用いた。
グループA(04-002、04-006、04-013、04-014、04-037、04-046、04-047、04-067、04-068、04-079、04-114、04-125、04-126、04-127、04-133、04-139、04-163)、グループB(04-021、04-115)、グループC(13-024、13-048)。各抗体の重鎖及び軽鎖の全長配列は、公知の定常領域の配列とつなぎ合わせることにより決定された。ラットの重鎖IgG2bの定常領域の塩基配列とアミノ酸配列はIMGT, the international ImMunoGeneTics information system(登録商標)に掲載されているAABR03048905(IGHG2B*01)の塩基配列とアミノ酸配列を参照した。また、ラットの軽鎖IgKの定常領域の塩基配列とアミノ酸配列は同システムに掲載されているV01241(IGKC*01)の塩基配列とアミノ酸配列を参照した。
マウスGPR20、N末端にFLAGタグを付加したヒトGPR20、ヒトGPR20の4つの細胞外領域(EC1、EC2、EC3、EC4)各々をマウスGPR20由来の配列に置換したヒト/マウスキメラGPR20に対する結合性をCell-ELISA法で測定することにより、抗ヒトGPR20抗体の結合部位の分類を行った。図21はヒトGPR20(NP_005284)、マウスGPR20(NP_775541)のアミノ酸配列の対照図であり、4つの細胞外領域EC1からEC4はヒトGPR20のアミノ酸配列番号で表されており、EC1は1-48、EC2は108-125、EC3は190-196、EC4は260-275であることを示す。
当業者に公知な方法に従いマウスGPR20のRef Seq配列NM_173365を基にcDNAを人工合成してpcDNA-DEST40発現ベクター(Invitrogen社)にクローニングすることで、マウスGPR20発現ベクターpcDNA-mGPR20を構築した。
4)-2-1
以下の発現ベクター構築においては、実施例1で作製した全長ヒトGPR20発現ベクターpcDNA3.1-hGPR20を鋳型とし、以下のプライマーセットを用いて各々PCR反応を行った。この反応にはKOD FX DNAポリメラーゼ(東洋紡社)を用い、98℃-10秒、58℃-30秒、68℃-7分、15サイクル又は10サイクルで反応を行った後、得られたPCR産物を制限酵素DpnIで処理した。このDpnI消化したDNAを大腸菌TOP10(Invitrogen社)に形質転換することで、N末端にFLAGタグを付加したヒトGPR20並びにEC2又はEC3又はEC4をマウスGPR20由来配列に置換したヒトGPR20の発現ベクターを構築した。各PCRに用いたプライマーセットは以下の通り。
5’―GACTACAAAGACGATGACGACAAGCCCTCTGTGTCTCCAGC―3’(NFLAG-1; 配列番号72)
5’―CTTGTCGTCATCGTCTTTGTAGTCCATGGTGGAGCCTGC―3’(NFLAG-2; 配列番号73)
PCRプライマーセット(EC2をマウスGPR20由来配列に置換したヒトGPR20用)
5’―ACGCGCTTCGCTGTGTTCTACGGCGCCAG―3’(mEC2-1; 配列番号74)
5’―CTGGCGCCGTAGAACACAGCGAAGCGCGT―3’(mEC2-2; 配列番号75)
PCRプライマーセット(EC3をマウスGPR20由来配列に置換したヒトGPR20用)
5’―TGTCGGTGCTGGGCGTGAAGTCGGGTGGACGATCATGCTGCCGTGTCTT―3’(mEC3-1;配列番号76)
5’―AAGACACGGCAGCATGATCGTCCACCCGACTTCACGCCCAGCACCGACA―3’(mEC3-2;配列番号77)
PCRプライマーセット(EC4をマウスGPR20由来配列に置換したヒトGPR20用)
5’―TGGCGCTGTGGCCCAACGTACCTAAGCACACGAGCCTCGTGGT―3’(mEC4-1;配列番号78)
5’―ACCACGAGGCTCGTGTGCTTAGGTACGTTGGGCCACAGCGCCA―3’(mEC4-2;配列番号79)
EC1をマウスGPR20由来配列に置換したヒトGPR20発現ベクター構築は、In-Fusion(登録商標) HD Cloning Kit(CLONTECH社)を用いて行った。即ち、実施例1で作製した全長ヒトGPR20発現ベクターpcDNA3.1-hGPR20を鋳型とし、以下のプライマーセットとKOD FX DNAポリメラーゼ(東洋紡社)を用いて、98℃-10秒、58℃-30秒、68℃-7分、10サイクルでPCR反応を行い、ベクター配列を含むDNA断片を増幅した。
PCRプライマーセット(EC1をマウスGPR20由来配列に置換したヒトGPR20用1)
5’―GCGCTGATGGCGGTGCACGGAGCCATCT―3’(mEC1-1;配列番号80)
5’―AGAGGGCATGGTGGAGCCTGCTTT―3’(mEC1-2;配列番号81)
PCRプライマーセット(EC1をマウスGPR20由来配列に置換したヒトGPR20用2)
5’―AGGCTCCACCATGCCCTCTGCGTTGTCTATGA―3’(mEC1-3;配列番号82)
5’―CACCGCCATCAGCGCTTGCCACAGGCTGGGGAAGGTGGCTTGCA―3’(mEC1-4;配列番号83)
上記2種のDNA断片を定法に従いアガロースゲル電気泳動し、目的サイズのDNA断片をQIAquick Gel Extraction Kit(プロメガ社)を用いて単離した。これら2つのDNA断片をIn-Fusion HD酵素(Clontech社)の反応により連結し、大腸菌TOP10(Invitrogen社)に形質転換することで、EC1をマウスGPR20由来配列に置換したヒトGPR20発現ベクターを構築した。
実施例1で示したCell-ELISA法と同様の方法で、各種GPR20発現ベクターを一過性に導入した293α細胞に対する抗GPR20抗体04-046、04-079、04-126、04-021、13-024、及び13-048の結合活性を調べた。測定結果を図22(a)及び図22(b)に示す。図22の横軸のEVはコントロール、human GPR20はヒト全長GPR20が発現した細胞、FLAG-huGPR20はN末端にFLAGタグを付加したヒトGPR20が発現した細胞、mouse GPR20はマウスGPR20が発現した細胞、hGPR20_mECD1はヒトGPR20のECD1をマウスのGPR20のECD1で置換したキメラGPR20が発現した細胞、、hGPR20_mECD2はヒトGPR20のECD2をマウスのGPR20のECD2で置換したキメラGPR20が発現した細胞、hGPR20_mECD3はヒトGPR20のECD3をマウスのGPR20のECD3で置換したキメラGPR20が発現した細胞、hGPR20_mECD4はヒトGPR20のECD4をマウスのGPR20のECD4で置換したキメラGPR20が発現した細胞を示す。
5)-1 ヒトキメラ化抗GPR20抗体の発現ベクターの構築
5)-1-1キメラ及びヒト化抗体軽鎖発現ベクターpCMA-LKの構築
プラスミドpcDNA3.3-TOPO/LacZ(Invitrogen社)を制限酵素XbaI及びPmeIで消化して得られる約5.4kbのフラグメントと、配列番号84に示すヒトκ鎖分泌シグナル及びヒトκ鎖定常領域をコードするDNA配列を含むDNA断片を、In-Fusion Advantage PCRクローニングキット(Clontech社)を用いて結合して、pcDNA3.3/LKを作製した。
プライマーセット
5’-TATACCGTCGACCTCTAGCTAGAGCTTGGC-3’(3.3-F1;配列番号85)
5’-GCTATGGCAGGGCCTGCCGCCCCGACGTTG-3’(3.3-R1;配列番号86)
pCMA-LKをXbaI及びPmeIで消化してκ鎖分泌シグナル及びヒトκ鎖定常領域を取り除いたDNA断片と、配列番号87に示すヒト重鎖シグナル配列及びヒトIgG1定常領域のアミノ酸をコードするDNA配列を含むDNA断片を、In-Fusion Advantage PCRクローニングキット(Clontech社)を用いて結合して、CMVプロモーターの下流にシグナル配列、クローニングサイト、ヒトIgG1重鎖定常領域をもつキメラ及びヒト化抗体IgG1タイプ重鎖発現ベクターpCMA-G1を構築した。
実施例3)-2で決定した配列番号3のラット抗GPR20抗体04-046の重鎖の可変領域のアミノ酸配列を基にヒトキメラ化抗体重鎖の発現ベクターを構築した。配列番号46に示すヒトキメラ化抗GPR20抗体04-046Chの重鎖のヌクレオチド配列のうち、可変領域をコードするDNA配列を含むヌクレオチド番号36乃至443に相当するDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をテンプレートとして、KOD-Plus-(TOYOBO社)と下記のプライマーセットでヒトキメラ化抗GPR20抗体の重鎖の可変領域をコードするDNA配列を含むDNA断片を増幅し、キメラ及びヒト化抗体IgG1タイプ重鎖発現ベクターpCMA-G1を制限酵素BlpIで切断した箇所にIn-Fusion HD PCRクローニングキット(Clontech社)を用いて挿入することによりヒトキメラ化抗GPR20抗体04-046Chの重鎖発現ベクターを構築した。得られた発現ベクターを「pCMA/04-046Ch-H」と命名した。ヒトキメラ化抗GPR20抗体04-046Chの重鎖のアミノ酸配列を配列番号44に示した。配列番号46のヌクレオチド配列及び配列番号44のアミノ酸配列は、図7にも記載されている。
プライマーセット
5’-AGCTCCCAGATGGGTGCTGAGC-3’(EG-Inf-F;配列番号88)
5’-GGGCCCTTGGTGGAGGCTGAGC-3’(EG1-Inf-R;配列番号89)
実施例3)-2で決定した配列番号8のラット抗GPR20抗体04-046の軽鎖の可変領域のアミノ酸配列を基にヒトキメラ化抗体軽鎖の発現ベクターを構築した。配列番号47に示すヒトキメラ化抗GPR20抗体04-046Chの軽鎖のヌクレオチド配列のうち、可変領域をコードするDNA配列を含むヌクレオチド番号38乃至399に相当するDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をテンプレートとして、KOD-Plus-(TOYOBO社)と下記のプライマーセットでヒトキメラ化抗GPR20抗体の軽鎖の可変領域をコードするDNA配列を含むDNA断片を増幅し、キメラ及びヒト化抗体軽鎖発現ベクターpCMA-LKを制限酵素BsiWIで切断した箇所にIn-Fusion HD PCRクローニングキット(Clontech社)を用いて挿入することによりヒトキメラ化抗GPR20抗体04-046Chの軽鎖発現ベクターを構築した。得られた発現ベクターを「pCMA/04-046Ch-L」と命名した。ヒトキメラ化抗GPR20抗体04-046Chの軽鎖のアミノ酸配列を配列番号45に示した。配列番号47のヌクレオチド配列及び配列番号45のアミノ酸配列は、図8にも記載されている。
プライマーセット
5’-CTGTGGATCTCCGGCGCGTACGGC-3’(CM-LKF;配列番号90)
5’-GGAGGGGGCGGCCACGGCTCTCTTCAGTTC-3’(046L-R;配列番号91)
5)-2 ヒトキメラ化抗GPR20抗体の発現、精製
5)-2-1 ヒトキメラ化抗GPR20抗体の発現
FreeStyle 293F細胞(Invitrogen社)はマニュアルに従い、継代、培養を行った。対数増殖期の1.2×109個のFreeStyle 293F細胞(Invitrogen社)を3L Fernbach Erlenmeyer Flask(CORNING社)に播種し、FreeStyle293 expression medium (Invitrogen社)で希釈して2.0×106細胞/mLに調製した後に、37℃、8%CO2インキュベーター内で90rpmで1時間振とう培養した。Polyethyleneimine(Polyscience #24765)1.8mgをOpti-Pro SFM培地(Invitrogen社)20mLに溶解し、次にNucleoBond Xtra(TaKaRa社)を用いて調製した重鎖発現ベクター(0.24mg)及び軽鎖発現ベクター(0.36mg)を20mLのOpti-Pro SFM培地(Invitrogen社)に添加した。Polyethyleneimine/Opti-Pro SFM混合液20mLに、発現ベクター/Opti-Pro SFM混合液20mLを加え穏やかに攪拌し、さらに5分間放置した後にFreeStyle 293F細胞に添加した。37℃、8%CO2インキュベーターで4時間、90rpmで振とう培養後に600mLのEX-CELL VPRO培地(SAFC Biosciences社)、18mLのGlutaMAX I(GIBCO社)、及び30mLのYeastolate Ultrafiltrate(GIBCO社)を添加し、37℃、8%CO2インキュベーターで7日間、90rpmで振とう培養して得られた培養上清をDisposable Capsule Filter (Advantec #CCS-045-E1H)でろ過した。
実施例5)-2-1で得られた培養上清から抗体をrProtein Aアフィニティークロマトグラフィー(4-6℃下)とセラミックハイドロキシアパタイト(室温下)の二段階工程で精製した。rProtein Aアフィニティークロマトグラフィー精製後とセラミックハイドロキシアパタイト精製後のバッファー置換工程は4-6℃下で実施した。PBSで平衡化したMabSelectSuRe(GE Healthcare Bioscience社、HiTrapカラム)に培養上清をアプライした。培養上清がカラムに全て入ったのち、カラム容量2倍以上のPBSでカラムを洗浄した。次に2Mアルギニン塩酸塩溶液(pH4.0)で溶出し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientific社、Slide-A-Lyzer Dialysis Cassette)によりPBSに置換した後、5mMリン酸ナトリウム/50mM MES/pH7.0のバッファーで5倍希釈した抗体溶液を、5mM NaPi/50mM MES/30mM NaCl/pH7.0のバッファーで平衡化されたセラミックハイドロキシアパタイトカラム(日本バイオラッド、Bio-Scale CHT Type―1 Hydroxyapatite Column)にアプライした。塩化ナトリウムによる直線的濃度勾配溶出を実施し、抗体の含まれる画分を集めた。その画分を透析(Thermo Scientificsha,Slide-A-Lyzer Dialysis Cassette)によりHBSor(25mM ヒスチジン/5% ソルビトール、pH6.0)への液置換を行った。Centrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社、4℃下)にて濃縮し、IgG濃度を20mg/mL以上に調製した。最後にMinisart-Plus filter(Sartorius社)でろ過し、精製サンプルとした。
作製したヒトキメラ化抗GPR20抗体04-046ChのGPR20結合性をフローサイトメトリー法により確認した。実施例1)-5-1と同様の方法でpcDNA3.1-hGPR20、又はpcDNA3.1をそれぞれ293T細胞にLipofectamine 2000を用いて一過性に導入し、37℃、5% CO2の条件下で一晩培養した後、細胞懸濁液を調製した。これらの細胞懸濁液にヒトキメラ化抗GPR20抗体04-046Ch又は陰性コントロールとしてヒトIgGを加えて4℃で1時間静置した後、5% FBS含有PBSで2回洗浄し、5% FBS含有PBSで320倍に希釈したPE標識F(ab’)2 Fragment抗ヒトIgG, Fcγ抗体(JACKSON IMMUNORESEARC社)を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、5% FBS含有PBSに再懸濁し、フローサイトメーター(FC500:BeckmanCoulter社)で検出を行った。データ解析はFlowjo(TreeStar社製)で行った。04-046Chは陰性コントロールであるpcDNA3.1導入293T細胞には結合せず(図23(b))、pcDNA3.1-hGPR20導入293T細胞に抗体濃度依存的に結合した(図23(a))。図23において横軸は抗体濃度を示し、縦軸は結合量をMeanFluorescent Intensity(平均蛍光強度)で示す。
6)-1 抗GPR20抗体04-046のヒト化体デザイン
6)-1-1 04-046の可変領域の分子モデリング
04-046の可変領域の分子モデリングは、ホモロジーモデリングとして公知の方法(Methods in Enzymology,203,121-153,(1991))によって実行した。Protein Data Bank(Nuc.Acid Res.35,D301-D303(2007))に登録されているヒト免疫グロブリンの可変領域の1次配列(X線結晶構造から誘導される三次元構造が入手可能である)を、実施例3)-2で決定された04-046の可変領域と比較した。1SY6と1LK3が04-046の重鎖と軽鎖の可変領域に対して最も高い配列同一性を有する配列として選択された。フレームワーク領域の三次元構造は、04-046の重鎖及び軽鎖に対応する1SY6と1LK3の座標を組み合わせて、「フレームワークモデル」を得ることによって作製された。次いで、それぞれのCDRについての代表的なコンホメーションがフレームワークモデルに組み込まれた。最後に、エネルギーの点で046の可変領域の可能性のある分子モデルを得るために、不利な原子間接触を除くためのエネルギー計算を行った。上記手順を、市販のタンパク質立体構造解析プログラムDiscovery Studio(Accelrys社)を用いて行った。
04-046をヒト化した抗体(以下、「ヒト化h046」という。)の構築を、CDRグラフティング(Proc.Natl.Acad.Sci.USA 86,10029-10033(1989))として一般的に公知の方法によって行った。アクセプター抗体は、フレームワーク領域内のアミノ酸同一性に基づいて選択された。
04-046のフレームワーク領域の配列を、KABAT et al. (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service National Institutes of Health, Bethesda,MD.(1991))において既定されるヒトのサブグループ・コンセンサス配列のフレームワーク領域と比較した。重鎖についてはヒトγ鎖サブグループ1のコンセンサス配列が、軽鎖についてはヒトκ鎖サブグループ1のコンセンサス配列が、そのフレームワーク領域において高い配列同一性を有することに基づいて、アクセプターとして選択された。アクセプターについてのフレームワーク領域のアミノ酸残基を、04-046についてのアミノ酸残基と整列させ、異なるアミノ酸が使用される位置を同定した。これらの残基の位置は、上で構築された04-046の三次元モデルを使用して分析され、そしてアクセプター上にグラフティングされるべきドナー残基が、Queen et al.(Proc.Natl.Acad.Sci.USA 86,10029-10033(1989))によって与えられる基準によって選択された。選択された幾つかのドナー残基をアクセプター抗体に移入することによって、ヒト化h046の配列を以下の実施例に記載されるように構築した。
6)-2-1 ヒト化h046-H4b タイプ重鎖
配列番号44に示されるヒトキメラ化抗体04-046Chの重鎖のうち、可変領域部分(配列番号44に示されるアミノ酸配列中の20乃至142番目のアミノ酸残基からなるアミノ酸配列)配列番号44におけるアミノ酸番号24のグルタミンをバリンに、30番のロイシンをバリンに、31番のアラニンをリジンに、35番のセリンをアラニンに、39番のイソロイシンをバリンに、57番のリジンをアルギニンに、40番59番)のスレオニンをアラニンに、60番)のスレオニンをプロリンに、67番のイソロイシンをメチオニンに、86番のリジンをアルギニンに、87番のアラニンをバリンに、89番のロイシンをイソロイシンに、91番のバリンをアラニンに、99番のフェニルアラニンをスレオニンに、101番のグルタミンをグルタミン酸に、106番のスレオニンをアルギニンに、107番のプロリンをセリンに、108番のアスパラギン酸をグルタミン酸に、120番のセリンをスレオニンに、122番のイソロイシンをバリンに、136番のバリンをスレオニンに、137番のメチオニンをロイシンに、置き換えることを伴い設計されたヒト化h046重鎖を「ヒト化h046-H4b タイプ重鎖」(「h046-H4b」と呼ぶこともある)と命名した。
配列番号44に示されるヒトキメラ化抗体04-046Chの重鎖のうち可変領域部分の配列番号44の20番のグルタミンをグルタミン酸に、配24番のグルタミンをバリンに、30番のロイシンをバリンに、31番のアラニンをリジンに、35番のセリンをアラニンに、39番のイソロイシンをバリンに、57番のリジンをアルギニンに、59番のスレオニンをアラニンに、60番のスレオニンをプロリンに、67番のイソロイシンをメチオニンに、86番のリジンをアルギニンに、68番87番のアラニンをバリンに、89番のロイシンをイソロイシンに、91番のバリンをアラニンに、99番のフェニルアラニンをスレオニンに、101番目のグルタミンをグルタミン酸に、106番)のスレオニンをアルギニンに、107番)のプロリンをセリンに、108番のアスパラギン酸をグルタミン酸に、120番のセリンをスレオニンに、122番のイソロイシンをバリンに、136番のバリンをスレオニンに、137番のメチオニンをロイシンに、それぞれ置き換えることを伴い設計されたヒト化h046重鎖を「ヒト化h046-H4e タイプ重鎖」(「h046-H4e」と呼ぶこともある)と命名した。
ヒト化h046-H4e タイプ重鎖のアミノ酸配列は、配列表の配列番号50に記載されている。配列番号50のアミノ酸配列の1乃至19番目のアミノ酸残基からなる配列はシグナル配列、20乃至142番目のアミノ酸残基からなる配列は重鎖可変領域、143乃至472番目のアミノ酸残基からなる配列は重鎖定常領域を示す。配列番号50のアミノ酸配列をコードするヌクレオチド配列は、配列表の配列番号51に記載されている。配列番号51のヌクレオチド配列の1乃至57番目のヌクレオチドからなる配列はシグナル配列、58乃至426番目のヌクレオチドからなる配列は重鎖可変領域の配列、427乃至1416番目のヌクレオチドからなる配列は重鎖定常領域の配列をコードしている。配列番号50のアミノ酸配列は、図10にも記載されている。
配列番号44に示されるヒトキメラ化抗体04-046Chの重鎖のうち、可変領域部分の配列番号44の24番のグルタミンをバリンに、30番のロイシンをバリンに、31番のアラニンをリジンに、35番のセリンをアラニンに、39番のイソロイシンをバリンに、57番のリジンをアルギニンに、59番のスレオニンをアラニンに、60番のスレオニンをプロリンに、67番のイソロイシンをメチオニンに、86番のリジンをアルギニンに、87番のアラニンをバリンに、89番のロイシンをイソロイシンに、91番のバリンをアラニンに、99番のフェニルアラニンをアスパラギンに、101番のグルタミンをグルタミン酸に、106番)のスレオニンをアルギニンに、107番のプロリンをセリンに、108番のアスパラギン酸をグルタミン酸に、120番のセリンをスレオニンに、122番のイソロイシンをバリンに、136番)のバリンをスレオニンに、137番ののメチオニンをロイシンに、それぞれ置き換えることを伴い設計されたヒト化h046重鎖を「ヒト化h046-H5b タイプ重鎖」(「h046-H5b」と呼ぶこともある)と命名した。
ヒト化h046-H5b タイプ重鎖のアミノ酸配列は、配列表の配列番号52に記載されている。配列番号52のアミノ酸配列の1乃至19番目のアミノ酸残基からなる配列はシグナル配列、20乃至142番目のアミノ酸残基からなる配列は重鎖可変領域、143乃至472番目のアミノ酸残基からなる配列は重鎖定常領域を示す。配列番号52のアミノ酸配列をコードするヌクレオチド配列は、配列表の配列番号53に記載されている。配列番号53のヌクレオチド配列の1乃至57番目のヌクレオチドからなる配列はシグナル配列、58乃至426番目のヌクレオチドからなる配列は重鎖可変領域配列、427乃至1416番目のヌクレオチドからなる配列は重鎖定常領域配列をコードしている。配列番号52のアミノ酸配列は、図11にも記載されている。
配列番号44に示されるヒトキメラ化抗体04-046Chの重鎖のうち可変領域部分のアミノ酸番号80目のフェニルアラニンをアスパラギンに、置き換えることを伴い設計されたヒト化h046重鎖を「ヒト化h046-H8 タイプ重鎖」(「h046-H8」と呼ぶこともある)と命名した。
ヒト化h046-H8 タイプ重鎖のアミノ酸配列は、配列表の配列番号54に記載されている。配列番号54のアミノ酸配列の1乃至19番目のアミノ酸残基からなる配列、20乃至142番目のアミノ酸残基からなる配列、143乃至472番目のアミノ酸残基からなる配列が、それぞれシグナル配列、重鎖可変領域、重鎖定常領域に相当する。配列番号54のアミノ酸配列をコードするヌクレオチド配列は、配列表の配列番号55に記載されている。配列番号55のヌクレオチド配列の1乃至57番目のヌクレオチドからなる配列、58乃至426番目のヌクレオチドからなる配列、427乃至1416番目のヌクレオチドからなる配列が、それぞれシグナル配列、重鎖可変領域配列、重鎖定常領域配列をコードしている。配列番号54のアミノ酸配列は、図12にも記載されている。
配列番号44に示されるヒトキメラ化抗体04-046Chの重鎖のうち可変領域部分の配列番号44の39番のイソロイシンをバリンに、99番のフェニルアラニンをアスパラギンに、それぞれ置き換えることを伴い設計されたヒト化h046重鎖を「ヒト化h046-H10 タイプ重鎖」(「h046-H10」と呼ぶこともある)と命名した。
6)-3-1 ヒト化h046-L1タイプ軽鎖
配列番号45に示されるヒトキメラ化抗体04-046Chの軽鎖のうち可変領域部分の配列番号45の22番のスレオニンをイソロイシンに、23番のバリンをグルタミンに、24番のロイシンをメチオニンに、29番のアラニンをセリンに、31番のアラニンをセリンに、32番のバリンをアラニンに、34番のロイシンをバリンに、36番のグルタミンをアスパラギン酸に、41番のセリンをスレオニンに、58番のアルギニンをリジンに、59番のセリンをプロリンに、61番のグルタミンをリジンに、62番のグルタミンをアラニンに、95番のアスパラギン酸をセリンに、96番のプロリンをセリンに、97番のバリンをロイシンに、98番のグルタミン酸をグルタミンに、100番)のアスパラギン酸をグルタミン酸に、102番のイソロイシンをフェニルアラニンに、104番のアスパラギンをスレオニンに、119番のアラニンをグルタミンに、123番のロイシンをバリンに、125番のロイシンをイソロイシンに、128番のアラニンをスレオニンに置き換え、29番と30番の間にセリンを挿入することを伴い設計されたヒト化h046軽鎖を「ヒト化h046-L1タイプ軽鎖」(「h046-L1)」と呼ぶこともある)と命名した。
ヒト化h046-L1タイプ軽鎖のアミノ酸配列は、配列表の配列番号58に記載されている。配列番号58のアミノ酸配列の1乃至20番目のアミノ酸残基からなる配列はシグナル配列、21乃至129番目のアミノ酸残基からなる配列は軽鎖可変領域、130乃至234番目のアミノ酸残基からなる配列は軽鎖定常領域を示す。配列番号58のアミノ酸配列をコードするヌクレオチド配列は、配列表の配列番号59に記載されている。配列番号58のヌクレオチド配列の1乃至60番目のヌクレオチドからなる配列はシグナル配列、61乃至387番目のヌクレオチドからなる配列は軽鎖可変領域配列、388乃至702番目のヌクレオチドからなる配列は軽鎖定常領域配列をコードしている。配列番号58のアミノ酸配列は、図14にも記載されている。
配列番号45に示されるヒトキメラ化抗体04-046Chの軽鎖のうち可変領域部分の配列番号45の22番のスレオニンをイソロイシンに、23番のバリンをグルタミンに、24番のロイシンをメチオニンに、29番のアラニンをセリンに、21番のアラニンをセリンに、32番)のバリンをアラニンに、34番目のロイシンをバリンに、36番のグルタミンをアスパラギン酸に、41番のセリンをスレオニンに、58番のアルギニンをリジンに、59番のセリンをプロリンに、61番のグルタミンをリジンに、95番のアスパラギン酸をセリンに、96番のプロリンをセリンに、97番のバリンをロイシンに、98番のグルタミン酸をグルタミンに、100番のアスパラギン酸をグルタミン酸に、102番のイソロイシンをフェニルアラニンに、104番のアスパラギンをスレオニンに、119番のアラニンをグルタミンに、123番のロイシンをバリンに、125番のロイシンをイソロイシンに、128番のアラニンをスレオニンに置き換え、29番目と30番目の間にセリンを挿入することを伴い設計されたヒト化h046軽鎖を「ヒト化h046-L2タイプ軽鎖」(「h046-L2)」と呼ぶこともある)と命名した。
ヒト化h046-L2タイプ軽鎖のアミノ酸配列は、配列表の配列番号60に記載されている。配列番号60のアミノ酸配列の1乃至20番目のアミノ酸残基からなる配列はシグナル配列、21乃至129番目のアミノ酸残基からなる配列は軽鎖可変領域、130乃至234番目のアミノ酸残基からなる配列は軽鎖定常領域に相当する。配列番号60のアミノ酸配列をコードするヌクレオチド配列は、配列表の配列番号61記載されている。配列番号61のヌクレオチド配列の1乃至60番目のヌクレオチドからなる配列はシグナル配列、61乃至387番目のヌクレオチドからなる配列は軽鎖可変領域配列、388乃至702番目のヌクレオチドからなる配列は軽鎖定常領域配列をコードしている。配列番号60のアミノ酸配列は、図15にも記載されている。
配列番号45に示されるヒトキメラ化抗体04-046Chの軽鎖のうち可変領域部分の配列番号45の23番のバリンをグルタミンに、29番のアラニンをセリンに、31番のアラニンをセリンに、32番のバリンをアラニンに、34番のロイシンをバリンに、36番のグルタミンをアスパラギン酸に、41番のセリンをスレオニンに、58番のアルギニンをリジンに、59番のセリンをプロリンに、61番のグルタミンをリジンに、72番のアスパラギンをアスパラギン酸に、73番のロイシンをアルギニンに、95番のアスパラギン酸をセリンに、96番のプロリンをセリンに、97番のバリンをロイシンに、98番のグルタミン酸をグルタミンに、100番のアスパラギン酸をグルタミン酸に、102番のイソロイシンをフェニルアラニンに、119番のアラニンをグルタミンに、123番のロイシンをバリンに、125番のロイシンをイソロイシンに、128番のアラニンをスレオニンに置き換え、29番目と30番目の間にセリンを挿入することを伴い設計されたヒト化h046軽鎖を「ヒト化h046-L6タイプ軽鎖」(「h046-L6)」と呼ぶこともある)と命名した。
配列番号62のアミノ酸配列は、図16にも記載されている。 6)-3-4 ヒト化h046-L7タイプ軽鎖
配列番号45に示されるヒトキメラ化抗体04-046Chの軽鎖のうち可変領域部分の23番のバリンをグルタミンに、29番のアラニンをセリンに、31番のアラニンをセリンに、32番のバリンをアラニンに、34番のロイシンをバリンに、36番のグルタミンをアスパラギン酸に、41番のセリンをスレオニンに、58番のアルギニンをリジンに、59番のセリンをプロリンに、61番のグルタミンをリジンに、71番のセリンをグリシンに、95番のアスパラギン酸をセリンに、96番のプロリンをセリンに、97番のバリンをロイシンに、98番のグルタミン酸をグルタミンに、100番のアスパラギン酸をグルタミン酸に、102番のイソロイシンをフェニルアラニンに、119番のアラニンをグルタミンに、123番のロイシンをバリンに、125番のロイシンをイソロイシンに、128番のアラニンをスレオニンに置き換え、29番目と30番目の間にセリンを挿入することを伴い設計されたヒト化h046軽鎖を「ヒト化h046-L7タイプ軽鎖」(「h046-L7)」と呼ぶこともある)と命名した。
配列番号64のアミノ酸配列は、図17にも記載されている。
ヒト化h046-H4e タイプ重鎖及びヒト化h046-L1タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4e/L1」(「h046-H4e/L1」と呼ぶこともある)と命名した。ヒト化h046-H4e タイプ重鎖及びヒト化h046-L2タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4e/L2」(「h046-H4e/L2」と呼ぶこともある)と命名した。ヒト化h046-H4e タイプ重鎖及びヒト化h046-L6タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4e/L6」(「h046-H4e/L6」と呼ぶこともある)と命名した。ヒト化h046-H4e タイプ重鎖及びヒト化h046-L7タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4e/L7」(「h046-H4e/L7」と呼ぶこともある)と命名した。ヒト化h046-H4b タイプ重鎖及びヒト化h046-L1タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4b/L1」(「h046-H4b/L1」と呼ぶこともある)と命名した。ヒト化h046-H4b タイプ重鎖及びヒト化h046-L2タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4b/L2」(「h046-H4b/L2」と呼ぶこともある)と命名した。ヒト化h046-H4b タイプ重鎖及びヒト化h046-L6タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4b/L6」(「h046-H4b/L6」と呼ぶこともある)と命名した。ヒト化h046-H4b タイプ重鎖及びヒト化h046-L7タイプ軽鎖からなる抗体を設計し「ヒト化h046-H4b/L7」(「h046-H4b/L7」と呼ぶこともある)と命名した。ヒト化h046-H5b タイプ重鎖及びヒト化h046-L1タイプ軽鎖からなる抗体を設計し「ヒト化h046-H5b/L1」(「h046-H5b/L1」と呼ぶこともある)と命名した。ヒト化h046-H5b タイプ重鎖及びヒト化h046-L2タイプ軽鎖からなる抗体を設計し「ヒト化h046-H5b/L2」(「h046-H5b/L2」と呼ぶこともある)と命名した。ヒト化h046-H5b タイプ重鎖及びヒト化h046-L6タイプ軽鎖からなる抗体を設計し「ヒト化h046-H5b/L6」(「h046-H5b/L6」と呼ぶこともある)と命名した。ヒト化h046-H5b タイプ重鎖及びヒト化h046-L7タイプ軽鎖からなる抗体を設計し「ヒト化h046-H5b/L7」(「h046-H5b/L7」と呼ぶこともある)と命名した。ヒト化h046-H8 タイプ重鎖及びヒト化h046-L1タイプ軽鎖からなる抗体を設計し「ヒト化h046-H8/L1」(「h046-H8/L1」と呼ぶこともある)と命名した。ヒト化h046-H8 タイプ重鎖及びヒト化h046-L2タイプ軽鎖からなる抗体を設計し「ヒト化h046-H8/L2」(「h046-H8/L2」と呼ぶこともある)と命名した。ヒト化h046-H8 タイプ重鎖及びヒト化h046-L6タイプ軽鎖からなる抗体を設計し「ヒト化h046-H8/L6」(「h046-H8/L6」と呼ぶこともある)と命名した。ヒト化h046-H8 タイプ重鎖及びヒト化h046-L7タイプ軽鎖からなる抗体を設計し「ヒト化h046-H8/L7」(「h046-H8/L7」と呼ぶこともある)と命名した。ヒト化h046-H10 タイプ重鎖及びヒト化h046-L1タイプ軽鎖からなる抗体を設計し「ヒト化h046-H10/L1」(「h046-H10/L1」と呼ぶこともある)と命名した。ヒト化h046-H10 タイプ重鎖及びヒト化h046-L2タイプ軽鎖からなる抗体を設計し「ヒト化h046-H10/L2」(「h046-H10/L2」と呼ぶこともある)と命名した。ヒト化h046-H10 タイプ重鎖及びヒト化h046-L6タイプ軽鎖からなる抗体を設計し「ヒト化h046-H10/L6」(「h046-H10/L6」と呼ぶこともある)と命名した。ヒト化h046-H10 タイプ重鎖及びヒト化h046-L7タイプ軽鎖からなる抗体を設計し「ヒト化h046-H10/L7」(「h046-H10/L7」と呼ぶこともある)と命名した。ヒトキメラc046重鎖及びヒト化h046-L1タイプ軽鎖からなる抗体を設計し「ヒトキメラc046/L1」(「h046-Hwt/L1」と呼ぶこともある)と命名した。ヒトキメラc046重鎖及びヒト化h046-L2タイプ軽鎖からなる抗体を設計し「ヒトキメラc046/L2」(「h046-Hwt/L2」と呼ぶこともある)と命名した。ヒトキメラc046重鎖及びヒト化h046-L6タイプ軽鎖からなる抗体を設計し「ヒトキメラc046/L6」(「h046-Hwt/L6」と呼ぶこともある)と命名した。ヒトキメラc046重鎖及びヒト化h046-L7タイプ軽鎖からなる抗体を設計し「ヒトキメラc046/L7」(「h046-Hwt/L7」と呼ぶこともある)と命名した。以上により設計した抗体は、実施例6)-5及び実施例6)-7に準じて作製し、実施例6)-6及び実施例8)、実施例9)に準じて評価することができる。
6)-5-1 ヒト化h046の重鎖発現ベクターの構築
6)-5-1-1 ヒト化h046-H4bタイプ重鎖発現ベクターの構築
配列番号94に示すDNA断片Aを合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を実施例5)-1-3と同様の方法でキメラ及びヒト化抗体IgG1タイプ重鎖発現ベクターpCMA-G1を制限酵素BlpIで切断した箇所に挿入しプラスミドAを構築した。プラスミドAをテンプレートとし、下記プライマーセットとKOD -Plus- Mutagenesis Kit(TOYOBO社)を用いて、変異を導入することによりヒト化h046-H4bタイプ重鎖発現ベクターを構築した。構築した発現ベクターを「pCMA/h046-H4b」と命名した。ヒト化h046-H4bタイプ重鎖のヌクレオチド配列を配列番号49に示し、アミノ酸配列を配列番号48に示した。
プライマーセット:
5’-ATCAACCCTGGCAGCGGCCACACCAACTAC-3’(Hb-F;配列番号95)
5’-GAAGCCCATGTACTTCAGGCCCTGTCCAGGGG-3’(Hb-R;配列番号96)
6)-5-1-2 ヒト化h046-H5bタイプ重鎖発現ベクターの構築
配列番号97に示すDNA断片Bを合成し(GENEART社 人工遺伝子合成サービス)、合成したDNA断片を実施例5)-1-3と同様の方法でキメラ及びヒト化抗体IgG1タイプ重鎖発現ベクターpCMA-G1を制限酵素BlpIで切断した箇所に挿入しプラスミドBを構築した。プラスミドBをテンプレートとし、6)-5-1-1と同様の方法で変異を導入することによりヒト化h046-H5bタイプ重鎖発現ベクターを構築した。構築した発現ベクターを「pCMA/h046-H5b」と命名した。ヒト化h046-H5bタイプ重鎖のヌクレオチド配列を配列番号53に示し、アミノ酸配列を配列番号52に示した。
実施例5)-1-3で構築したpCMA/04-046Ch-Hをテンプレートとして、下記プライマーセットとKOD -Plus- Mutagenesis Kit(TOYOBO社)を用いて、変異を導入することによりヒト化h046-H8タイプ重鎖発現ベクターを構築した。構築した発現ベクターを「pCMA/h046-H8」と命名した。ヒト化h046-H8タイプ重鎖のヌクレオチド配列を配列番号55に示し、アミノ酸配列を配列番号54に示した。
プライマーセット:
5’-AACATGCAGCTGTCCAGCCTGACCCCCGACGAC-3’(H08-F;配列番号98)
5’-GGCGGTGCTGCTGCTCTTGTCCACGGTCAG-3’(H08-R;配列番号99)
6)-5-1-4 ヒト化h046-H10タイプ重鎖発現ベクターの構築
実施例6)-5-1-3で構築したpCMA/h046-H8をテンプレートとして、下記プライマーセットとKOD -Plus- Mutagenesis Kit(TOYOBO社)を用いて、変異を導入することによりヒト化h046-H10タイプ重鎖発現ベクターを構築した。構築した発現ベクターを「pCMA/h046-H10」と命名した。ヒト化h046-H10タイプ重鎖のヌクレオチド配列を配列番号57に示し、アミノ酸配列を配列番号56に示した。
5’-GTGAGCTGCAAGGCCAGCGGCTACACCTTCACC-3’(H10-F;配列番号100)
5’-CTTCACGCTGCTGCCAGGCTTGGCCAGTTC-3’(H10-R;配列番号101)
6)-5-2-1 ヒト化h046-L1タイプ軽鎖発現ベクターの構築
配列番号59に示すヒト化h046-L1のヌクレオチド配列のうち、可変領域をコードするDNA配列を含むヌクレオチド番号37乃至402に相当するDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をテンプレートとして、KOD-Plus-(TOYOBO社)と下記のプライマーセットでヒト化h046-L1の可変領域をコードするDNA配列を含むDNA断片を増幅し、実施例5)-1-1で構築したキメラ及びヒト化抗体軽鎖発現ベクターpCMA-LKを制限酵素BsiWIで切断した箇所にIn-Fusion HD PCRクローニングキット(Clontech社)を用いて挿入することによりヒト化h046-L1発現ベクターを構築した。得られた発現ベクターを「pCMA/h046-L1」と命名した。ヒト化h046-L1のアミノ酸配列を配列番号58に示した。
プライマーセット
5’-CTGTGGATCTCCGGCGCGTACGGC-3’(CM-LKF;配列番号90)
5’-GGAGGGGGCGGCCACCGTACG-3’(KCL-Inf-R;配列番号102)
配列番号61に示すヒト化h046-L2のヌクレオチド配列のヌクレオチド番号37乃至402に示されるヒト化h046-L2の可変領域をコードするDNA配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。実施例6)-5-2-1と同様の方法でヒト化h046-L2発現ベクターを構築した。得られた発現ベクターを「pCMA/h046-L2」と命名した。ヒト化h046-L2のアミノ酸配列を配列番号60に示した。
配列番号63に示すヒト化h046-L6のヌクレオチド配列のヌクレオチド番号37乃至402に示されるヒト化h046-L6の可変領域をコードするDNA配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。実施例6)-5-2-1と同様の方法でヒト化h046-L6発現ベクターを構築した。得られた発現ベクターを「pCMA/h046-L6」と命名した。ヒト化h046-L6のアミノ酸配列を配列番号62に示した。
配列番号65に示すヒト化h046-L7のヌクレオチド配列のヌクレオチド番号37乃至402に示されるヒト化h046-L2の可変領域をコードするDNA配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。実施例6)-5-2-1と同様の方法でヒト化h046-L7発現ベクターを構築した。得られた発現ベクターを「pCMA/h046-L7」と命名した。ヒト化h046-L7のアミノ酸配列を配列番号64に示した。
FreeStyle 293F細胞(Invitrogen社)はマニュアルに従い、継代、培養を行った。対数増殖期の1×107個のFreeStyle 293F細胞(Invitrogen社)をFreeStyle293 expression medium (Invitrogen社)で9.6mLに希釈した後に、30mL Square Storage Bottle(Nalgene社)に播種し、37℃、8%CO2インキュベーター内で90rpmで1時間振とう培養した。Polyethyleneimine(Polyscience #24765)30μgをOpti-Pro SFM(Invitrogen社)200μLに溶解し、次にNucleoBond Xtra(TaKaRa社)を用いて調製した軽鎖発現ベクター(6μg)及び重鎖発現ベクター(4μg)を200μLのOpti-Pro SFM(Invitrogen社)に添加した。Polyethyleneimine/Opti-Pro SFM混合液200μLに、発現ベクター/Opti-Pro SFM混合液200μLを加え穏やかに攪拌し、さらに5分間放置した後にFreeStyle 293F細胞に添加した。37℃、8%CO2インキュベーターで7日間、90rpmで振とう培養して得られた培養上清をMinisart-Plus filter(Sartorius社)ろ過し、評価用のサンプルとした。
6)-6-1 ヒト化抗GPR20抗体の結合性評価
実施例6)-5-3で作製したヒト化抗GPR20抗体のGPR20結合性をフローサイトメトリー法により評価した。実施例1)-5-1と同様の方法でpcDNA3.1-hGPR20を293T細胞にLipofectamine 2000を用いて一過性に導入し、37℃、5% CO2の条件下で一晩培養した後、細胞懸濁液を調製した。これらの細胞懸濁液にヒト化抗GPR20抗体を加えて4℃で1時間静置した後、5% FBS含有PBSで2回洗浄し、5% FBS含有PBSで320倍に希釈したPE標識F(ab’)2 Fragment抗ヒトIgG, Fcγ抗体(JACKSON IMMUNORESEARC社)を加えて懸濁し、4℃で1時間静置した。5% FBS含有PBSで2回洗浄した後、5% FBS含有PBSに再懸濁し、フローサイトメーター(BD FACSCant II:BD バイオサイエンス社)で検出を行った。データ解析はFlowjo(TreeStar社)で行った。その結果、作製したヒト化抗GPR20抗体はpcDNA3.1-hGPR20導入293T細胞に結合することを確認した。図24にヒトGPR20に特異的に結合したヒト化抗GPR20抗体の結果を示す。図24のヒストグラムにおいて、横軸は抗体結合量を表すPEの蛍光強度、縦軸は細胞数を示す。網掛けは、抗GPR20抗体を反応させていない陰性コントロールであり、白抜きは、各抗GPR20抗体を反応させた際に、細胞表面のGPR20に抗体が結合したことによって蛍光強度が増強したことを示す。
実施例6)-5-3で作製したヒト化抗GPR20抗体の内在化活性は、1)-6と同様の方法でタンパク質合成を阻害する毒素(サポリン)を結合させた抗ヒトIgG抗体試薬Hum-ZAP(ADVANCED TARGETING SYSTEMS)を用いて評価した。すなわち、ヒトGPR20のC末端側にEGFPをつないだGPR20-EGFPタンパク質を安定発現するHEK293細胞を2.5x103cells/wellで播種して37℃、5% CO2の条件下で一晩培養後、ヒト化抗GPR20抗体(終濃度:0.015から1.27μg/mL)とHum-ZAP(終濃度:1μg/mL)を添加し、4日間培養後に生存細胞数をCellTiter-Glo(登録商標) Luminescent Cell Viability AssayによるATPの定量で測定した。その結果、実施例6)-5-3で作製したヒト化抗GPR20抗体である、ヒト化h046-H4b/L7、ヒト化h046-H5b/L2、ヒト化h046-Hwt/L6、ヒト化h046-H8/L1、ヒト化h046-H10/L1及びヒト化h046-H10/L6において、抗体内在化による細胞増殖抑制作用を示した。
6)-7-1 ヒト化h046の重鎖発現ベクターの構築
6)-7-1-1 ヒト化h046-H4bタイプ重鎖発現ベクターの構築
配列表の配列番号103に示されるヒト化h046_H4bタイプ重鎖のヌクレオチド配列(2)のヌクレオチド番号58乃至1416に示されるヒト化h046_H4bタイプ重鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を用い、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じてヒト化h046_H4bタイプ重鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_H4b」と命名した。
配列表の配列番号104に示されるヒト化h046_H5bタイプ重鎖のヌクレオチド配列(2)のヌクレオチド番号58乃至1416に示されるヒト化h046_H5bタイプ重鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を用い、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じてヒト化h046_H5bタイプ重鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_H5b」と命名した。
配列表の配列番号105に示されるヒト化h046_Hwtタイプ重鎖のヌクレオチド配列(2)のヌクレオチド番号58乃至1416に示されるヒト化h046_Hwtタイプ重鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を用い、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じてヒト化h046_Hwtタイプ重鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_Hwt」と命名した。
配列表の配列番号106に示されるヒト化h046_H8タイプ重鎖のヌクレオチド配列(2)のヌクレオチド番号58乃至1416に示されるヒト化h046_H8タイプ重鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を用い、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じてヒト化h046_H8タイプ重鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_H8」と命名した。
配列表の配列番号107に示されるヒト化h046_H10タイプ重鎖のヌクレオチド配列(2)のヌクレオチド番号58乃至1416に示されるヒト化h046_H10タイプ重鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を用い、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じてヒト化h046_H10タイプ重鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_H10」と命名した。
配列表の配列番号51に示されるヒト化h046_H4eタイプ重鎖のヌクレオチド配列のヌクレオチド番号58乃至1416に示されるヒト化h046_H4eタイプ重鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片を用い、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じてヒト化h046_H4eタイプ重鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_H4e」と命名した。
6)-7-2-1 ヒト化h046-L1タイプ軽鎖発現ベクターの構築
配列表の配列番号108に示されるヒト化h046_L1タイプ軽鎖のヌクレオチド配列(2)のヌクレオチド番号61乃至702に示されるヒト化h046_L1タイプ軽鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をLonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046_L1タイプ軽鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_L1」と命名した。
配列表の配列番号109に示されるヒト化h046_L2タイプ軽鎖のヌクレオチド配列(2)のヌクレオチド番号61乃至702に示されるヒト化h046_L2タイプ軽鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をLonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046_L2タイプ軽鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_L2」と命名した。
配列表の配列番号110に示されるヒト化h046_L6タイプ軽鎖のヌクレオチド配列(2)のヌクレオチド番号61乃至702に示されるヒト化h046_L6タイプ軽鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をLonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046_L6タイプ軽鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_L6」と命名した。
配列表の配列番号111に示されるヒト化h046_L7タイプ軽鎖のヌクレオチド配列のヌクレオチド番号61乃至702に示されるヒト化h046_L7タイプ軽鎖をコードする配列を含むDNA断片を合成した(GENEART社 人工遺伝子合成サービス)。合成したDNA断片をLonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046_L7タイプ軽鎖発現ベクターを構築した。構築した発現ベクターを「GSV-h046_L7」と命名した。
6)-7-3-1 ヒト化h046-H4b/L7発現ベクターの構築
構築した発現ベクター「GSV-h046_H4b」及び「GSV-h046_L7」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-H4b/L7発現ベクターを構築した。得られた発現ベクターを「DGV-h046_H4bL7-GS」と命名した。
構築した発現ベクター「GSV-h046_H5b」及び「GSV-h046_L2」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-H5b/L2発現ベクターを構築した。得られた発現ベクターを「DGV-h046_H5bL2-GS」と命名した。
構築した発現ベクター「GSV-h046_Hwt」及び「GSV-h046_L6」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-Hwt/L6発現ベクターを構築した。得られた発現ベクターを「DGV-h046_HwtL6-GS」と命名した。
構築した発現ベクター「GSV-h046_H8」及び「GSV-h046_L1」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-H8/L1発現ベクターを構築した。得られた発現ベクターを「DGV-h046_H8L1-GS」と命名した。
構築した発現ベクター「GSV-h046_H10」及び「GSV-h046_L1」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-H10/L1発現ベクターを構築した。得られた発現ベクターを「DGV-h046_H10L1-GS」と命名した。
構築した発現ベクター「GSV-h046_H10」及び「GSV-h046_L6」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-H10/L6発現ベクターを構築した。得られた発現ベクターを「DGV-h046_H10L6-GS」と命名した。
構築した発現ベクター「GSV-h046_H4e」及び「GSV-h046_L7」から、Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、ヒト化h046-H4e/L7発現ベクターを構築した。得られた発現ベクターを「DGV-h046_H4eL7-GS」と命名した。
6)-7-4-1 ヒト化h046-H4b/L7生産細胞の作製
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-1で構築したヒト化h046-H4b/L7発現ベクター、DGV-h046_H4bL7-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-H4b/L7生産株を構築した。得られた生産株を「GPR1-12」と命名した。
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-2で構築したヒト化h046-H5b/L2発現ベクター、DGV-h046_H5bL2-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-H5b/L2生産株を構築した。得られた生産株を「GPR2-15」と命名した。
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-3で構築したヒト化h046-Hwt/L6発現ベクター、DGV-h046_HwtL6-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-Hwt/L6生産株を構築した。得られた生産株を「GPR3-2」と命名した。
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-4で構築したヒト化h046-H8/L1発現ベクター、DGV-h046_H8L1-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-H8/L1生産株を構築した。得られた生産株を「GPR4-1」と命名した。
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-5で構築したヒト化h046-H10/L1発現ベクター、DGV-h046_H10L1-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-H10/L1生産株を構築した。得られた生産株を「GPR5-10」と命名した。
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-6で構築したヒト化h046-H10/L6発現ベクター、DGV-h046_H10L6-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-H10/L6生産株を構築した。得られた生産株を「GPR6-7」と命名した。
Lonza社のGS Gene Expression System(登録商標)のプロトコールに準じて、実施例6)-7-3-7で構築したヒト化h046-H4e/L7発現ベクター、DGV-h046_H4eL7-GSをCHOK1SV細胞(Lonza社)にトランスフェクションし、ヒト化h046-H4e/L7生産株を構築した。得られた生産株を「GPE-23」と命名した。
6)-7-5-1 ヒト化h046-H4b/L7生産細胞の培養
実施例6)-7-4-1で作製したヒト化h046-H4b/L7生産株「GPR1-12」の培養は、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いて実施した。生産株「GPR1-12」をC36(JXエネルギー社)培地を用いて解凍し、37℃、5% CO2インキュベーター内にて120rpmで培養した。得られた培養液をC36培地で希釈し、37℃、5% CO2インキュベーター内にて120rpmで拡大培養した。得られた培養液を細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、37℃、5% CO2、エアー供給量0.3L/分、揺動18-24rpm、角度6-8°で10日間培養した。培養開始3日目よりFM4Ae2培地(自家調製)を1日あたり、仕込み量の6%分を毎日添加した。得られた培養液をデプスフィルターMillistak MC0HC054H1(Merck Millipore社)で粗ろ過後、Flexboy Bagsに付属の0.22μmフィルター(Sartorius社)でろ過した。このろ過液を「h046-H4b/L7 培養上清」と命名した。
実施例6)-7-5-1と同様に、実施例6)-7-4-2で作製したヒト化h046-H5b/L2生産株「GPR2-15」を培養、拡大し、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いてフェッドバッチ培養を実施した。細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、11日間培養した。得られた培養液をろ過し、このろ過液を「h046-H5b/L2 培養上清」と命名した。
実施例6)-7-5-1と同様に、実施例6)-7-4-3で作製したヒト化h046-Hwt/L6生産株「GPR3-2」を培養、拡大し、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いてフェッドバッチ培養を実施した。細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、14日間培養した。得られた培養液をろ過し、このろ過液を「h046-Hwt/L6 培養上清」と命名した。
実施例6)-7-5-1と同様に、実施例6)-7-4-4で作製したヒト化h046-H8/L1生産株「GPR4-1」を培養、拡大し、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いてフェッドバッチ培養を実施した。細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、11日間培養した。得られた培養液をろ過し、このろ過液を「h046-H8/L1 培養上清」と命名した。
実施例6)-7-5-1と同様に、実施例6)-7-4-5で作製したヒト化h046-H10/L1生産株「GPR5-10」を培養、拡大し、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いてフェッドバッチ培養を実施した。細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、10日間培養した。得られた培養液をろ過し、このろ過液を「h046-H10/L1 培養上清」と命名した。
実施例6)-7-5-1と同様に、実施例6)-7-4-6で作製したヒト化h046-H10/L6生産株「GPR6-7」を培養、拡大し、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いてフェッドバッチ培養を実施した。細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、12日間培養した。得られた培養液をろ過し、このろ過液を「h046-H10/L6 培養上清」と命名した。
実施例6)-7-5-1と同様に、実施例6)-7-4-7で作製したヒト化h046-H4e/L7生産株「GPE-23」を培養、拡大し、培養装置Wave reactor(GEヘルスケア・ジャパン社)を用いてフェッドバッチ培養を実施した。細胞密度30×104cells/mLとなるようにC36培地で希釈してWAVE CELLBAG(GE Healthcare Bioscience社)に仕込み、11日間培養した。得られた培養液をろ過し、このろ過液を「h046-H4e/L7 培養上清」と命名した。
6)-7-6-1 ヒト化h046-H4b/L7の精製
実施例6)-7-5-1で得られた「h046-H4b/L7 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、グラスファイバーフィルター AP20(Merck Millipore社)で粗ろ過後、0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
次に、AEX精製プールを、酢酸緩衝液で平衡化した陽イオン交換クロマト樹脂にアプライした。アプライ液がカラムに全て入ったのち、酢酸緩衝液でカラムを洗浄した。次に高濃度のNaClを含む酢酸緩衝液を用いて溶出し、280nmの吸収ピークを回収した。回収液を0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをCEX精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度20mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046_H4b/L7」と命名した。
実施例6)-7-5-2で得られた「h046-H5b/L2 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、グラスファイバーフィルター AP20(Merck Millipore社)で粗ろ過後、0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度40mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046_H5b/L2」と命名した。
実施例6)-7-5-3で得られた「h046-Hwt/L6 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、0.5/0.2μmのフィルターであるMillipore Express SHC(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
次に、AEX精製プールを、酢酸緩衝液で平衡化した陽イオン交換クロマト樹脂にアプライした。アプライ液がカラムに全て入ったのち、酢酸緩衝液でカラムを洗浄した。次に高濃度のNaClを含む酢酸緩衝液を用いて溶出し、280nmの吸収ピークを回収した。回収液を0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをCEX精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度40mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046-Hwt/L6」と命名した。
実施例6)-7-5-4で得られた「h046-H8/L1 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
次に、AEX精製プールを、酢酸緩衝液で平衡化した陽イオン交換クロマト樹脂にアプライした。アプライ液がカラムに全て入ったのち、酢酸緩衝液でカラムを洗浄した。次に高濃度のNaClを含む酢酸緩衝液を用いて溶出し、280nmの吸収ピークを回収した。回収液を0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをCEX精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度40mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046-H8/L1」と命名した。
実施例6)-7-5-5で得られた「h046-H10/L1 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、グラスファイバーフィルター AP20(Merck Millipore社)で粗ろ過後、0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
次に、AEX精製プールを、酢酸緩衝液で平衡化した陽イオン交換クロマト樹脂にアプライした。アプライ液がカラムに全て入ったのち、酢酸緩衝液でカラムを洗浄した。次に高濃度のNaClを含む酢酸緩衝液を用いて溶出し、280nmの吸収ピークを回収した。回収液を0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをCEX精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度40mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046-H10/L1」と命名した。
実施例6)-7-5-6で得られた「h046-H10/L6 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
次に、AEX精製プールを、酢酸緩衝液で平衡化した陽イオン交換クロマト樹脂にアプライした。アプライ液がカラムに全て入ったのち、酢酸緩衝液でカラムを洗浄した。次に高濃度のNaClを含む酢酸緩衝液を用いて溶出し、280nmの吸収ピークを回収した。回収液を0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをCEX精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度40mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046-H10/L6」と命名した。
実施例6)-7-5-7で得られた「h046-H4e/L7 培養上清」を、rProteinAアフィニティークロマトグラフィー、陰イオン交換クロマトグラフィー、陽イオン交換クロマトグラフィーの三段階工程で精製した。最初に、培養上清を、PBSで平衡化したrProteinAアフィニティークロマト樹脂にアプライした。培養液がカラムに全て入ったのち、PBS、アルギニンを含む緩衝液、PBSでカラムを洗浄した。次に、酢酸緩衝液で溶出し、280nmの吸収ピークを回収した。回収液をTris緩衝液で中和し、0.5/0.2μmのフィルターであるMillipore Express SHC(Merck Millipore社)でろ過したものをrProteinA精製プールとした。
次に、AEX精製プールを、酢酸緩衝液で平衡化した陽イオン交換クロマト樹脂にアプライした。アプライ液がカラムに全て入ったのち、酢酸緩衝液でカラムを洗浄した。次に高濃度のNaClを含む酢酸緩衝液を用いて溶出し、280nmの吸収ピークを回収した。回収液を0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものをCEX精製プールとした。
CEX精製プールを、Pellicon 3 Cassette 30kDa(Merck Millipore社)にて抗体濃度40mg/mLに濃縮した後、ヒスチジンバッファー(25mM Histidine、5% Sorbitol、pH6.0)に置換した。最後に0.22μmフィルターであるステリカップ-GV(Merck Millipore社)でろ過したものを精製サンプルとした。精製サンプルを「h046-H4e/L7」と命名した。
7)-1 抗体-薬物コンジュゲートの作製(1)
抗体の還元:実施例5)-2にて作製した04-046Chを、製造方法1に記載した共通操作B(280nm吸光係数として1.47mLmg-1cm-1を使用)及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(3.40mL)に9.4mM TCEP(東京化成工業株式会社)水溶液(0.104mL;抗体一分子に対して5.0当量)及び1Mリン酸水素二カリウム水溶液(Nacalai Tesque,Inc.;0.170mL)を加えた。本溶液のpHが7.4±0.1内であることを確認した後に、37℃で1時間インキュベートすることによって、抗体内鎖間部のジスルフィド結合を還元した。
抗体濃度:2.26mg/mL,抗体収量:31.6mg(93%),抗体一分子あたりの薬物平均結合数(n):5.6
7)-2 抗体-薬物コンジュゲート(2)の作製
抗体の還元:実施例5)-2にて作製した04-046Chを、製造方法1に記載した共通操作B(280nm吸光係数として1.47mLmg-1cm-1を使用)及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(3.40mL)に9.4mM TCEP(東京化成工業株式会社)水溶液(0.104mL;抗体一分子に対して5.0当量)及び1Mリン酸水素二カリウム水溶液(Nacalai Tesque,Inc.;0.0509mL)を加えた。本溶液のpHが7.0±0.1内であることを確認した後に、37℃で1時間インキュベートすることによって、抗体内鎖間部のジスルフィド結合を還元した。
抗体濃度:2.23mg/mL,抗体収量:31.2mg(92%),抗体一分子あたりの薬物平均結合数(n):5.6
7)-3 抗体-薬物コンジュゲート(3)の作製
抗体の還元:実施例6)-7-6-1にて作製したh046-H4b/L7を、製造方法1に記載した共通操作B(280nm吸光係数として1.31mLmg-1cm-1を使用)及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(6.25mL)に10mM TCEP(東京化成工業株式会社)水溶液(0.237mL;抗体一分子に対して5.5当量)及び1Mリン酸水素二カリウム水溶液(Nacalai Tesque,Inc.;0.0940mL)を加えた。本溶液のpHが7.0±0.1内であることを確認した後に、37℃で1時間インキュベートすることによって、抗体内鎖間部のジスルフィド結合を還元した。
特性評価:製造方法1に記載した共通操作E及びF((εD,280=5178、εD,370=20217を使用)を使用して、下記の特性値を得た。
抗体濃度:2.03mg/mL,抗体収量:61.0mg(97%),共通操作Eにて測定された抗体一分子あたりの薬物平均結合数(n):5.5;共通操作Fにて測定された抗体一分子あたりの薬物平均結合数(n):7.8。
抗体の還元:実施例6)-7-6-1にて作製したh046-H4b/L7を、製造方法1に記載した共通操作B(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(300mL)をポリカーボネート製の1000mL三角フラスコ容器に入れ、マグネティックスターラー撹拌下、室温で1Mリン酸水素二カリウム水溶液(4.80mL)を加えた後、10mM TCEP水溶液(11.3mL;抗体一分子に対して5.5当量)を加えた。本溶液のpHが7.0±0.1内であることを確認した後に撹拌を停止し、37℃で2時間インキュベートすることにより抗体内鎖間部のジスルフィド結合を還元した。
実施例6)-7-6-2にて作製したh046-H5b/L2(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7-3工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-H5b/L2-ADC1」を得た。
実施例6)-7-6-3にて作製したh046-Hwt/L6(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7-3工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-Hwt/L6-ADC1」を得た。
抗体の還元:実施例6)-7-6-1にて作製したh046-H4b/L7を、製造方法1に記載した共通操作B(280nm吸光係数として1.31mLmg-1cm-1を使用)及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(5.00mL)に10mM TCEP(東京化成工業株式会社)水溶液(0.193mL;抗体一分子に対して5.5当量)及び1Mリン酸水素二カリウム水溶液(Nacalai Tesque,Inc.;0.0752mL)を加えた。本溶液のpHが7.0±0.1内であることを確認した後に、37℃で1時間インキュベートすることによって、抗体内鎖間部のジスルフィド結合を還元した。
抗体濃度:2.07mg/mL,抗体収量:49.6mg(99%),共通操作Eにて測定された抗体一分子あたりの薬物平均結合数(n):6.1;共通操作Fにて測定された抗体一分子あたりの薬物平均結合数(n):7.6。
抗体の還元:実施例6)-7-6-2にて作製したh046-H5b/L2を、製造方法1に記載した共通操作B(280nm吸光係数として1.31mLmg-1cm-1を使用)及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(6.25mL)に10mM TCEP(東京化成工業株式会社)水溶液(0.237mL;抗体一分子に対して5.5当量)及び1Mリン酸水素二カリウム水溶液(Nacalai Tesque,Inc.;0.0940mL)を加えた。本溶液のpHが7.0±0.1内であることを確認した後に、37℃で1時間インキュベートすることによって、抗体内鎖間部のジスルフィド結合を還元した。
特性評価:製造方法1に記載した共通操作E及びF(εD,280=4964、εD,370=18982を使用)を使用して、下記の特性値を得た。
抗体濃度:2.07mg/mL,抗体収量:62.2mg(99%),共通操作Eにて測定された抗体一分子あたりの薬物平均結合数(n):6.0;共通操作Fにて測定された抗体一分子あたりの薬物平均結合数(n):7.7。
実施例6)-7-6-3にて作製したh046-Hwt/L6(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7)-8工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-Hwt/L6-ADC2」を得た。
実施例6)-7-6-4にて作製したh046-H8/L1(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7)-3工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-H8/L1-ADC1」を得た。
実施例6)-7-6-5にて作製したh046-H10/L1(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7)-3工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-H10/L1-ADC1」を得た。
実施例6)-7-6-6にて作製したh046-H10/L6(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7)-3工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-H10/L6-ADC1」を得た。
抗体の還元:実施例6)-7-6-7にて作製したh046-H4e/L7を、製造方法1に記載した共通操作B(280nm吸光係数として1.31mLmg-1cm-1を使用)及びCを用いて、PBS6.0/EDTAにて10mg/mLに調製した。本溶液(11.0mL)に10mM TCEP(東京化成工業株式会社)水溶液(0.412mL;抗体一分子に対して5.5当量)及び1Mリン酸水素二カリウム水溶液(Nacalai Tesque,Inc.;0.165mL)を加えた。本溶液のpHが7.0±0.1内であることを確認した後に、37℃で1時間インキュベートすることによって、抗体内鎖間部のジスルフィド結合を還元した。
特性評価:製造方法1に記載した共通操作E及びF(εD,280=5178、εD,370=20217を使用)を使用して、下記の特性値を得た。
抗体濃度:3.01mg/mL,抗体収量:104mg(95%),共通操作Eにて測定された抗体一分子あたりの薬物平均結合数(n):5.6;共通操作Fにて測定された抗体一分子あたりの薬物平均結合数(n):7.7。
実施例6)-7-6-7にて作製したh046-H4e/L7(280nm吸光係数として1.31mLmg-1cm-1を使用)を用いて、実施例7-13工程1と同様の方法により、標記抗体-薬物コンジュゲート「h046-H4e/L7-ADC1」を得た。
8)-1-1 ヒト化抗GPR20抗体及び抗体-薬物コンジュゲートの結合性評価
実施例6)-7-6で作製したヒト化抗GPR20抗体、並びに実施例7で作製した抗体-薬物コンジュゲートのGPR20結合性をフローサイトメトリー法により評価した。実施例6)-6-1と同様の方法でpcDNA3.1-hGPR20を一過性に導入した293T細胞に対する結合を、フローサイトメーター(BD FACSCant II:BD バイオサイエンス社)で解析した結果、抗体濃度依存的な結合性を確認した。図25にその代表的な反応例を示す。図25において横軸は抗体濃度(nM)、縦軸はMFI(mean fluorescence intensity)による抗体結合量を示す。図25に示す通り、ヒト化抗GPR20抗体h046-H4b/L7及びh046-H4e/L7、並びに抗体-薬物コンジュゲート(4)、(13)は、pcDNA3.1-hGPR20導入293T細胞に対して濃度依存的に結合量が増加した。
GIST-T1/GPR20安定発現細胞株は、GIST-T1細胞(コスモバイオ社より入手可能)にヒトGPR20発現用組換えレトロウイルスを感染させることにより作製した。
ヒトGPR20発現レトロウイルスベクターは、In-Fusion HD Cloning Kit (CLONTECH社)を用いて作製した。即ち、実施例1で作製したヒトGPR20発現ベクターpcDNA3.1-hGPR20を鋳型とし、以下のプライマーセットを用いてPCR反応を行った。この反応にはKOD FX DNAポリメラーゼ(東洋紡社)を用い、98℃-10秒、58℃-30秒、68℃-2分、30サイクルで反応を行った後、得られたGPR20 cDNA断片を含むPCR産物をアガロースゲル電気泳動し、QIAquick Gel Extraction Kit(QIAGEN社)で目的サイズのDNAを抽出した。このDNA断片と、制限酵素EcoRI,NotIで消化したレトロウイルスベクターpLPCX(CLONTECH社)、In-Fusion HD Enzyme premixとを混合し、連結反応をした後、大腸菌TOP10(Invitrogen社)に形質転換することで、ヒトGPR20発現レトロウイルスベクターpLPCX-GPR20を構築した。PCRに用いたプライマーセットは以下の通り。
5’―CTCAAGCTTCGAATTCACCATGCCCTCTGTGTCTCCA―3’(LPCX-1;配列番号112)
5’―TTGGCCGAGGCGGCCTCCTAAGCCTCGGGCCCATTAG―3’(LPCX-1;配列番号113)
8)-2-2 GIST-T1/GPR20安定発現細胞株の樹立
Lipofectamine 2000(インビトロジェン社)を用いてレトロウイルスパッケージング細胞293-10A1にpLPCX-GPR20を一過性に導入し、72時間後に組換えレトロウイルスを含む培養上清を回収し、GIST-T1細胞培養系に添加することで同細胞に感染させた。感染3日後にGIST-T1細胞を1 cell/wellで96ウェルプレートに播種し、0.3から1μg/mLのPuromycinを添加した培地で37℃、5% CO2の条件下で長期間培養することでGPR20を安定的に発現する細胞株GIST-T1/GPR20を樹立した。
GIST-T1/GPR20細胞を10% FBS含有DMEM培地中2.5x103細胞/100μL/wellになるよう96ウェルプレートに播種し、37℃、5% CO2の条件下で一晩培養した。実施例7で作製した抗体-薬物コンジュゲートを終濃度0.032から100 nMとなるように添加した。7乃至11日間培養後に生存細胞数をCellTiter-Glo(登録商標) Luminescent Cell Viability AssayによるATPの定量で測定した。図26にヒトキメラ化抗体から作製した抗体-薬物コンジュゲート(1)、図27にヒト化抗体から作製した抗体-薬物コンジュゲート(7)、(8)、(9)を各々添加した際の濃度依存的な細胞増殖抑制活性を示す。本実験におけるhIgG-ADC2は、GPR20とは無関係な抗原を認識するヒトIgGから作製した抗体-薬物コンジュゲートであり、陰性コントロールとして使用した。
抗体-薬物コンジュゲートの抗腫瘍効果は、ヒト消化管間質腫瘍由来の細胞を免疫不全マウスに移植した動物モデルを用いて評価した。5-6週齢の雌BALB/c ヌードマウス(CAnN.Cg-Foxnlnu/CrlCrlj、日本チャールス・リバー社)を実験使用前にSPF条件化で4乃至7日間馴化した。マウスには滅菌した固形飼料(FR-2,Funabashi Farms Co.,Ltd)を給餌し、滅菌した水道水(5-15ppm次亜塩素酸ナトリウム溶液を添加して調製)を与えた。移植した腫瘍の長径及び短径を電子式デジタルノギス(CD-15CX,Mitutoyo Corp.)で1週間に1乃至2回測定し、以下に示す計算式により腫瘍体積を算出した。
腫瘍体積(mm3)=1/2×長径(mm)×[短径(mm)]2
抗体-薬物コンジュゲートは全てABS緩衝液(10mM-Acetate Buffer, 5% Sorbitol, pH5.5)(NACALAI)で希釈し、10mL/kgの液量を尾静脈内投与した。また、コントロール群(Vehicle群)としてABS緩衝液を同様に投与した。1群あたり5乃至6匹のマウスを実験に用いた。
8)-1-2で作製したGIST-T1/GPR20細胞をマトリゲル(コーニング社)に懸濁し、5×106cellsを雌ヌードマウスの右側腹部に皮下移植し(Day0)、Day14に無作為に群分けを実施した。抗体-薬物コンジュゲート(1)、(2)をDay14に10mg/kgの用量で尾静脈内投与した。陰性対照としてヒトIgGを用いて作製した抗体-薬物コンジュゲートを10mg/kgの用量で同様に投与した。抗体-薬物コンジュゲート(1)、(2)の投与によって腫瘍体積が著しく減少し、いずれも腫瘍を縮退させる効果を発揮した(図28)。なお、図中、横軸は日数、縦軸は腫瘍体積を示す。hIgG-ADC1及びhIgG-ADC2は、GPR20とは無関係な抗原を認識するヒトIgGから作製した抗体-薬物コンジュゲートであり、陰性コントロールとして使用した。
2×107cellsのヒト消化管間質腫瘍細胞株GIST430(Brigham Women’s Hospitalより入手)を雌ヌードマウスの右側腹部に皮下移植して(Day0)、Day29に無作為に群分けを実施した。抗体-薬物コンジュゲート(1)、(2)をDay29、36、43に10mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(1)、(2)の投与によって腫瘍体積がコントロール群に比べて著しく減少し、いずれも腫瘍増殖抑制効果を発揮した(図29)。
実施例9)-1と同様にGIST-T1/GPR20細胞を雌ヌードマウスに皮下移植し(Day0)、Day24に無作為に群分けを実施した。抗体-薬物コンジュゲート(6)、(9)をDay24に10mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(6)、(9)の投与によって腫瘍体積が著しく減少し、いずれも腫瘍を縮退させる効果を発揮した(図30)。
実施例9)-1と同様にGIST-T1/GPR20細胞を雌ヌードマウスに皮下移植し(Day0)、Day17に無作為に群分けを実施した。抗体-薬物コンジュゲート(3)、(5)、(6)、(10)、(11)、(12)をDay17に全て1mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(3)、(5)、(6)、(10)、(11)、(12)の投与によって腫瘍体積が著しく減少し、いずれも腫瘍増殖抑制効果を発揮した(図31)。
実施例9)-1と同様にGIST-T1/GPR20細胞を雌ヌードマウスに皮下移植し(Day0)、Day17に無作為に群分けを実施した。抗体-薬物コンジュゲート(13)をDay17に0.3、1、3mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(13)の投与によって用量依存的に腫瘍体積が減少し、1mg/kg以上の用量で腫瘍を退縮させる効果を発揮した(図32)。
小腸の消化管間質腫瘍患者より摘出した腫瘍を免疫不全マウスの皮下にブロック移植することにより継代維持したGIST020(医薬基盤・健康・栄養研究所より入手)を雌ヌードマウスの右側腹部に皮下移植して(Day0)、Day55に無作為に群分けを実施した。抗体-薬物コンジュゲート(4)、(13)をDay55、75に10mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(4)、(13)の投与によって腫瘍体積がコントロール群に比べて著しく減少し、いずれも腫瘍増殖抑制効果を発揮した(図33)。
食道の消化管間質腫瘍患者より摘出した腫瘍を免疫不全マウスの皮下にブロック移植することにより継代維持したGIST1(富山大学より入手)を雌ヌードマウスの右側腹部に皮下移植して(Day0)、Day38に無作為に群分けを実施した。抗体-薬物コンジュゲート(13)をDay38、59に3、10mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(13)の投与によって腫瘍体積がコントロール群に比べて著しく減少し、いずれも腫瘍増殖抑制効果を発揮した(図34)。
HER2 分子を強発現しているがGPR20を発現していない胃癌細胞株 NCI-N87を1×107cellsを雌ヌードマウスの右側腹部に皮下移植して(Day0)、Day6に無作為に群分けを実施した。抗体-薬物コンジュゲート(14)をDay6に3、10mg/kgの用量で尾静脈内投与したが、抗体-薬物コンジュゲート(14)は、顕著な腫瘍増殖抑制効果を示さなかった。一方、抗HER2抗体から作製した-薬物コンジュゲートの投与によって、腫瘍体積がコントロール群に比べて著しく減少し、腫瘍増殖抑制効果を発揮した(図35)。
9)-9 抗腫瘍効果(9)
Regorafenib治療に不応答性となった胃の消化管間質腫瘍患者由来腫瘍を免疫不全マウスの皮下にブロック移植することにより継代維持されたGIST074(医薬基盤・健康・栄養研究所より入手)を雌ヌードマウスの右側腹部に皮下移植し(Day0)、Day29に無作為に群分けを実施した。抗体-薬物コンジュゲート(14)をDay29、50に10mg/kgの用量で尾静脈内投与した。抗体-薬物コンジュゲート(14)の投与により、腫瘍増殖が完全に抑制された(図36)。一方、Imatinib, Sunitinib, Regorafenibを各々90、30、4 mg/kgで図36の三角印で示した日に一日1回経口投与した場合には、腫瘍の増殖は完全には抑制されなかった。このことから、抗体-薬物コンジュゲート(14)は、標準治療薬である3種のチロシンキナーゼ阻害剤に抵抗性を示す消化管間質腫瘍に対しても抗腫瘍効果を発揮した。
KIT遺伝子に V654AのImatinib耐性変異を有するヒト消化管間質腫瘍細胞株GIST430/654(Brigham Women’s Hospitalより入手)モデルにおいて、Sunitinibとの併用効果を評価した。2×107cellsのGIST430/654を雌ヌードマウスの右側腹部に皮下移植して(Day0)、Day21に無作為に群分けを実施した。図37に示すように、抗体-薬物コンジュゲート(3)はDay21に10mg/kgの用量で尾静脈内に1回投与した。Imatinib及びSunitinibは各々150、40 mg/kgの用量で三角印をした日に一日1回経口投与した。GIST430/654モデルにおいて、Imatinibは腫瘍増殖を全く抑制しなかったが、抗体-薬物コンジュゲート(3)及びSunitinibを投与した群では、腫瘍増殖の抑制が観察された。抗体-薬物コンジュゲート(3)とSunitinibの併用群では、単剤より更に強い薬効が観察され、腫瘍の縮退が認められた。
配列番号45:04-046Ch軽鎖のアミノ酸配列
配列番号46:04-046Ch抗体重鎖のヌクレオチド配列
配列番号47:04-046Ch軽鎖のヌクレオチド配列
配列番号48:h046-H4bのアミノ酸配列
配列番号49:h046-H4bのヌクレオチド配列(1)
配列番号50:h046-H4eのアミノ酸配列
配列番号51:h046-H4eのヌクレオチド配列
配列番号52:h046-H5bのアミノ酸配列
配列番号53:h046-H5bのヌクレオチド配列(1)
配列番号54:h046-H8のアミノ酸配列
配列番号55:h046-H8のヌクレオチド配列(1)
配列番号56:h046-H10のアミノ酸配列
配列番号57:h046-H10のヌクレオチド配列(1)
配列番号58:h046-L1のアミノ酸配列
配列番号59:h046-L1のヌクレオチド配列(1)
配列番号60:h046-L2のアミノ酸配列
配列番号61:h046-L2のヌクレオチド配列(1)
配列番号62:h046-L6のアミノ酸配列
配列番号63:h046-L6のヌクレオチド配列(1)
配列番号64:h046-L7のアミノ酸配列
配列番号65:h046-L7のヌクレオチド配列(1)
配列番号66:PCRプライマー Nhe-polyC-S
配列番号67:PCRプライマー rIgγ-AS1
配列番号68:PCRプライマー rIgγ-AS2
配列番号69:PCRプライマー rIgκ-AS
配列番号70:PCRプライマー rIgγ-seq
配列番号71:PCRプライマー rIgκ-seq
配列番号72:PCRプライマー NFLAG-1
配列番号73:PCRプライマー NFLAG-2
配列番号74:PCRプライマー mEC2-1
配列番号75:PCRプライマー mEC2-2
配列番号76:PCRプライマー mEC3-1
配列番号77:PCRプライマー mEC3-2
配列番号78:PCRプライマー mEC4-1
配列番号79:PCRプライマー mEC4-2
配列番号80:PCRプライマー mEC1-1
配列番号81:PCRプライマー mEC1-2
配列番号82:PCRプライマー mEC1-3
配列番号83:PCRプライマー mEC1-4
配列番号85:PCRプライマー 3.3-F1
配列番号86:PCRプライマー 3.3-R1
配列番号88:PCRプライマー EG-Inf-F
配列番号89:PCRプライマー EG1-Inf-R
配列番号90:PCRプライマー CM-LKF
配列番号91:PCRプライマー 046L-R
配列番号92:h046-L6のCDRL2のアミノ酸配列
配列番号93:h046-L7のCDRL2のアミノ酸配列
配列番号94:DNA断片A
配列番号95:PCRプライマー Hb-F
配列番号96:PCRプライマー Hb-R
配列番号97:DNA断片B
配列番号98:H08-F
配列番号99:H08-R
配列番号100:H10-F
配列番号101:H10-R
配列番号102:PCRプライマー KCL-Inf-R
配列番号103:化h046-H4bのヌクレオチド配列(2)
配列番号104:化h046-H5bのヌクレオチド配列(2)
配列番号105:化h046-Hwtのヌクレオチド配列(2)
配列番号106:化h046-H8のヌクレオチド配列(2)
配列番号107:化h046-H10のヌクレオチド配列(2)
配列番号108:化h046-L1のヌクレオチド配列(2)
配列番号109:化h046-L2のヌクレオチド配列(2)
配列番号110:化h046-L6のヌクレオチド配列(2)
配列番号111:化h046-L7のヌクレオチド配列(2)
配列番号112:PCRプライマー LPCX-1
配列番号113:PCRプライマー LPCX-2
Claims (63)
- 以下の(a)及び(b)に記載の特性を有することを特徴とする抗体又は当該抗体の機能性断片;
(a)GPR20に特異的に結合する、及び
(b)GPR20に結合後、細胞内に取り込まれる内在化能を有する。 - GPR20が配列番号1に記載のアミノ酸配列からなる分子である請求項1に記載の抗体又は当該抗体の機能性断片。
- 配列番号1に記載のアミノ酸配列からなるポリペプチドには特異的に結合し、アミノ酸番号113のアミノ酸をY(チロシン)から他のアミノ酸に置換したポリペプチドには特異的に結合しない、請求項1又は2に記載の抗体又は当該抗体の機能性断片。
- 配列番号1に記載のアミノ酸配列からなるポリペプチドには特異的に結合し、アミノ酸番号113のアミノ酸をY(チロシン)からF(フェニルアラニン)に置換したポリペプチドには特異的に結合しない、請求項1乃至3のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- 配列番号1においてアミノ酸番号1乃至48に記載のアミノ酸配列及びアミノ酸番号108乃至125に記載のアミノ酸配列からなる高次構造に特異的に結合する請求項1乃至4のいずれか1項に記載の抗体又は当該抗体の機能性断片
- 配列番号1においてアミノ酸番号1乃至48に記載のアミノ酸配列から選択される少なくとも1つのアミノ酸及びアミノ酸番号108乃至125に記載のアミノ酸配列から選択される少なくとも1つのアミノ酸に特異的に結合する、請求項1乃至5のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- 特異的に結合するアミノ酸の少なくとも1つが、配列番号1のアミノ酸番号113のアミノ酸である、請求項1乃至6のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- GPR20への結合に対して、以下の(a)乃至(c)からなる群から選択されるいずれか1項に記載の抗体と競合阻害活性を有する請求項1乃至7のいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号2のアミノ酸番号20乃至475に示されるアミノ酸配列からなる重鎖及び配列番号7のアミノ酸番号21乃至233に示されるアミノ酸配列からなる軽鎖を有する抗体、
(b)配列番号12のアミノ酸番号20乃至475に示されるアミノ酸配列からなる重鎖及び配列番号17のアミノ酸番号21乃至233に示されるアミノ酸配列からなる軽鎖を有する抗体、及び、
(c)配列番号22のアミノ酸番号20乃至475に示されるアミノ酸配列からなる重鎖及び配列番号27のアミノ酸番号21乃至233に示されるアミノ酸配列からなる軽鎖を有する抗体。 - 以下の(a)乃至(c)からなる群から選択されるいずれか1項に記載のCDRH1、CDRH2及びCDRH3、並びに(d)乃至(h)からなる群から選択されるいずれか1項に記載のCDRL1、CDRL2及びCDRL3を含む、請求項1乃至8のいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、
(b)配列番号14に記載のアミノ酸配列からなるCDRH1、配列番号15に記載のアミノ酸配列からなるCDRH2及び配列番号16に記載のアミノ酸配列からなるCDRH3、及び、
(c)配列番号24に記載のアミノ酸配列からなるCDRH1、配列番号25に記載のアミノ酸配列からなるCDRH2及び配列番号26に記載のアミノ酸配列からなるCDRH3、
(d)配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号10に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(e)配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号92に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(f)配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号93に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(g)配列番号19に記載のアミノ酸配列からなるCDRL1、配列番号20に記載のアミノ酸配列からなるCDRL2及び配列番号21に記載のアミノ酸配列からなるCDRL3、及び
(h)配列番号29に記載のアミノ酸配列からなるCDRL1、配列番号30に記載のアミノ酸配列からなるCDRL2及び配列番号31に記載のアミノ酸配列からなるCDRL3。 - 以下の(a)乃至(e)からなる群から選択されるいずれか1項に記載のCDRH1、CDRH2及びCDRH3、並びにCDRL1、CDRL2及びCDRL3を含む、請求項1乃至9のいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、及び配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号10に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(b)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、及び配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号92に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3、
(c)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3、及び配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号93に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3
(d)配列番号14に記載のアミノ酸配列からなるCDRH1、配列番号15に記載のアミノ酸配列からなるCDRH2及び配列番号16に記載のアミノ酸配列からなるCDRH3、及び配列番号19に記載のアミノ酸配列からなるCDRL1、配列番号20に記載のアミノ酸配列からなるCDRL2及び配列番号21に記載のアミノ酸配列からなるCDRL3、及び
(e)配列番号24に記載のアミノ酸配列からなるCDRH1、配列番号25に記載のアミノ酸配列からなるCDRH2及び配列番号26に記載のアミノ酸配列からなるCDRH3、及び配列番号29に記載のアミノ酸配列からなるCDRL1、配列番号30に記載のアミノ酸配列からなるCDRL2及び配列番号31に記載のアミノ酸配列からなるCDRL3。 - 以下の(a)乃至(c)からなる群から選択されるいずれか1項に記載の重鎖の可変領域、及び(d)乃至(h)から選択されるいずれか1項に記載の軽鎖の可変領域を有する請求項1乃至10のいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、
(b)配列番号13に記載のアミノ酸配列からなる重鎖の可変領域、及び、
(c)配列番号23に記載のアミノ酸配列からなる重鎖の可変領域、からなる群から選択される重鎖の可変領域、
(d)配列番号8に記載のアミノ酸配列からなる軽鎖の可変領域、
(e)配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(f)配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(g)配列番号18に記載のアミノ酸配列からなる軽鎖の可変領域、及び、
(h)配列番号28に記載のアミノ酸配列からなる軽鎖の可変領域。 - 以下の(a)乃至(e)からなる群から選択されるいずれか1項に記載の重鎖可変領域及び軽鎖可変領域を含む請求項1乃至11のいずれか1項に記載の抗体又は当該抗体の機能性断片:
(a)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号8に記載のアミノ酸配列からなる軽鎖の可変領域、
(b)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(c)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域、
(d)配列番号13に記載のアミノ酸配列からなる重鎖の可変領域、及び配列番号18に記載のアミノ酸配列からなる軽鎖の可変領域、
(e)配列番号23に記載のアミノ酸配列からなる重鎖の可変領域、及び、配列番号28に記載のアミノ酸配列からなる軽鎖の可変領域。 - 定常領域がヒト由来定常領域である請求項1乃至12のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- 配列番号44に記載のアミノ酸配列からなる重鎖及び配列番号45に記載のアミノ酸配列からなる軽鎖を含む請求項13に記載の抗体又は当該抗体の機能性断片。
- ヒト化されている請求項1乃至14のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- 以下の(a)乃至(h)からなる群から選択されるいずれか1項に記載のアミノ酸配列からなる重鎖の可変領域、及び(i)乃至(o)からなる群から選択されるいずれか1項に記載のアミノ酸配列からなる軽鎖の可変領域を有する請求項15に記載の抗体又は当該抗体の機能性断片:
(a)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(b)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(c)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(d)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(e)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(f)配列番号44においてアミノ酸番号20乃至142に記載のアミノ酸配列、
(g)(a)乃至(f)の配列において各CDR 配列以外のフレームワーク領域の配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、
(h)(a)乃至(g)の配列における各CDR 配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列、
(i)配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(j)配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(k)配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(l)配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列、
(m)配列番号45においてアミノ酸番号21乃至128に記載のアミノ酸配列、
(n)(i)乃至(m)の配列において各CDR 配列以外のフレームワーク領域の配列に対して少なくとも95%以上の相同性を有するアミノ酸配列、及び
(o) (i)乃至(n)の配列における各CDR 配列以外のフレームワーク領域の配列において1又は数個のアミノ酸が欠失、置換又は付加されたアミノ酸配列。 - 以下の(a)乃至(t)からなる群から選択されるいずれか1項に記載の重鎖可変領域及び軽鎖可変領域を含む請求項16に記載の抗体又は抗体の機能性断片:
(a)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域および配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変流域、
(b)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる、重鎖可変領域および配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(c)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域および配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(d)配列番号48においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域および配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域からなる軽鎖可変領域、
(e)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(f)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(g)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(h)配列番号50においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(i)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(j)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(k)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(l)配列番号52においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(m)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(n)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(o)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(p)配列番号54においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(q)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号58においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(r)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号60においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、
(s)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域、及び、
(t)配列番号56においてアミノ酸番号20乃至142に記載のアミノ酸配列からなる重鎖可変領域、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖可変領域。 - 以下の(a)乃至(x)からなる群から選択されるいずれか1項に記載の重鎖及び軽鎖を含む請求項16又は17に記載の抗体又は抗体の機能性断片:
(a)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(b)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる、重鎖および配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(c)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(d)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(e)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖および配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(f)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(g)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(h)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(i)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(j)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(k)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(l)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(m)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(n)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(o)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(p)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(q)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(r)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(s)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(t)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(u)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(v)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(w)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(x)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖。 - 機能性断片がFab、F(ab)2、Fab’及びFvからなる群から選択される請求項1乃至18のいずれか1項に記載の抗体の機能性断片。
- 請求項1乃至19のいずれか1項に記載の抗体又は当該抗体の機能性断片をコードするポリヌクレオチド。
- 以下の(a)乃至(e)からなる群から選択されるいずれか1項に記載のポリヌクレオチドを含む請求項20に記載のポリヌクレオチド:
(a)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号10に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRLをコードするポリヌクレオチド、
(b)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号92に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、
(c)配列番号4に記載のアミノ酸配列からなるCDRH1、配列番号5に記載のアミノ酸配列からなるCDRH2及び配列番号6に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号9に記載のアミノ酸配列からなるCDRL1、配列番号93に記載のアミノ酸配列からなるCDRL2及び配列番号11に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド
(d)配列番号14に記載のアミノ酸配列からなるCDRH1、配列番号15に記載のアミノ酸配列からなるCDRH2及び配列番号16に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号19に記載のアミノ酸配列からなるCDRL1、配列番号20に記載のアミノ酸配列からなるCDRL2及び配列番号21に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド、及び、
(e)配列番号24に記載のアミノ酸配列からなるCDRH1、配列番号25に記載のアミノ酸配列からなるCDRH2及び配列番号26に記載のアミノ酸配列からなるCDRH3をコードするポリヌクレオチド、並びに配列番号29に記載のアミノ酸配列からなるCDRL1、配列番号30に記載のアミノ酸配列からなるCDRL2及び配列番号31に記載のアミノ酸配列からなるCDRL3をコードするポリヌクレオチド。 - 以下の(a)乃至(e)からなる群から選択されるいずれか1項に記載のポリヌクレオチドを含む請求項20又は21に記載のポリヌクレオチド:
(a)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号8に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(b)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号62においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(c)配列番号3に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号64においてアミノ酸番号21乃至129に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、
(d)配列番号13に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号18に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド、及び、
(e)配列番号23に記載のアミノ酸配列からなる重鎖の可変領域をコードするポリヌクレオチド、及び配列番号28に記載のアミノ酸配列からなる軽鎖の可変領域をコードするポリヌクレオチド。 - 請求項20乃至22のいずれか1項に記載のポリヌクレオチドを含有する発現ベクター。
- 請求項23に記載の発現ベクターにより形質転換された宿主細胞。
- 宿主細胞が真核細胞である請求項23に記載の宿主細胞。
- 請求項24又は25に記載の宿主細胞を培養する工程、及び当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程を含むことを特徴とする当該抗体又は当該抗体の機能性断片の製造方法。
- 請求項26に記載の製造方法により得られることを特徴とする抗体又は当該抗体の機能性断片。
- 機能性断片がFab、F(ab)2、Fab’及びFvからなる群から選択される請求項27に記載の抗体の機能性断片。
- N-結合への糖鎖付加、O-結合への糖鎖付加、N末のプロセッシング、C末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、N末にメチオニン残基の付加、プロリン残基のアミド化及びカルボキシル末端において1つ又は2つのアミノ酸が欠失した重鎖からなる群より選択される1又は2以上の修飾を含む請求項1乃至19、27及び28のいずれか1項に記載の抗体又は当該抗体の機能性断片。
- 重鎖のカルボキシル末端において1つ又は2つのアミノ酸が欠失している請求項29に記載の抗体。
- 2本の重鎖の双方でカルボキシル末端において1つのアミノ酸が欠失している請求項30に記載の抗体。
- 重鎖のカルボキシル末端のプロリン残基が更にアミド化されている請求項29乃至231のいずれか1項に記載の抗体。
- 抗体依存性細胞傷害活性を増強させるために糖鎖修飾が調節されている請求項1乃至19、及び26乃至31から選択されるいずれか1項に記載の抗体又は当該抗体の機能性断片。
- 請求項1乃至19、及び27乃至33のいずれか1項に記載の抗体又は当該抗体の機能性断片に薬物が結合している抗体-薬物コンジュゲート。
- 薬物が抗腫瘍性化合物である、請求項34に記載の抗体-薬物コンジュゲート。
- 抗体と抗腫瘍性化合物が、次式(a)乃至(f):(a) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(b) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(c) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
(d) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
(e) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-、
(f) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。
からなる群から選択されるいずれかの構造のリンカーを介して、結合している請求項35又は36に記載の抗体-薬物コンジュゲート。
(ここで、抗体は、-(Succinimid-3-yl-N)の末端において結合する。抗腫瘍性化合物は、1位のアミノ基の窒素原子を結合部位として、-(CH2)n2-C(=O)-部分のカルボニル基に結合する。上記式中GGFGは、グリシン-グリシン-フェニルアラニン-グリシンからなるペプチド結合でつながっているアミノ酸配列を示す。
-(Succinimid-3-yl-N)-は次式:
- リンカーが以下の(a)乃至(c)からなる群から選択されるいずれかの式で示される請求項34乃至37のいずれか1項に記載の抗体-薬物コンジュゲート:(a) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
(b) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2CH2-O-CH2-C(=O)-、
(c) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。 - リンカーが以下の(a)又は(b)の式で示される請求項34乃至38のいずれか1項に記載の抗体-薬物コンジュゲート:(a) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-、
(b) -(Succinimid-3-yl-N)-CH2CH2-C(=O)-NH-CH2CH2O-CH2CH2O-CH2CH2-C(=O)-GGFG-NH-CH2CH2CH2-C(=O)-。 - リンカーが以下の(a)の式で示される、請求項34乃至39のいずれか1項に記載の抗体-薬物コンジュゲート:
(a) -(Succinimid-3-yl-N)-CH2CH2CH2CH2CH2-C(=O)-GGFG-NH-CH2-O-CH2-C(=O)-。 - 抗体が以下の(a)乃至(x)からなる群から選択されるいずれか1項に記載の重鎖及び軽鎖を含む抗体又は当該抗体の機能性断片である、請求項41乃至43のいずれか1項に記載の抗体-薬物コンジュゲート:(a)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(b)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる、重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(c)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(d)配列番号48においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(e)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(f)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(g)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(h)配列番号50においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(i)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(j)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(k)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(l)配列番号52においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(m)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(n)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(o)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(p)配列番号54においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(q)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(r)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(s)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(t)配列番号56においてアミノ酸番号20乃至474に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(u)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号58においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(v)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号60においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(w)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号62においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖、
(x)配列番号44においてアミノ酸番号20乃至472に記載のアミノ酸配列からなる重鎖、及び配列番号64においてアミノ酸番号21乃至234に記載のアミノ酸配列からなる軽鎖。 - 重鎖がN-結合への糖鎖付加、O-結合への糖鎖付加、N末のプロセッシング、C末のプロセッシング、脱アミド化、アスパラギン酸の異性化、メチオニンの酸化、N末にメチオニン残基の付加、プロリン残基のアミド化及びカルボキシル末端において1つ又は2つのアミノ酸が欠失した重鎖からなる群より選択される1又は2以上の修飾を含む抗体である、請求項44に記載の抗体-薬物コンジュゲート。
- 選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が1から10個の範囲である請求項34乃至45のいずれか1項に記載の抗体-薬物コンジュゲート。
- 選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が2から8個の範囲である請求項34乃至46のいずれか1項に記載の抗体-薬物コンジュゲート。
- 選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が3から8個の範囲である請求項34乃至47のいずれか1項に記載の抗体-薬物コンジュゲート。
- 選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が7から8個の範囲である請求項34乃至48のいずれか1項に記載の抗体-薬物コンジュゲート。
- 選択された1種の薬物-リンカー構造の1抗体あたりの平均結合数が8である請求項34乃至49のいずれか1項に記載の抗体-薬物コンジュゲート。
- 請求項1乃至19、27乃至33に記載の抗体又は当該抗体の機能性断片、請求項34乃至50に記載の抗体-薬物コンジュゲートから選択されるいずれか、その塩、又はそれらの水和物を含むことを特徴とする医薬組成物。
- 抗腫瘍薬であることを特徴とする、請求項51に記載の医薬組成物。
- 腫瘍がGPR20が発現している腫瘍であることを特徴とする、請求項52に記載の抗腫瘍薬。
- 腫瘍が消化管間質腫瘍であることを特徴とする、請求項50又は51に記載の抗腫瘍薬。
- 更に、他の抗腫瘍薬を含むことからなる、請求項49乃至54のいずれか1項に記載の医薬組成物又は抗腫瘍薬。
- 請求項1乃至19、及び27乃至33に記載の抗体又は当該抗体の機能性断片、請求項34乃至50に記載の抗体-薬物コンジュゲート、その塩、又はそれらの水和物から選択されるいずれかを個体に投与することを特徴とする腫瘍の治療方法。
- 腫瘍が、消化管間質腫瘍であることを特徴とする、請求項56に記載の治療方法。
- 腫瘍が、チロシンキナーゼ阻害剤に抵抗性を示す腫瘍であることを特徴とする、請求項56又は57に記載の治療方法。
- 請求項1乃至19、及び27乃至33に記載の抗体又は当該抗体の機能性断片、請求項34乃至50に記載の抗体-薬物コンジュゲート、その塩、又はそれらの水和物から選択される少なくとも一つ、を含むことからなる医薬組成物及び少なくとも一つの抗腫瘍薬を、同時に、別々に又は連続して個体に投与することを特徴とする腫瘍の治療方法。
- 抗腫瘍薬が、チロシンキナーゼ阻害剤であることを特徴とする、請求項59に記載の腫瘍の治療方法。
- チロシンキナーゼ阻害剤が、スニチニブ(Sunitinib)、イマチニブ(Imatinib)、レゴラフェニブ(Regorafenib)から選択される少なくとも1つである、請求項60に記載の腫瘍の治療方法、
- 腫瘍が、チロシンキナーゼ阻害剤に抵抗性を示す消化管間質腫瘍であることを特徴とする、請求項56乃至61のいずれか1項に記載の腫瘍の治療方法。
- 請求項24又は25に記載の宿主細胞を培養する工程、当該工程で得られた培養物から目的の抗体又は当該抗体の機能性断片を採取する工程、及び当該工程で得られた抗体又は当該抗体の機能性断片と薬物-リンカー中間化合物を反応させる工程を含むことを特徴とする、抗体-薬物コンジュゲートの製造方法。
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PH12019501663A1 (en) | 2020-02-24 |
EP3572428A4 (en) | 2020-12-30 |
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SG11201906554RA (en) | 2019-08-27 |
CN110382535A (zh) | 2019-10-25 |
MX2019008059A (es) | 2019-12-11 |
JP2020099351A (ja) | 2020-07-02 |
IL268102A (en) | 2019-09-26 |
AU2018210081A1 (en) | 2019-08-08 |
KR102537651B1 (ko) | 2023-05-26 |
US20190225686A1 (en) | 2019-07-25 |
TW201831214A (zh) | 2018-09-01 |
EP3572428A1 (en) | 2019-11-27 |
BR112019012847A2 (pt) | 2019-12-10 |
JP2021105063A (ja) | 2021-07-26 |
RU2019123616A3 (ja) | 2021-06-02 |
JP6875575B2 (ja) | 2021-05-26 |
US10906974B2 (en) | 2021-02-02 |
IL268102B1 (en) | 2024-08-01 |
JPWO2018135501A1 (ja) | 2019-11-07 |
CA3050668C (en) | 2023-08-15 |
US20200362032A1 (en) | 2020-11-19 |
US11434289B2 (en) | 2022-09-06 |
CA3050668A1 (en) | 2018-07-26 |
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