WO2017002776A1 - 抗体-薬物コンジュゲートの選択的製造方法 - Google Patents
抗体-薬物コンジュゲートの選択的製造方法 Download PDFInfo
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- WO2017002776A1 WO2017002776A1 PCT/JP2016/069068 JP2016069068W WO2017002776A1 WO 2017002776 A1 WO2017002776 A1 WO 2017002776A1 JP 2016069068 W JP2016069068 W JP 2016069068W WO 2017002776 A1 WO2017002776 A1 WO 2017002776A1
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- A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
- A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
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Definitions
- the present invention relates to a method for producing an antibody-drug conjugate composition in which the number and position of drug binding are controlled, and to an antibody-drug conjugate composition in which the number and position of drug binding are controlled.
- Antibody-drug conjugate (Antibody-Drug Conjugate; hereinafter referred to as “ADC”) in which a cytotoxic drug is bound to an antibody that binds to an antigen that is expressed on the surface of a cancer cell and can be internalized in the cell. Can be expected to accumulate the drug in the cancer cell and kill the cancer cell by being able to selectively deliver the drug to the cancer cell (see Non-Patent Documents 1 to 3).
- An antibody-drug conjugate an antibody-drug conjugate obtained by binding exatecan, which is a camptothecin derivative, to an anti-B7-H3 antibody is known (Patent Document 1).
- the antibody has four interchain disulfides, and is more easily reduced because it is closer to the solvent than other disulfides, and can be used as a binding site for the drug (or drug linker) in the antibody-drug conjugate.
- An antibody-drug conjugate in which 2 to 8 drugs are bound per antibody molecule is prepared by reducing the interchain disulfide of the antibody and binding the drug to the resulting thiol group.
- An antibody-drug conjugate in which 8 drugs are bound per molecule of antibody may have safety problems such as side effects and toxicity even though it has an excellent antitumor effect.
- antibody-drug conjugates having an average number of drug bonds of less than 8 may be used.
- An antibody-drug conjugate having an average drug binding number of less than 8 can be obtained by, for example, controlling the amount of drug per antibody molecule to react, and the reaction product has a drug binding number.
- compositions of antibody-drug conjugates of 2, 4, 6 and 8. Thus, even antibody-drug conjugate compositions with the same average drug binding number may have different therapeutic efficacy and toxicity if the distribution of each drug binding number is different.
- an antibody-drug conjugate composition having an average drug bond number of 4 and an antibody-drug conjugate content of 0 and 8 drug bonds the antibody-drug conjugate having a drug bond number of 4 is high. Compared with the case where the content of is high, the therapeutic efficacy may be reduced, and toxicity may be strongly developed. In addition, even antibody-drug conjugates with the same number of drug bonds may have different therapeutic efficacy and toxicity due to differences in drug binding positions. Therefore, in the production of an antibody-drug conjugate composition, a method for producing an antibody-drug conjugate composition in which the number and position of drug binding are controlled is required.
- an antibody-drug conjugate composition in which the number and position of drug binding are controlled by a simpler operation. That is, by reducing the antibody with a reducing agent in a buffer solution at ⁇ 10 to 10 ° C., and reacting the drug linker intermediate with the obtained antibody having a thiol group, the average drug binding number is 3.5. It was found that an antibody-drug conjugate composition with a content of an antibody-drug conjugate of ⁇ 4.5 and having 4 drug linkers bonded to the heavy chain-light chain thiol is 50% or more can be produced.
- the antibody-drug conjugate composition of the present invention is an antibody-drug conjugate composition produced by a conventional production method (average drug binding number is 3.5 to 4.5, and a drug is added to the thiol between heavy chain and light chain). It was found that the antibody-drug conjugate composition having an antibody-drug conjugate content of 35% or less having 4 linkers bound thereto has superior safety, and the present invention has been completed.
- the present invention (1) (I) reacting the antibody with a reducing agent in a buffer to reduce interchain disulfide; and (Ii) reacting a drug linker intermediate with the antibody having a thiol group obtained in (i);
- a method for producing an antibody-drug conjugate composition comprising: The reaction temperature in step (i) is ⁇ 10 to 10 ° C .; The antibody-drug conjugate composition produced has an average number of drug bonds of 3.5 to 4.5, and a content ratio of antibody-drug conjugate in which four drug linkers are bonded to the thiol between the heavy chain and the light chain is 50% or more.
- the manufacturing method characterized by the above-mentioned.
- the production method according to (1), wherein the antibody-drug conjugate composition produced has an average drug binding number of 4.0 to 4.1.
- the content of the antibody-drug conjugate in which four drug linkers are bonded to the heavy chain-light chain thiol is in the range of 50 to 90%, (1) or ( The manufacturing method as described in 2).
- the antibody-drug conjugate composition produced has a content of antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-light chain thiol in the range of 50 to 80%, according to (3) Manufacturing method.
- the antibody-drug conjugate composition to be produced has a content of antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-light chain thiol in the range of 50 to 70%. Manufacturing method.
- the antibody-drug conjugate composition produced has a content of antibody-drug conjugate in which four drug linkers are bonded to a thiol between the heavy chain and light chain in the range of 50 to 60%. Manufacturing method.
- the content of the antibody-drug conjugate in which four drug linkers are bonded to the heavy chain-heavy chain thiol is 5% or less, (1) to (6) The manufacturing method of any one of Claims.
- the production method according to (7) wherein the content of the antibody-drug conjugate in which four drug linkers are bonded to the heavy chain-heavy chain thiol of the antibody-drug conjugate composition to be produced is 1% or less. .
- the antibody-drug conjugate composition to be produced contains an antibody-drug conjugate in which two drug linkers are bonded to the heavy chain-heavy chain thiol and two drug linkers are bonded to the heavy chain-light chain thiol.
- the antibody-drug conjugate composition to be produced contains an antibody-drug conjugate in which two drug linkers are bonded to the heavy chain-heavy chain thiol and two drug linkers are bonded to the heavy chain-light chain thiol.
- (11) The production method according to any one of (1) to (10), wherein the reaction temperature in the step (i) is from -5 to 5 ° C.
- (12) The production method according to (11), wherein the reaction temperature in the step (i) is -3 to 3 ° C.
- the production method according to (12), wherein the reaction temperature in the step (i) is 0 to 2 ° C.
- the production method according to (22), wherein the antibody is an anti-TROP2 antibody.
- the production method according to (22), wherein the antibody is an anti-CD98 antibody.
- the production method according to (22), wherein the antibody is an anti-B7-H3 antibody.
- the production method according to (22), wherein the antibody is an anti-HER2 antibody.
- the drug linker intermediate is
- the content of the antibody-drug conjugate in which two drug linkers are bonded to the heavy chain-heavy chain thiol and two drug linkers are bonded to the heavy chain-light chain thiol is 5% or less, (31) to (38) The antibody-drug conjugate composition according to any one of (38). (40) (39) The content of the antibody-drug conjugate in which two drug linkers are bonded to the heavy chain-heavy chain thiol and two drug linkers are bonded to the heavy chain-light chain thiol is 1% or less.
- a drug linker is any one of (31) to (40), wherein the antibody is an anti-TROP2 antibody, an anti-CD98 antibody, an anti-B7-H3 antibody, or an anti-HER2 antibody.
- A represents the binding position with the antibody, and -GGFG- represents a tetrapeptide residue consisting of glycine-glycine-phenylalanine-glycine.
- the antibody according to (46) A drug conjugate composition.
- (48) (30) A pharmaceutical composition comprising the antibody-drug conjugate composition according to any one of (47). (49) The pharmaceutical composition according to (48), for the treatment of tumors and / or cancers.
- (51) (30) A method for treating a tumor and / or cancer, comprising administering the antibody-drug conjugate composition according to any one of (47).
- a method of producing an antibody having a thiol group comprising a step of reacting an antibody with a reducing agent in a buffer solution to reduce interchain disulfide, The reaction temperature is ⁇ 10 to 10 ° C.,
- the manufactured antibody having a thiol group has an average drug bond number of 3.5 to 4.5, and the content of an antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-light chain thiol is 50% or more.
- Used to produce an antibody-drug conjugate composition The manufacturing method characterized by the above-mentioned.
- the production method according to (54) which is used for producing
- the antibody-drug conjugate composition, wherein the antibody having a thiol group produced has a content of an antibody-drug conjugate in which 4 drug linkers are bonded to a heavy chain-light chain thiol in the range of 50 to 80%.
- the antibody-drug conjugate composition, wherein the antibody having the thiol group produced has a content of antibody-drug conjugate in which 4 drug linkers are bonded to the heavy chain-light chain thiol in the range of 50 to 70%.
- the manufacturing method according to (56), which is used to manufacture (58) The antibody-drug conjugate composition in which the produced antibody-drug conjugate has a content of antibody-drug conjugate in which four drug linkers are bonded to the heavy chain-light chain thiol in the range of 50-60%.
- An antibody-drug conjugate composition in which an antibody having a thiol group to be produced has a content of an antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-heavy chain thiol is 5% or less (53) to (58), the production method according to any one of (53) to (58).
- An antibody-drug conjugate composition in which an antibody having a thiol group to be produced has an antibody-drug conjugate content of 1% or less in which four drug linkers are bonded to a heavy chain-heavy chain thiol. The manufacturing method according to (59), which is used for the purpose.
- the produced antibody having a thiol group has a content ratio of an antibody-drug conjugate in which two drug linkers are bonded to a heavy chain-heavy chain thiol and two drug linkers are bonded to a heavy chain-light chain thiol.
- the production method according to any one of (53) to (60), which is used for producing an antibody-drug conjugate composition that is 5% or less.
- the produced antibody having a thiol group has a content ratio of an antibody-drug conjugate in which two drug linkers are bonded to a heavy chain-heavy chain thiol and two drug linkers are bonded to a heavy chain-light chain thiol.
- the present invention provides a method for producing an antibody-drug conjugate composition in which the number and position of drug binding are controlled, and an antibody-drug conjugate composition in which the number and position of drug binding are controlled.
- the antibody-drug conjugate composition of the present invention is excellent in safety and useful as a medicament for the treatment of tumors and / or cancers.
- an antibody-drug conjugate composition aggregated at a certain number of drug bonds and binding positions can be obtained, it is excellent in terms of quality control and is preferable.
- cancer and “tumor” are used interchangeably.
- gene includes not only DNA but also its mRNA, cDNA and its cRNA.
- polynucleotide is used in the same meaning as nucleic acid, and includes DNA, RNA, probe, oligonucleotide, and primer.
- polypeptide and “protein” are used interchangeably.
- cell includes a cell in an animal individual and a cultured cell.
- interchain disulfide refers to a disulfide between two heavy chains (heavy chain-to-heavy chain disulfide) in an antibody, or a disulfide between a heavy chain and a light chain (heavy chain—light chain). Interchain disulfide).
- interchain thiol refers to a thiol group obtained by reduction of an interchain disulfide of an antibody.
- “heavy chain-heavy chain thiol” refers to a thiol group obtained by reduction of an antibody heavy chain-heavy chain disulfide.
- heavy chain-light chain thiol refers to a thiol group obtained by reduction of an antibody heavy chain-light chain disulfide.
- tumor-associated antigen refers to an antigen that is expressed in normal cells and tumor cells but is relatively limited to tumor cells.
- tumor-specific antigen refers to an antigen specific to tumor cells.
- TROP2 is used in the same meaning as TROP2 protein.
- CD98 is used interchangeably with CD98 protein. Since CD98 consists of a heavy chain and a light chain, “CD98 heavy chain” and “CD98 light chain” are used interchangeably with CD98 heavy chain protein and CD98 light chain protein, respectively. Further, in this specification, “CD98” is interchangeable with “CD98 heavy chain” and “CD98 light chain” or “CD98 heavy chain” or “CD98 light chain” unless otherwise specified. Used for.
- anti-TROP2 antibody refers to an antibody that can bind to TROP2.
- anti-CD98 antibody refers to an antibody that can bind to CD98.
- anti-B7-H3 antibody refers to an antibody that can bind to B7-H3.
- anti-HER2 antibody refers to an antibody that can bind to HER2.
- cytotoxicity refers to a pathological change in cells in some form, and is not limited to direct trauma, but also includes DNA cleavage, base dimer formation, chromosomal This refers to any structural or functional damage to cells such as cutting, damage to cell division equipment, or reduction of various enzyme activities.
- cytotoxic activity refers to causing the above-mentioned cytotoxicity.
- antibody-dependent cytotoxic activity means “antibody dependent cellular cytotoxicity (ADCC) activity”, and the activity of NK cells to damage target cells such as tumor cells via antibodies. means.
- ADCC antibody dependent cellular cytotoxicity
- complement-dependent cytotoxic activity means “complement-dependent cytotoxicity (CDC) activity”, which is an activity in which complement damages target cells such as tumor cells via antibodies. Means.
- epitope means a partial peptide or a partial three-dimensional structure of an antigen to which a specific antibody binds.
- the epitope which is a partial peptide of the antigen can be determined by a method well known to those skilled in the art such as immunoassay, for example, the following method.
- various partial structures of the antigen are prepared. In producing the partial structure, a known oligopeptide synthesis technique can be used.
- the epitope can be determined by synthesizing shorter peptides and examining their reactivity with those peptides.
- an epitope which is a partial three-dimensional structure of an antigen to which a specific antibody binds can be determined by specifying amino acid residues of the antigen adjacent to the antibody by X-ray structural analysis.
- an antibody that binds to the same epitope means different antibodies that bind to a common epitope. If the second antibody binds to the partial peptide or partial conformation 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 and the antigen), a specific epitope is determined. Even if the sequence or structure of is not determined, it can be determined that the first antibody and the second antibody bind to the same epitope. Furthermore, 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, the second antibody is expected to have the same activity. it can.
- CDR means a complementarity determining region (CDR). It is known that there are three CDRs in each of the heavy and light chains of the antibody molecule. CDRs, also called hypervariable domains, are sites in the variable region of the heavy and light chains of an antibody that have particularly high primary structure variability and are heavy and light chain polypeptide chains. In the primary structure, each is separated into three locations.
- CDRH1, CDRH2, CDRH3 from the N-terminal side of the heavy chain amino acid sequence
- CDRL1 from the N-terminal side of the light chain amino acid sequence. Indicated as CDRL2 and CDRL3. These sites are close to each other on the three-dimensional structure and determine the specificity for the antigen to be bound.
- “several” means 2 to 10 pieces. Preferably, it is 2-9, more preferably 2-8, more preferably 2-7, more preferably 2-6, more preferably 2-5, More preferably, the number is 2 to 4, more preferably 2 to 3, and still more preferably 2.
- antibody-drug conjugate composition means an antibody-drug conjugate in which two drug linkers are bonded, an antibody-drug conjugate in which four drug linkers are bonded, and six drug linkers are bonded. It means a composition comprising an antibody-drug conjugate, an antibody-drug conjugate to which 8 drug linkers are bound, and an antibody to which no drug linker is bound in any proportion.
- ADC composition an antibody-drug conjugate composition
- the “content” means the content (mol% based on the antibody) of the antibody-drug conjugate having a specific drug binding number and binding position in the antibody-drug conjugate composition. To do.
- Antibodies The antibodies used in the present invention can be raised against a target antigen, such as a tumor specific antigen (TAA) or a tumor associated antigen (TSA).
- TAA tumor specific antigen
- TSA tumor associated antigen
- Such antibodies have the property of recognizing tumor cells, the property of being able to bind to such tumor cells, and the property of being taken up and internalized within such tumor cells.
- the target antigen is not particularly limited as long as it is an antigen related to tumor cells.
- the antibody used in the present invention can be obtained, for example, by the method described in WO2009 / 091048, WO2011 / 027808, or WO2012 / 133572. That is, a non-human animal is immunized with a target antigen, lymphoid fluid, lymphoid tissue, blood cell sample or bone marrow-derived cells are collected from the immunized animal, and the plasma cells of the non-human animal that specifically bind to the target antigen and / or Or a plasmablast is selected. An antibody gene against the target antigen is collected from the obtained plasma cells and / or plasma blast cells, the base sequence thereof is identified, and the antibody or antibody fragment can be obtained based on the base sequence of the identified gene. The obtained antibodies can be screened for antibodies applicable to human diseases by testing the binding to the antigen of interest.
- Hybridomas can be established by fusing antibody-producing cells that produce antibodies against the target antigen and myeloma cells to obtain monoclonal antibodies. Specific examples of such a method are described in WO2009 / 048072 (published on April 16, 2009) and WO2010 / 117011 (published on October 14, 2010).
- the antibodies used in the present invention include genetically modified antibodies that have been artificially modified for the purpose of reducing the heteroantigenicity to humans, such as chimeric antibodies, humanized antibodies, and human antibodies. Etc. are also included. 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 heterologous to 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. USA, 81, 6851-6855, (1984))
- Humanized antibodies include antibodies in which only CDRs are incorporated into human-derived antibodies (see Nature (1986) 321, pp.522-525). By CDR grafting, in addition to CDR sequences, amino acid residues of some frameworks Examples of the antibody include an antibody (International Publication No. WO90 / 07861 pamphlet) transplanted to a human antibody.
- Preferred antibodies of the present invention include anti-TROP2 antibody, anti-CD98 antibody, anti-B7-H3 antibody, or anti-HER2 antibody.
- humanized anti-TROP2 antibodies include (1) an amino acid sequence consisting of amino acid residues 20 to 140 of SEQ ID NO: 2, and (2) at least 95% homology to the amino acid sequence of (1) above. And (3) a heavy chain comprising a heavy chain variable region 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) an amino acid sequence consisting of amino acid residues 21 to 129 of SEQ ID NO: 4, (5) an amino acid sequence having at least 95% homology with the amino acid sequence of (4) above, and (6) Examples include any combination of light chains comprising a light chain variable region consisting of 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. Kill.
- a preferred combination antibody of the above heavy chain and light chain consists of a heavy chain consisting of amino acid sequences consisting of amino acid residues 20 to 470 of SEQ ID NO: 2 and 21st to 234th amino acid residues of SEQ ID NO: 4.
- An antibody (hTINA1-H1L1) consisting of a light chain consisting of an amino acid sequence can be mentioned.
- Humanized anti-TROP2 antibodies include CDRH1 (TAGMQ) consisting of the amino acid sequence shown in SEQ ID NO: 5 in the sequence listing, CDRH2 (WINTHSGVPKYAEDFKG) consisting of the amino acid sequence shown in SEQ ID NO: 6, and the amino acid sequence shown in SEQ ID NO: 7.
- CDRL3 (SGFGSSYWYFDV), CDRL1 (KASQDVSTAVA) consisting of the amino acid sequence shown in SEQ ID NO: 8 in the sequence listing, CDRL2 (SASYRYT) consisting of the amino acid sequence shown in SEQ ID NO: 9, and the amino acid sequence shown in SEQ ID NO: 10
- CDRL3 QQHYITPLT
- humanized anti-CD98 antibodies include (1) an amino acid sequence consisting of amino acid residues 20 to 135 of SEQ ID NO: 12, and (2) at least 95% identity to the amino acid sequence of (1) above. And (3) a heavy chain comprising a heavy chain variable region 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) an amino acid sequence consisting of amino acid residues 21 to 135 of SEQ ID NO: 14, (5) an amino acid sequence having at least 95% identity to the amino acid sequence of (4) above, and (6) List any combination of light chains comprising a light chain variable region consisting of any one of the amino acid sequences in which one or several amino acids are deleted, substituted or added in the amino acid sequence of (4) above It can be.
- a preferred combination antibody of the above heavy chain and light chain consists of a heavy chain consisting of the amino acid sequence of amino acid residues 20 to 465 of SEQ ID NO: 12 and 21st to 240th amino acid residues of SEQ ID NO: 14.
- An antibody (hM23-H1L1) consisting of a light chain consisting of an amino acid sequence can be mentioned.
- Humanized anti-CD98 antibodies include CDRH1 (NYLIE) consisting of the amino acid sequence shown in SEQ ID NO: 15, CDRH2 (VINPGSGVTNYNEKFKG) consisting of the amino acid sequence shown in SEQ ID NO: 16, CDRH3 consisting of the amino acid sequence shown in SEQ ID NO: 17 ( AEAWFAY), CDRL1 (KSSQSLLYSSNQKNYLA) consisting of the amino acid sequence shown in SEQ ID NO: 18 in the sequence listing, CDRL2 (WASTRES) consisting of the amino acid sequence shown in SEQ ID NO: 19, and CDRL3 (QRYYGYPWT consisting of the amino acid sequence shown in SEQ ID NO: 20) ) Is not limited to a specific humanized antibody.
- humanized anti-B7-H3 antibodies include (1) an amino acid sequence consisting of amino acid residues 20 to 141 of SEQ ID NO: 25, (2) at least 95% or more of the amino acid sequence of (1) above.
- An amino acid sequence having identity and (3) a heavy chain comprising a heavy chain variable region consisting of 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 (4) an amino acid sequence consisting of amino acid residues 21 to 128 of SEQ ID NO: 26, (5) an amino acid sequence having at least 95% identity to the amino acid sequence of (4) above, and ( 6) List any combination of light chains comprising a light chain variable region consisting of any one of the amino acid sequences wherein one or several amino acids have been deleted, substituted or added in the amino acid sequence of (4) above. It can be.
- a preferred combination antibody of the above heavy chain and light chain consists of a heavy chain consisting of amino acid sequences consisting of amino acid residues 20 to 471 of SEQ ID NO: 25 and 21st to 233rd amino acid residues of SEQ ID NO: 26.
- An antibody (M30-H1-L4) consisting of a light chain consisting of an amino acid sequence can be mentioned.
- CDRH1 (NYVMH) consisting of the amino acid sequence shown in SEQ ID NO: 27, CDRH2 (YINPYNDDVKYNEKFKG) consisting of the amino acid sequence shown in SEQ ID NO: 28, and amino acid sequence shown in SEQ ID NO: 29 CDRH3 (WGYYGSPLYYFDY), CDRL1 (RASSRLIYMH) consisting of the amino acid sequence shown in SEQ ID NO: 30 of the sequence listing, CDRL2 (ATSNLAS) consisting of the amino acid sequence shown in SEQ ID NO: 31, and CDRL3 consisting of the amino acid sequence shown in SEQ ID NO: 32 As long as it retains (QQWNSNPPT), it is not limited to a specific humanized antibody.
- humanized anti-HER2 antibodies include a heavy chain consisting of amino acid sequences 1 to 449 of SEQ ID NO: 33 and an amino acid sequence consisting of amino acids 1 to 214 of SEQ ID NO: 34
- An antibody consisting of a light chain (trastuzumab; US Pat. No. 5,721,337) can be mentioned.
- CDR-modified humanized antibodies in which 1 to 3 amino acid residues in each CDR are substituted with other amino acid residues are also included in the antibodies used in the present invention as long as they have binding activity to tumor cells.
- antibodies used in the present invention include human antibodies.
- the human antibody is produced by 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 vol.10, p. 69-73 (Kitagawa, Y., Matsuda, T. and Iijima, S. eds.), Kluwer Academic Publishers, 1999 .; Tomizuka, K. et. Al., Proc. Natl. Acad. Sci. USA (2000) 97, p.722-727, etc.).
- 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; menCarmen, S . Et. Al., Briefings in Functional Genomics and Proteomics (2002), 1 (2), p.189-203; Siriwardena, D. et. Al., Ophthalmology (2002) 109 (3), p.427-431 Etc.) are also known.
- a phage display method (Nature ⁇ ⁇ ⁇ Biotechnology (2005), 23, ⁇ ⁇ ⁇ ⁇ (9), p.1105) that expresses the variable region of a human antibody as a single chain antibody (scFv) on the phage surface and selects phages that bind to the antigen. -1116) can be used.
- the DNA sequence encoding the variable region of the human antibody that binds to the antigen can be determined.
- an expression vector having the sequence is prepared, and human antibodies can be obtained by introducing it into an appropriate host and expressing it (WO92 / 01047, WO92 / 20791, WO93 / 06213, WO93 / 11236, WO93 / 19172, WO95 / 01438, WO95 / 15388, Annu. Rev. Immunol (1994) 12, p.433-455, Nature Biotechnology (2005) 23 (9), p. 1105-1116).
- the above-mentioned antibody can be selected for a suitable antibody by evaluating the binding to the target antigen.
- Evaluation of binding to the target antigen is not limited to the use of Biacore T200, but KinExA (Sapidyne Instruments Inc.) based on detection principle based on the detection principle of surface plasmon resonance (SPR) and Kinetic Exclusion Assay It is also possible to use the BLItz system (Pall) or the ELISA (Enzyme-Linked ImmunoSorbent Assay) method based on the detection principle of Bio-Layer Interferometry.
- the activity internalized in cells is as follows: (1) An assay that visualizes antibodies taken into cells using a secondary antibody (fluorescent label) that binds to a therapeutic antibody with a fluorescence microscope (Cell Death and Differentiation (2008) 15, 751-761), (2) Assays that measure the amount of fluorescence incorporated into cells using secondary antibodies (fluorescent labels) that bind to therapeutic antibodies (MolecularMBiology of the Cell Vol. 15, 5268-5282, -5December 2004), or (3) Mab-ZAP assay that inhibits cell proliferation when released into cells using immunotoxins that bind to therapeutic antibodies (BioTechniques 28: 162-165, January 2000 ). As the immunotoxin, a diphtheria toxin catalytic domain and a protein G recombinant complex protein can also be used.
- An example of another index for comparing the properties of antibodies is antibody stability.
- DSC Differential scanning calorimetry
- Tm thermal denaturation midpoint
- a suitable antibody can be selected using stability as an index.
- Other indicators for selecting antibodies include high yields in appropriate 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. .
- WO99 / 54342, WO2000 / 61739, WO2002 / 31140, and the like are known as techniques for regulating antibody sugar chain modification, but are not limited thereto.
- 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.
- eukaryotic cells are used as hosts, animal cells, plant cells, and eukaryotic microorganisms can be used.
- animal cells include mammalian cells such as COS cells (Gluzman, Y., Cell (1981) 23, p.175-182, ATCC CRL-1650), mouse fibroblasts NIH3T3 (ATCC No. CRL-1658) and Chinese hamster ovary cells (CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad. Sci. USA (1980) 77, p.4126-4220).
- COS cells Gluzman, Y., Cell (1981) 23, p.175-182, ATCC CRL-1650
- mouse fibroblasts NIH3T3 ATCC No. CRL-1658
- Chinese hamster ovary cells CHO cells, ATCC CCL-61) dihydrofolate reductase-deficient strains (Urlaub, G. and Chasin, L. A. Proc. Natl. Acad.
- 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. From the antibodies having equivalent binding activity, those that can be easily produced as pharmaceuticals can be selected using the yield as an index.
- IgG IgG1, IgG2, IgG3, IgG4
- IgM IgA (IgA1, IgA2)
- IgD IgD
- IgE preferably IgG or IgM, more preferably IgG1, IgG2 or IgG3 can be mentioned.
- antibody functions include antigen-binding activity, activity that neutralizes antigen activity, activity that enhances antigen activity, antibody-dependent cytotoxic activity, complement-dependent cytotoxic activity, complement-dependent activity Mention may be made of cellular cytotoxic activity and internalization ability.
- the antibody used in the present invention may be a multispecific antibody having specificity for at least two different antigens.
- a molecule binds to two types of antigens (ie, bispecific antibodies), but the “multispecific antibody” in the present invention is more than that (for example, three types). It includes an antibody having specificity for the antigens.
- the antibody used in the present invention may be an antibody having 80% to 99% identity (or homology) as compared to the heavy chain and / or light chain of the above antibody.
- sequences having high homology with the above heavy chain amino acid sequence and light chain amino acid sequence, it is possible to select an antibody having the same antigen binding ability and internalization ability as each of the above antibodies.
- homology is generally 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 99%. It is the above homology.
- amino acid sequence in which 1 to several amino acid residues are substituted, deleted and / or added to the heavy chain and / or light chain amino acid sequences various effects equivalent to those of the above-described antibodies can be obtained.
- the number of amino acid residues to be substituted, deleted and / or added is generally 10 amino acid residues or less, preferably 9 amino acid residues or less, more preferably 8 amino acid residues or less, and more Preferably it is 7 amino acid residues or less, more preferably 6 amino acid residues or less, more preferably 5 amino acid residues or less, more preferably 4 amino acid residues or less, more preferably 3 amino acid residues. Or less, more preferably 2 amino acid residues or less, and most preferably 1 amino acid residue.
- the present invention also includes antibodies having such modifications, including deletions in which 1 or 2 amino acids have been deleted at the heavy chain carboxyl terminus, and such a deletions that have been amidated (for example, at the carboxyl terminus site).
- Heavy chain in which a proline residue is amidated is not limited to the above type.
- the two heavy chains constituting the antibody according to the present invention may be any one of the full length and the heavy chain selected from the group consisting of the above-mentioned deletion forms, 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 a carboxyl group in both of the two heavy chains. A case where the terminal amino acid residue is deleted can be mentioned.
- Blast algorithm version 2.2.2 (Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.Lipman (1997 ), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25: 3389-3402).
- Blast algorithm can also be used by accessing www.ncbi.nlm.nih.gov/blast on the Internet.
- the Blast ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ ⁇ algorithm calculates two types of percentage values, Identity (or Identities) and Positivity (or Positivities).
- the former is a value when amino acid residues match between two types of amino acid sequences for which homology is to be obtained, and the latter is a numerical value considering amino acid residues having similar chemical structures.
- the value of Identity (identity) when amino acid residues are coincident is used as the value of homology.
- the obtained antibody can be purified to homogeneity. Separation and purification of antibodies may be carried out using separation and purification methods used for ordinary proteins. For example, 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. (Strategies 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 D )) Is not limited to these.
- chromatography examples include affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, reverse phase chromatography, and adsorption chromatography.
- Examples of columns used for affinity chromatography include protein A columns and protein G columns.
- Drug The drug used in the present invention 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 part 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.
- a drug is bound to an antibody via a linker moiety having a specific structure.
- a drug linker including the drug and the linker moiety may be referred to as a drug.
- antitumor compound examples include calicheamicin, doxorubicin, daunorubicin, mitomycin C, bleomycin, cyclocytidine, vincristine, vinblastine, methotrexate, cisplatin or a derivative thereof, auristatin or a derivative thereof, maytansine or a derivative thereof, taxol or a derivative thereof Derivatives, camptothecin or derivatives thereof can be mentioned, and exatecan or monomethyl auristatin E is preferred.
- Exatecan ((1S, 9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H, 12H-benzo [de] pyrano [3 '), a camptothecin derivative , 4 ′: 6,7] indolidino [1,2-b] quinoline-10,13 (9H, 15H) -dione) is a compound represented by the following formula:
- Linker The drug used in the present invention can be bound to an antibody via a linker.
- the linker used in the present invention preferably has an N-substituted maleimidyl group.
- the linker can include a cleavable linker and a non-cleavable linker.
- Examples of the cleavable linker include peptide linkers that are cleaved by intracellular proteases such as lysosomal protease and endosomal protease.
- the linker used in the present invention is preferably
- GGFG is a tetrapeptide residue consisting of glycine-glycine-phenylalanine-glycine.
- the linker used in the present invention can be prepared, for example, according to the method described in Example 58 of WO2014 / 057687.
- Drug Linker Intermediate used in the present invention can be produced by reacting the carboxyl group of the linker compound with the amino group of the antitumor compound using a condensing agent.
- the drug linker intermediate used in the method of the present invention is not particularly limited as long as it is a compound that is subjected to a reaction with an interchain thiol of an antibody, but is preferably a compound having an N-substituted maleimidyl group, more preferably Is
- the drug linker intermediate used in the present invention can be prepared, for example, according to the method described in Example 58, Example 43 and Example 14 of WO2014 / 057687.
- the antibody and the drug can be bound via a linker by reacting with an interchain thiol generated by reduction of the interchain disulfide of the antibody. it can.
- a compound having an N-substituted maleimidyl group is preferable as the drug linker intermediate, but is not limited thereto, and the production of the present invention is not limited as long as it has a functional group capable of proceeding with an interchain thiol of an antibody. Can be applied to the method. 5).
- Antibody-Drug Conjugate In the antibody-drug conjugate, the number of drugs bound to one antibody molecule is an important factor affecting the effectiveness and safety. Since there are four interchain disulfides in an antibody, and the disulfide is composed of two thiol groups, the number of drugs bound to one antibody molecule is 2, 4, 6, or 8.
- An antibody-drug conjugate in which two drugs are bound per antibody molecule is an antibody-drug in which two drug linkers are bound to a thiol between a heavy chain and a light chain.
- Conjugate hereinafter also referred to as “D2-1”
- D2-2 antibody-drug conjugate
- An antibody-drug conjugate in which 4 drugs are bound per antibody molecule is an antibody-drug in which 4 drug linkers are bound to a thiol between the heavy chain and the light chain.
- Conjugate hereinafter also referred to as “D4-1”
- antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-heavy chain thiol hereinafter also referred to as “D4-2”.
- D4-3 antibody-drug conjugate in which two drug linkers are bonded to the heavy chain-light chain thiol and two drug linkers are bonded to the heavy chain-heavy chain thiol.
- D4-1 antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-heavy chain thiol
- D4-3 antibody-drug conjugate in which two drug linkers are bonded to the heavy chain-light chain thiol and two drug linkers are bonded to the heavy chain-heavy chain thiol.
- an antibody-drug conjugate (hereinafter also referred to as “D6”) in which 6 drugs are bound per antibody molecule, 4 drug linkers bind to the thiol between the heavy chain and the light chain, and the heavy chain—
- An antibody-drug conjugate (hereinafter also referred to as “D6-1”) in which two drug linkers are bonded to the heavy chain thiol, and two drug linkers are bonded to the heavy chain-light chain thiol.
- D6-2 antibody-drug conjugate in which four drug linkers are bonded to a heavy chain-heavy chain thiol.
- an antibody-drug conjugate (hereinafter also referred to as “D8”) in which 8 drugs are bound per antibody molecule, 4 drug linkers bind to the thiol between the heavy chain and the light chain, and the heavy chain— There are antibody-drug conjugates in which four drug linkers are attached to the inter-heavy chain thiol.
- the interchain thiol of the antibody binds to, for example, the 3-position of the N-substituted maleimidyl group of the drug linker intermediate by forming a thioether. That is, the binding portion between the antibody and the drug linker can be represented, for example, by the following formula:
- antibody-S- is derived from an antibody
- a in the formula represents the binding position with the antibody. More preferably,
- a in the formula represents the binding position with the antibody.
- Antibody-drug conjugates are manufactured by defining reaction conditions such as the amount of raw materials and reagents to be reacted so that the number of drug bonds can be controlled. Unlike chemical reactions of low-molecular compounds, It is usually obtained as a mixture in which different numbers of drugs are 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 production method of the present invention is a method of producing an antibody-drug conjugate composition in which the number of drug bonds and the position of the drug are controlled, and selectively reduces the heavy chain-light chain disulfide of the antibody to a thiol group.
- the first step of conversion consists of a second step of producing an antibody-drug conjugate composition in which the number of drug bonds and the position of the drug are controlled by reacting an antibody having a thiol group with a drug linker intermediate.
- An antibody having a thiol group can be produced by reacting an antibody with a reducing agent in a buffer solution at ⁇ 10 to 10 ° C.
- the reaction temperature is preferably -5 to 5 ° C, more preferably -3 to 3 ° C, still more preferably 0 to 2 ° C, and even more preferably 0 to 1 ° C.
- the amount of the reducing agent is 1 to 4 molar equivalents, preferably 2 to 3 molar equivalents, per antibody molecule.
- the reducing agent for example, tris (2-carboxyethyl) phosphine or a salt thereof, dithiothreitol, 2-mercaptoethanol and the like can be used, preferably tris (2-carboxyethyl) phosphine or a salt thereof, More preferred is tris (2-carboxyethyl) phosphine hydrochloride.
- histidine buffer phosphate buffer, borate buffer, acetic acid buffer, HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid) buffer, etc.
- histidine buffer preferably histidine buffer. is there.
- the buffer preferably contains a chelating agent.
- a chelating agent for example, ethylenediaminetetraacetic acid (hereinafter sometimes referred to as “EDTA”) or diethylenetriaminepentaacetic acid can be used, and ethylenediaminetetraacetic acid is preferable. These may be used at a concentration of 1 to 20 mM.
- EDTA ethylenediaminetetraacetic acid
- diethylenetriaminepentaacetic acid diethylenetriaminepentaacetic acid
- the reaction time is preferably 1 to 15 hours, more preferably 3 to 10 hours, and even more preferably 5 to 7 hours.
- the pH during the reaction is pH 5-9, preferably pH 6-8, more preferably pH 6.8-7.2.
- An antibody-drug conjugate composition can be produced by reacting the drug linker intermediate with the antibody having the thiol group obtained in the first step. .
- the amount of the drug linker intermediate to be used is 2 to 10 molar equivalents, preferably 4 to 6 molar equivalents, per antibody molecule.
- a solution in which the drug linker intermediate is dissolved may be added to the buffer solution containing the antibody having a thiol group obtained in the first step and reacted.
- Solvents that dissolve the drug linker intermediate include 50% aqueous acetone, 80% aqueous ethanol, 80% aqueous methanol, 80% aqueous isopropanol, 80% aqueous dimethyl sulfoxide, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), dimethyl
- An organic solvent such as acetamide (DMA) or N-methyl-2-pyrrolidone (NMP) can be used, and a 50% aqueous acetone solution or an 80% aqueous dimethyl sulfoxide solution is preferred.
- the organic solvent solution in which the drug linker intermediate is dissolved may be reacted by adding 1 to 20% v / v to a buffer solution containing an antibody having a thiol group.
- the reaction temperature is preferably -10 to 10 ° C, more preferably -5 to 5 ° C, and still more preferably 0 to 2 ° C.
- the reaction time is preferably 0.5 to 2 hours.
- the conjugation reaction can be terminated by deactivating the reactivity of the unreacted drug linker intermediate with a thiol group-containing reagent.
- the thiol group-containing reagent for example, cysteine or N-acetyl-L-cysteine can be used. More specifically, the reaction can be terminated by adding 1 to 2 molar equivalents of N-acetyl-L-cysteine to the drug linker intermediate used and reacting at room temperature for 10 to 30 minutes.
- the ultrafiltration membrane may be a suitable one, but may be 1 kDa to 100 kDa, and preferably a 30 kDa ultrafiltration membrane.
- Antibody concentration measurement Using a UV measuring device (Nanodrop 1000, Thermo Fisher Scientific Inc.), antibody concentration can be measured according to the method specified by the manufacturer. At that time, a different 280 nm extinction coefficient (1.3 mLmg ⁇ 1 cm ⁇ 1 to 1.8 mLmg ⁇ 1 cm ⁇ 1 ) is used for each antibody.
- This preparative fraction is placed on the NAP-25 column again, and the gel filtration purification procedure for elution with a buffer solution is repeated 2 to 3 times in total, so that unbound drug linker intermediates and low molecular weight compounds (Tris (2-carboxyl An antibody-drug conjugate composition is obtained without (ethyl) phosphine hydrochloride, N-acetyl-L-cysteine and dimethyl sulfoxide).
- HPLC system Shimadzu Science HPLC system Detector: UV absorbance meter (measurement wavelength: 280nm)
- Mobile phase A 25 mM phosphate buffer (pH 7.0) aqueous solution containing 1.5 M ammonium sulfate
- Mobile phase B Mixed solution containing 75% 25 mM phosphate buffer (pH 7.0) and 25% isopropyl alcohol Gradient program: 20% -60% (0-20 minutes), 20% -80% (20-20.1 minutes), 80% -80% (20.1-23 minutes), 80% -20% (23-23.1 minutes), 20% -20% (23.1-40 minutes) Sample injection volume: 2 ⁇ L
- the content of the antibody-drug conjugate having 4 drug bonds is 50% or more.
- the content of D4-1 in the antibody-drug conjugate composition produced by the production method of the present invention is 50% or more, or 50 to 90%, 50 to 80%, 50 to 70%, or 50 to 60. % Range.
- the content of D4-2 in the antibody-drug conjugate composition produced by the production method of the present invention is preferably 5% or less, more preferably 1% or less.
- the content of D4-3 in the antibody-drug conjugate composition produced by the production method of the present invention is preferably 5% or less, more preferably 1% or less.
- the bound drug concentration in the antibody-drug conjugate composition can be calculated by measuring the UV absorbance at two wavelengths of 280 nm and 370 nm of the antibody-drug conjugate aqueous solution, and then performing the following calculation.
- the total absorbance at a certain wavelength is equal to the sum of the absorbances of all the absorbing species present in the system [addition of absorbance], so that the molar extinction coefficient of the antibody and drug changes before and after conjugation of the antibody and drug.
- the antibody concentration and the drug concentration in the antibody-drug conjugate composition are represented by the following relational expression.
- a Formula (2)
- 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 represents the absorbance of the antibody at 370 nm
- AD, 280 represents the absorbance of the conjugate precursor at 280 nm
- AD, 370 represents the absorbance of the conjugate precursor at 370 nm
- ⁇ A, 280 represents the absorbance at 280 nm.
- ⁇ A, 370 indicates the molar extinction coefficient of the antibody at 370 nm
- ⁇ D, 280 indicates the molar extinction coefficient of the conjugate precursor at 280 nm
- ⁇ D, 370 indicates the conjugate at 370 nm. shows the molar extinction coefficient of the precursor
- C a antibody - indicates antibody concentration in the drug conjugate composition
- C D antibody - shows the drug concentration in the drug conjugate composition.
- ⁇ A, 280 , ⁇ A, 370 , ⁇ D, 280 and ⁇ 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.
- 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 mean a drug binding per antibody determined by the dividing in C A.
- HPLC system Agilent 1290 HPLC system (Agilent Technologies) Detector: UV absorbance meter (measurement wavelength: 280nm)
- Mobile phase A 0.04% trifluoroacetic acid (TFA) aqueous solution
- Mobile phase B acetonitrile solution containing 0.04% TFA Gradient program: 29% -36% (0 min-12.5 min), 36% -42% (12.5-15 min) ), 42% -29% (15 min-15.1 min), 29% -29% (15.1 min-25 min)
- 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.
- the molar extinction coefficient (280 nm) of the drug linker is the measured molar extinction coefficient of the compound obtained by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine and converting the N-substituted maleimidyl group to succinimide thioether. (280 nm) can be used.
- L 1 and H 1 are preferentially generated, it can be assumed that the heavy chain-light chain disulfide is selectively reduced, and if L 0 and H 2 are preferentially generated, the heavy chain— It can be presumed that the inter-heavy chain disulfide is selectively reduced.
- the average drug binding number in the antibody-drug conjugate composition is calculated according to the following formula.
- Average drug binding number (L 0 peak area ratio ⁇ 0 + L 1 peak area ratio ⁇ 1 + H 0 peak area ratio ⁇ 0 + H 1 peak area ratio ⁇ 1 + H 2 peak area ratio ⁇ 2 + H 3 peak area ratio ⁇ 3) / 100 ⁇ 2
- the average drug binding number of the antibody-drug conjugate composition produced by the production method of the present invention is preferably 3.5 to 4.5, more preferably 4.0 to 4.1.
- Medicament Containing Antibody-Drug Conjugate Composition The antibody-drug conjugate composition obtained by the present invention is moved into a tumor cell and then the linker moiety is cleaved to release the drug in the tumor cell.
- the antibody-drug conjugate composition obtained by the present invention exhibits cytotoxic activity against cancer cells, it can be used as an active ingredient of a pharmaceutical composition for treating and / or preventing cancer.
- the antibody-drug conjugate composition obtained according to the present invention can be selected and used as a drug for chemotherapy, which is the main therapeutic method for cancer treatment, and as a result, the growth of cancer cells is delayed. , Can suppress proliferation and even destroy cancer cells. By these, in cancer patients, it is possible to achieve relief from cancer symptoms and improvement of QOL, and to achieve the therapeutic effect while maintaining the lives of cancer patients. Even when cancer cells are not destroyed, long-term survival can be achieved while achieving higher QOL in cancer patients by suppressing or controlling the growth of cancer cells.
- drugs alone in such drug therapy, they can also be used as drugs combined with other therapies in adjuvant therapy, and can be combined with surgery, radiation therapy, hormone therapy, and the like. Furthermore, it can also be used as a drug for pharmacotherapy in neoadjuvant therapy.
- the antibody-drug conjugate composition obtained by the present invention can be administered to a patient as a systemic therapy, or locally administered to a cancer tissue and expected to have a therapeutic effect.
- the types of cancer include lung cancer, renal cancer, urothelial cancer, colon cancer, prostate cancer, glioblastoma multiforme, ovarian cancer, pancreatic cancer, breast cancer, melanoma, liver cancer, bladder cancer, stomach cancer, cervical cancer, uterus
- Examples include, but are not limited to, body cancer, head and neck cancer, esophageal cancer, biliary tract cancer, thyroid cancer, lymphoma, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, multiple myeloma and the like.
- the antibody-drug conjugate composition obtained by the present invention can be used as an active ingredient of a pharmaceutical composition for treating an autoimmune disease or a pharmaceutical composition for suppressing rejection to transplantation.
- the pharmaceutical composition containing the antibody-drug conjugate composition obtained by the present invention is systemic or local when administered to a mammal (eg, human, horse, cow, pig, etc., preferably human). In particular, it can be administered parenterally.
- a mammal eg, human, horse, cow, pig, etc., preferably human.
- it can be administered parenterally.
- Parenteral routes of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous routes.
- Examples of the administration method include injection, bolus injection and the like, preferably injection.
- the pharmaceutical composition of the present invention can be prepared by selecting an appropriate form according to the administration method and preparing various preparations usually used.
- antibody-drug conjugate compositions obtained according to the present invention and sterilized liquids eg, water and oil (petroleum, animal, plant, or synthetic sources) such as those described in “Remington's Pharmaceutical Sciences” by EW Martin) Oil (including peanut oil, soybean oil, mineral oil, sesame oil, etc.)), solvents such as saline solution, dextrose solution, glycerol solution, wetting agents, emulsifiers, pH buffering agents, etc.
- the pharmaceutical composition of the present invention can be prepared.
- the pharmaceutical composition of the present invention may also contain a solubilizer, a local anesthetic (eg, lignocaine) for relieving pain at the injection site, and the like.
- a solubilizer e.g, lignocaine
- the pharmaceutical composition of the present invention may be supplied in a form in which the active ingredient and the solvent are contained in separate containers.
- the pharmaceutical composition of the invention when administered by infusion, it may be administered, for example, in an infusion bottle containing the active ingredient and sterile pharmaceutical grade water or saline.
- the active ingredient may be mixed with sterile water for injection or saline before administration.
- the pharmaceutical composition of the present invention may comprise an antibody-drug conjugate composition and at least one other therapeutic agent for cancer.
- the antibody-drug conjugate composition obtained by 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 an individual simultaneously or separately with the antibody-drug conjugate composition, or may be administered at different intervals.
- cancer therapeutic agents include carboplatin, cisplatin, gemcitabine, irinotecan (CPT-11), paclitaxel, pemetrexed, sorafenib, vinblastin, drugs described in International Publication No.
- the pharmaceutical composition of the present invention may be provided as a lyophilized formulation or a liquid formulation.
- a lyophilized formulation it may be a formulation containing appropriate formulation additives used in this field.
- liquid preparations may be preparations containing appropriate preparation additives used in this field.
- the antibody-drug conjugate composition contained in the pharmaceutical composition of the present invention has an affinity for the antigen of the antibody-drug conjugate. That is, in terms of the dissociation constant (Kd value) for the antigen, the higher the affinity (the lower the Kd value), the more effective the drug effect can be achieved. Therefore, in determining the dose of the antibody-drug conjugate composition, the dose can be set based on the affinity state between the antibody-drug conjugate and the antigen.
- Kd value dissociation constant
- the dose can be set based on the affinity state between the antibody-drug conjugate and the antigen.
- Example 1 Construction of humanized anti-TROP2 antibody expression vector and production of antibody
- i Construction of humanized anti-TROP2 antibody heavy chain (hTINA1-H1) expression vector
- hTINA1-H1 humanized anti-TROP2 antibody heavy chain
- a DNA fragment containing the DNA sequence encoding the variable region of the humanized anti-TROP2 antibody heavy chain (hTINA1-H1) is amplified with KOD-Plus- (TOYOBO) and the following primer set, Humanized anti-TROP2 antibody heavy chain (hTINA1) by inserting the chimeric and humanized antibody IgG1-type heavy chain expression vector pCMA-G1 into the site cut with the restriction enzyme BlpI using the In-Fusion HD PCR cloning kit (CLONTECH) -H1) An expression vector was constructed. The resulting expression vector was named “pCMA-G1 / hTINA1-H1”.
- a DNA fragment containing the DNA sequence encoding the variable region of the humanized anti-TROP2 antibody light chain (hTINA1-L1) is amplified with KOD-Plus- (TOYOBO) and the following primer set,
- the humanized anti-TROP2 antibody light chain (hTINA1-L1) is inserted into the chimeric and humanized antibody light chain expression vector pCMA-LK using the In-Fusion HD PCR Cloning Kit (CLONTECH) after cutting with the restriction enzyme BsiWI. )
- An expression vector was constructed.
- the obtained expression vector was named “pCMA-LK / hTINA1-L1”.
- the humanized anti-TROP2 antibody obtained by combining pCMA-G1 / hTINA1-H1 and pCMA-LK / hTINA1-L1 was named “hTINA1-H1L1”.
- Example 3 Preparation of humanized anti-TROP2 antibody ADC composition
- Example 3-1 Preparation of humanized anti-TROP2 antibody (hTINA1-H1L1)
- ADC composition by a conventional method
- i Reduction of antibody
- a humanized anti-TROP2 antibody (hTINA1-H1L1) (15 mL: equivalent to 327 mg, concentration 21.8 mg / mL; 25 mM histidine buffer) is placed in a glass reaction vessel, and further 25 mM histidine buffer (18 mL, pH 5.0).
- 25 mM histidine buffer (18 mL, pH 5.0).
- sample for HPLC analysis (reduction of antibody-drug conjugate)
- the antibody-drug conjugate solution (about 1 mg / mL, 60 ⁇ L) was mixed with an aqueous dithiothreitol (DTT) solution (100 mM, 15 ⁇ L).
- DTT dithiothreitol
- HPLC analysis HPLC analysis was performed under the following measurement conditions.
- HPLC system Shimadzu Science HPLC system Detector: UV absorbance meter (measurement wavelength: 280nm)
- Mobile phase A 0.05% trifluoroacetic acid (TFA) aqueous solution
- Mobile phase B acetonitrile solution containing 0.04% TFA Gradient program: 29% -36% (0 min-12.5 min), 36% -42% (12.5-15 min) ), 42% -29% (15 minutes-15.1 minutes), 29%-29% (15.1 minutes-25 minutes)
- the light chain (L 0 ) and heavy chain (H 0 ) of the antibody to which the drug is not bound are compared to the light chain to which the drug is bound (light chain to which one drug is bound: L 1 ) and the heavy chain.
- the chains (heavy chain with one drug attached: H 1 , heavy chain with two drug attached: H 2 , heavy chain with three drug attached: H 3 ) are hydrophobic in proportion to the number of drugs attached Since the property increases and the retention time increases, elution is performed in the order of L 0 , L 1 , H 0 , H 1 , H 2 , H 3 .
- the detection peak was assigned to any one of L 0 , L 1 , H 0 , H 1 , H 2 , and H 3 by comparing the retention times with L 0 and H 0 .
- the peak area value was corrected according to the following formula using the molar extinction coefficient of the light chain, H 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). The value estimated from the sequence was used.
- a humanized anti-TROP2 antibody (hTINA1-H1L1) 27640 was used as the molar extinction coefficient of the light chain and 83810 as the estimated molar extinction coefficient of the heavy chain according to its amino acid sequence.
- the molar extinction coefficient (280 nm) of the drug linker is the measured molar extinction coefficient of the compound obtained by reacting each drug linker intermediate with mercaptoethanol or N-acetylcysteine and converting the N-substituted maleimidyl group to succinimide thioether. (280 nm) was used.
- Each chain peak area ratio (%) with respect to the total peak area correction value was calculated according to the following formula.
- the average number of drug bonds per antibody molecule in the antibody-drug conjugate composition was calculated according to the following formula.
- Average number of drug bindings (L 0 peak area ratio x 0 + L 1 peak area ratio x 1 + H 0 peak area ratio x 0 + H 1 peak area ratio x 1 + H 2 peak area ratio x 2 + H 3 peak area Ratio x 3) / 100 x 2
- the antibody concentration was 16.49 mg / mL, the antibody yield was 290 mg (86%), and the average drug binding number per antibody molecule (n) measured in common operation A was 4.4.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- HPLC system Shimadzu Science HPLC system Detector: UV absorbance meter (measurement wavelength: 280nm)
- Mobile phase A 25 mM phosphate buffer (pH 7.0) aqueous solution containing 1.5 M ammonium sulfate
- Mobile phase B Mixed solution containing 75% 25 mM phosphate buffer (pH 7.0) and 25% isopropyl alcohol Gradient program: 20% -60% (0-20 minutes), 20% -80% (20-20.1 minutes), 80% -80% (20.1-23 minutes), 80% -20% (23-23.1 minutes), 20% -20% (23.1-40 minutes) Sample injection volume: 2 ⁇ L
- Example 3-2 Preparation of humanized anti-TROP2 antibody (hTINA1-H1L1)
- ADC composition by the method of the present invention (i) Reduction of antibody Humanized anti-TROP2 antibody (hTINA1-H1L1) (22.9 mL: equivalent to 500 mg, concentration 21.8 mg / mL; 25 mM histidine buffer) is placed in a glass reaction vessel, and further 25 mM histidine buffer (22 mL, pH 5). .0) was added.
- Antibody concentration 19.63 mg / mL, antibody yield: 463 mg (92%), average drug binding number per antibody molecule (n) measured in common operation A: 4.1.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- the area value of the drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution state of the drug binding number was D0: 1.9%, D2: 19.5%, D4-1: 53.3%, D6: 18.5%, D8: 5.5% (FIG. 8).
- Example 4 Construction of humanized anti-CD98 antibody expression vector and production of antibody
- i Construction of humanized anti-CD98 antibody heavy chain (hM23-H1) expression vector Humanization shown in nucleotide numbers 58 to 405 of the nucleotide sequence of humanized anti-CD98 antibody heavy chain (hM23-H1) shown in SEQ ID NO: 11
- a DNA fragment (nucleotide number 36 to 422) containing a DNA sequence encoding the variable region of the anti-CD98 antibody heavy chain (hM23-H1) was synthesized (GENEART Artificial Gene Synthesis Service).
- a DNA fragment containing the DNA sequence encoding the variable region of hM23-H1 is amplified with KOD-Plus- (TOYOBO) and the following primer set, and chimeric and humanized antibody IgG1-type heavy chain
- the humanized anti-CD98 antibody heavy chain (hM23-H1) expression vector was constructed by inserting the expression vector pCMA-G1 into the site cut with the restriction enzyme BlpI using the In-Fusion HD PCR cloning kit (CLONTECH). The obtained expression vector was designated as “pCMA-G1 / hM23-H1”.
- a DNA fragment containing the DNA sequence encoding the variable region of the humanized anti-CD98 antibody light chain (hM23-L1) with KOD-Plus- (TOYOBO) and the following primer set is amplified.
- the humanized anti-CD98 antibody light chain (hM23-L1) was inserted into the chimeric and humanized antibody light chain expression vector pCMA-LK using the In-Fusion HD PCR cloning kit (CLONTECH) after being cleaved with the restriction enzyme BsiWI. )
- An expression vector was constructed.
- the obtained expression vector was designated as “pCMA-LK / hM23-L1”.
- Primer set 5'-CTGTGGATCTCCGGCGCGTACGGC-3 '(SEQ ID NO: 23: Primer CM-LKF) 5'-GGAGGGGGCGGCCACCGTACG-3 '(SEQ ID NO: 24: primer KCL-Inf-R)
- Example 5 Preparation of humanized anti-CD98 antibody ADC composition
- Example 5-1 Preparation of humanized anti-CD98 antibody (hM23-H1L1)
- ADC composition by a conventional method (i) Reduction of antibody Humanized anti-CD98 antibody (hM23-H1L1) (12 mL: equivalent to 480 mg, concentration 40 mg / mL; 25 mM histidine buffer) is placed in a glass reaction vessel, and further 25 mM histidine buffer (36 mL, pH 5.0) was added.
- Antibody concentration 20.8 mg / mL, antibody yield: 449 mg (91%), average drug binding number per antibody molecule (n) measured in common operation A: 4.0.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- the area value of the drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution of the number of drug bonds was D0: 4.2%, D2: 24.2%, D4-1: 27.8%, D4-2: 13.3%, D6: 20.8%, D8: 7.6% (FIG. 10).
- Example 5-2 Preparation of humanized anti-CD98 antibody (hM23-H1L1)
- ADC composition by the method of the present invention (i) Reduction of antibody Humanized anti-CD98 antibody (hM23-H1L1) (12.5 mL: equivalent to 500 mg, concentration 40 mg / mL; 25 mM histidine buffer) is placed in a glass reaction vessel, and further 25 mM histidine buffer (27.5 mL, pH 5. 0) was added.
- the reaction mixture was cooled and stirred at an internal temperature of 0 to 1 ° C., and a 1.00 mg / mL tris (2-carboxyethyl) phosphine hydrochloride (Nacalai Tesque, Inc.) aqueous solution (2.75 mL; 2.80 equivalents per antibody molecule)
- the mixture was stirred for 6 hours so that the internal temperature was 0 to 1 ° C., and the interchain disulfide of the antibody was reduced.
- Antibody concentration 19.91 mg / mL, antibody yield: 470 mg (94%), average drug binding number per antibody molecule (n) measured in common operation A: 4.1.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- the area value of the drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution state of the number of drug bonds was D0: 2.2%, D2: 18.1%, D4-1: 51.0%, D6: 20.6%, D8: 7.6% (FIG. 12).
- Example 6 Preparation of humanized anti-B7-H3 antibody ADC composition (Example 6-1) Preparation of humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition by conventional methods (i) Reduction of antibody Humanized anti-B7-H3 antibody (M30-H1-L4) (prepared according to the method of Reference Example 1 of WO2014 / 057687, 12.4 mL: equivalent to 250 mg, concentration: 20.1 mg / mL; 25 mM citrate buffer) was placed in a glass reaction vessel, and 25 mM histidine buffer (18 mL, pH 7.5) was further added.
- Antibody concentration 18.4 mg / mL, antibody yield: 241 mg (94%), average drug binding number per antibody molecule (n) measured in common operation A: 3.8.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- the area value of the drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution state of the drug binding number was D0: 6.5%, D2: 30.5%, D4-1: 27.9%, D4-2: 12.3%, D6: 17.7%, D8: 4.9% (FIG. 16).
- Example 6-2 Production of humanized anti-B7-H3 antibody (M30-H1-L4) ADC composition by the method of the present invention (i) Reduction of antibody Humanized anti-B7-H3 antibody (M30-H1-L4) ) (Prepared according to the method of Reference Example 1 of WO2014 / 057687, 27.1 mL: equivalent to 500 mg, concentration: 18.5 mg / mL; 10 mM histidine buffer) was placed in a glass reaction vessel, and 10 mM histidine aqueous solution (25 mL) was further added.
- the reaction mixture was cooled and stirred at an internal temperature of 0 to 1 ° C., and a 1.00 mg / mL aqueous solution of tris (2-carboxyethyl) phosphine hydrochloride (Nacalai Tesque, Inc.) (2.08 mL; 2.13 equivalents per antibody molecule)
- the mixture was stirred for 5.5 hours so that the internal temperature was 0 to 1 ° C., and the interchain disulfide of the antibody was reduced.
- Antibody concentration 19.4 mg / mL, antibody yield: 455 mg (89%), average drug binding number per antibody molecule (n) measured in common operation A: 4.1.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- the area value of the drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution of the number of drug bonds was D0: 2.7%, D2: 22.3%, D4-1: 58.4%, D6: 14.1%, D8: 2.4% (FIG. 18).
- Example 7-1 Preparation of humanized anti-HER2 antibody ADC composition by conventional methods (i) Reduction of antibody A humanized anti-HER2 antibody (trastuzumab; US Pat. No. 5,811,337) (22.3 mL: equivalent to 500 mg, concentration 22.4 mg / mL; 25 mM histidine buffer) was placed in a glass reaction vessel, and further 25 mM histidine buffer ( 27 mL, pH 5.0) was added. After adding 0.5M EDTA aqueous solution (0.034mL; 5 equivalents to antibody) to this reaction solution, 0.1g / mL polysorbate 20 aqueous solution (0.050mL; 0.01% to antibody) was added, and then 0.3M phosphoric acid was added.
- a humanized anti-HER2 antibody (trastuzumab; US Pat. No. 5,811,337) (22.3 mL: equivalent to 500 mg, concentration 22.4 mg / mL; 25 mM histidine buffer) was placed in a glass
- Antibody concentration 20.39 mg / mL, antibody yield: 462 mg (90%), average drug binding number per antibody molecule (n) measured by common operation A IV: 3.9.
- An HPLC chromatograph showing the peak area ratio (%) of each chain is shown in FIG.
- the area value of the drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution state of the drug binding number was D0: 3.6%, D2: 26.1%, D4-1: 34.1%, D4-2: 13.6%, D6: 17.6%, D8: 5.0% (FIG. 22).
- Example 7-2 Production of humanized anti-HER2 antibody ADC composition by the method of the present invention
- Antibody concentration 21.2 mg / mL, antibody yield: 459 mg (89%), average drug binding number per antibody molecule (n) measured in common operation A: 4.0.
- the area value of each antibody-drug binding number was measured in the same manner as in the common operation B described in Example 3-1 (iv).
- the distribution of the number of bonds was D0: 2.8%, D2: 23.8%, D4-1: 55.2%, D6: 15.0%, D8: 3.3% (FIG. 24).
- mice 5-6 week old female BALB / c-nu / nu mice (Nippon Charles River Co., Ltd.) were acclimated for 4-7 days under SPF conditions before use in experiments. Mice were fed a sterilized solid feed (FR-2, Funabashi Farms Co., Ltd) and given sterilized tap water (prepared by adding 5-15 ppm sodium hypochlorite solution).
- Measurement and calculation formula The major axis and minor axis of the tumor were measured twice or more a week with an electronic digital caliper (CD-15C, Mitutoyo Corp.), and the tumor volume (mm 3 ) was calculated.
- the calculation formula is as follows.
- Tumor volume (mm 3 ) 1/2 ⁇ major axis (mm) ⁇ [minor axis (mm)] 2
- All antibody-drug conjugate compositions were diluted with Acetate-Buffered Saline (pH 5.5) (Nacalai Tesque) and administered at a volume of 10 mL / kg.
- the therapeutic efficacy was judged using the minimum dose (Regression dose) that can reduce the tumor volume as an index.
- the regression dose of the humanized anti-TROP2 antibody ADC composition prepared by the conventional method is 1 mg / kg
- the regression dose of the humanized anti-TROP2 antibody ADC composition prepared by the method of the present invention is also 1 mg / kg. (FIG. 19).
- the antibody-drug conjugate composition produced by the production method of the present invention has the same therapeutic efficacy as the antibody-drug conjugate composition produced by the conventional production method. .
- a humanized anti-TROP2 antibody ADC composition (Example 3-1) produced by a conventional method and a humanized anti-TROP2 antibody ADC composition (Example 3-2) produced by the method of the present invention, Each was administered to cross-species cynomolgus monkeys three times at intervals of once every three weeks. Observation was made until the day after the last administration, and the maximum dose (HNSTD) that did not cause serious toxicity was examined. As a result, the HNSTD of the humanized anti-TROP2 antibody ADC composition produced by the conventional method was 10 mg / kg, whereas the HNSTD of the humanized anti-TROP2 antibody ADC composition produced by the method of the present invention was 30 mg / kg. kg. Thus, it was shown that the antibody-drug conjugate composition produced by the production method of the present invention has superior safety than the antibody-drug conjugate composition produced by the conventional production method. .
- the average number of drug bonds is 3.5 to 4.5, and the content of the antibody-drug conjugate in which four drug linkers are bonded to the heavy chain-light chain thiol is 50%. It has been shown that antibody-drug conjugate compositions that are greater than or equal to% can be selectively produced.
- the antibody-drug conjugate composition produced by the production method of the present invention has better safety than the antibody-drug conjugate composition produced by the conventional production method. Indicated.
- the antibody-drug conjugate composition of the present invention (average drug bond number is 3.5 to 4.5, and the content ratio of the antibody-drug conjugate in which four drug linkers are bonded to the heavy chain-light chain thiol is 50 %
- Antibody-drug conjugate composition is an antibody-drug conjugate composition produced by a conventional production method (average drug binding number is 3.5 to 4.5, and drug is added to the heavy chain-light chain thiol). It was shown that the antibody-drug conjugate composition having an antibody-drug conjugate content of 35% or less in which four linkers are bonded has superior safety.
- SEQ ID NO: 1 Nucleotide sequence of humanized anti-TROP2 antibody heavy chain (hTINA1-H1)
- SEQ ID NO: 2 Amino acid sequence of humanized anti-TROP2 antibody heavy chain (hTINA1-H1)
- SEQ ID NO: 3 Humanized anti-TROP2 antibody light chain ( nucleotide sequence of hTINA1-L1)
- SEQ ID NO: 4 amino acid sequence of humanized anti-TROP2 antibody light chain (hTINA1-L1)
- SEQ ID NO: 6 anti-TROP2 antibody (TINA1 ) CDRH2 amino acid sequence
- SEQ ID NO: 9 anti-TROP2 antibody (TINA1) CDRL2 amino acid
Abstract
Description
(1)
(i)緩衝液中、抗体を還元剤と反応させ、鎖間ジスルフィドを還元する工程;および、
(ii)(i)で得られたチオール基を有する抗体に対して、薬物リンカー中間体を反応させる工程;
を含む、抗体-薬物コンジュゲート組成物の製造方法であって、
(i)の工程の反応温度が、-10~10℃であり、
製造される抗体-薬物コンジュゲート組成物の、平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である、
ことを特徴とする、製造方法。
(2)
製造される抗体-薬物コンジュゲート組成物の、平均薬物結合数が4.0~4.1である、(1)に記載の製造方法。
(3)
製造される抗体-薬物コンジュゲート組成物の、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~90%の範囲である、(1)または(2)に記載の製造方法。
(4)
製造される抗体-薬物コンジュゲート組成物の、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~80%の範囲である、(3)に記載の製造方法。
(5)
製造される抗体-薬物コンジュゲート組成物の、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~70%の範囲である、(4)に記載の製造方法。
(6)
製造される抗体-薬物コンジュゲート組成物の、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~60%の範囲である、(5)に記載の製造方法。
(7)
製造される抗体-薬物コンジュゲート組成物の、重鎖-重鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が5%以下である、(1)~(6)のいずれか1項に記載の製造方法。
(8)
製造される抗体-薬物コンジュゲート組成物の、重鎖-重鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が1%以下である、(7)に記載の製造方法。
(9)
製造される抗体-薬物コンジュゲート組成物の、重鎖-重鎖間チオールに薬物リンカーが2個結合し、重鎖-軽鎖間チオールに薬物リンカーが2個結合した抗体-薬物コンジュゲートの含有率が5%以下である、(1)~(8)のいずれか1項に記載の製造方法。
(10)
製造される抗体-薬物コンジュゲート組成物の、重鎖-重鎖間チオールに薬物リンカーが2個結合し、重鎖-軽鎖間チオールに薬物リンカーが2個結合した抗体-薬物コンジュゲートの含有率が1%以下である、(1)~(8)のいずれか1項に記載の製造方法。
(11)
(i)の工程の反応温度が、-5~5℃である、(1)~(10)のいずれか1項に記載の製造方法。
(12)
(i)の工程の反応温度が、-3~3℃である、(11)に記載の製造方法。
(13)
(i)の工程の反応温度が、0~2℃である、(12)に記載の製造方法。
(14)
(i)の工程の反応温度が、0~1℃である、(13)に記載の製造方法。
(15)
(ii)の工程の反応温度が、0~2℃である、(1)~(14)のいずれか1項に記載の製造方法。
(16)
還元剤が抗体一分子あたり2~3モル当量で用いられる、(1)~(15)のいずれか1項に記載の製造方法。
(17)
還元剤がトリス(2-カルボキシエチル)ホスフィンまたはその塩である、(1)~(16)のいずれか1項に記載の製造方法。
(18)
トリス(2-カルボキシエチル)ホスフィンの塩がトリス(2-カルボキシエチル)ホスフィン塩酸塩である、(17)に記載の製造方法。
(19)
緩衝液が、ヒスチジンバッファーである、(1)~(18)のいずれか1項に記載の製造方法。
(20)
緩衝液が、キレート剤を含む、(1)~(19)のいずれか1項に記載の製造方法。
(21)
キレート剤がエチレンジアミン四酢酸である、(20)に記載の製造方法。
(22)
抗体が、抗TROP2抗体、抗CD98抗体、抗B7-H3抗体、または抗HER2抗体である、(1)~(21)のいずれか1項に記載の製造方法。
(23)
抗体が、抗TROP2抗体である、(22)に記載の製造方法。
(24)
抗体が、抗CD98抗体である、(22)に記載の製造方法。
(25)
抗体が、抗B7-H3抗体である、(22)に記載の製造方法。
(26)
抗体が、抗HER2抗体である、(22)に記載の製造方法。
(27)
薬物リンカー中間体が、N-置換マレイミジル基を有する、(1)~(26)のいずれか1項に記載の製造方法。
(28)
薬物リンカー中間体が、
(29)
薬物リンカー中間体が、
(30)
(1)~(29)のいずれか1項に記載の製造方法によって製造される、抗体-薬物コンジュゲート組成物。
(31)
平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である、抗体-薬物コンジュゲート組成物。
(32)
平均薬物結合数が4.0~4.1である、(31)に記載の抗体-薬物コンジュゲート組成物。
(33)
重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~90%の範囲である、(31)または(32)に記載の抗体-薬物コンジュゲート組成物。
(34)
重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~80%の範囲である、(33)に記載の抗体-薬物コンジュゲート組成物。
(35)
重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~70%の範囲である、(34)に記載の抗体-薬物コンジュゲート組成物。
(36)
重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~60%の範囲である、(35)に記載の抗体-薬物コンジュゲート組成物。
(37)
重鎖-重鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が5%以下である、(31)~(36)のいずれか1項に記載の抗体-薬物コンジュゲート組成物。
(38)
重鎖-重鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が1%以下である、(37)に記載の抗体-薬物コンジュゲート組成物。
(39)
重鎖-重鎖間チオールに薬物リンカーが2個結合し、重鎖-軽鎖間チオールに薬物リンカーが2個結合した抗体-薬物コンジュゲートの含有率が5%以下である、(31)~(38)のいずれか1項に記載の抗体-薬物コンジュゲート組成物。
(40)
重鎖-重鎖間チオールに薬物リンカーが2個結合し、重鎖-軽鎖間チオールに薬物リンカーが2個結合した抗体-薬物コンジュゲートの含有率が1%以下である、(39)に記載の抗体-薬物コンジュゲート組成物。
(41)
抗体が、抗TROP2抗体、抗CD98抗体、抗B7-H3抗体、または抗HER2抗体である、(31)~(40)のいずれか1項に記載の抗体-薬物コンジュゲート組成物。
(42)
抗体が、抗TROP2抗体である、(41)に記載の抗体-薬物コンジュゲート組成物。
(43)
抗体が、抗CD98抗体である、(41)に記載の抗体-薬物コンジュゲート組成物。
(44)
抗体が、抗B7-H3抗体である、(41)に記載の抗体-薬物コンジュゲート組成物。
(45)
抗体が、抗HER2抗体である、(41)に記載の抗体-薬物コンジュゲート組成物。
(46)
薬物リンカーが、
(47)
薬物リンカーが、
(48)
(30)~(47)のいずれか1項に記載の抗体-薬物コンジュゲート組成物を含有する、医薬組成物。
(49)
腫瘍および/または癌の治療のための、(48)に記載の医薬組成物。
(50)
肺癌、腎癌、尿路上皮癌、大腸癌、前立腺癌、多型神経膠芽腫、卵巣癌、膵癌、乳癌、メラノーマ、肝癌、膀胱癌、胃癌、子宮頸癌、子宮体癌、頭頸部癌、食道癌、胆道癌、甲状腺癌、リンパ腫、急性骨髄性白血病、急性リンパ性白血病、慢性骨髄性白血病、および/または多発性骨髄腫の治療のための、(49)に記載の医薬組成物。
(51)
(30)~(47)のいずれか1項に記載の抗体-薬物コンジュゲート組成物を投与することを特徴とする、腫瘍および/または癌の治療方法。
(52)
肺癌、腎癌、尿路上皮癌、大腸癌、前立腺癌、多型神経膠芽腫、卵巣癌、膵癌、乳癌、メラノーマ、肝癌、膀胱癌、胃癌、子宮頸癌、子宮体癌、頭頸部癌、食道癌、胆道癌、甲状腺癌、リンパ腫、急性骨髄性白血病、急性リンパ性白血病、慢性骨髄性白血病、および/または多発性骨髄腫の治療方法である、(51)に記載の治療方法。
(53)
緩衝液中、抗体を還元剤と反応させ、鎖間ジスルフィドを還元する工程、を含む、チオール基を有する抗体の製造方法であって、
反応温度が、-10~10℃であり、
製造されるチオール基を有する抗体が、平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である、抗体-薬物コンジュゲート組成物を製造するために用いられる、
ことを特徴とする、製造方法。
(54)
製造されるチオール基を有する抗体が、平均薬物結合数が4.0~4.1である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(53)に記載の製造方法。
(55)
製造されるチオール基を有する抗体が、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~90%の範囲である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(53)または(54)に記載の製造方法。
(56)
製造されるチオール基を有する抗体が、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~80%の範囲である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(55)に記載の製造方法。
(57)
製造されるチオール基を有する抗体が、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~70%の範囲である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(56)に記載の製造方法。
(58)
製造されるチオール基を有する抗体が、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50~60%の範囲である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(57)に記載の製造方法。
(59)
製造されるチオール基を有する抗体が、重鎖-重鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が5%以下である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(53)~(58)のいずれか1項に記載の製造方法。
(60)
製造されるチオール基を有する抗体が、重鎖-重鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が1%以下である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(59)に記載の製造方法。
(61)
製造されるチオール基を有する抗体が、重鎖-重鎖間チオールに薬物リンカーが2個結合し、重鎖-軽鎖間チオールに薬物リンカーが2個結合した抗体-薬物コンジュゲートの含有率が5%以下である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(53)~(60)のいずれか1項に記載の製造方法。
(62)
製造されるチオール基を有する抗体が、重鎖-重鎖間チオールに薬物リンカーが2個結合し、重鎖-軽鎖間チオールに薬物リンカーが2個結合した抗体-薬物コンジュゲートの含有率が1%以下である、抗体-薬物コンジュゲート組成物を製造するために用いられる、(61)に記載の製造方法。
(63)
反応温度が、-5~5℃である、(53)~(62)のいずれか1項に記載の製造方法。
(64)
反応温度が、-3~3℃である、(63)に記載の製造方法。
(65)
反応温度が、0~2℃である、(64)に記載の製造方法。
(66)
反応温度が、0~1℃である、(65)に記載の製造方法。
(67)
還元剤が抗体一分子あたり2~3モル当量で用いられる、(53)~(66)のいずれか1項に記載の製造方法。
(68)
還元剤がトリス(2-カルボキシエチル)ホスフィンまたはその塩である、(53)~(67)のいずれか1項に記載の製造方法。
(69)
トリス(2-カルボキシエチル)ホスフィンの塩がトリス(2-カルボキシエチル)ホスフィン塩酸塩である、(68)に記載の製造方法。
(70)
緩衝液が、ヒスチジンバッファーである、(53)~(69)のいずれか1項に記載の製造方法。
(71)
緩衝液が、キレート剤を含む、(53)~(70)のいずれか1項に記載の製造方法。
(72)
キレート剤がエチレンジアミン四酢酸である、(71)に記載の製造方法。
(73)
抗体が、抗TROP2抗体、抗CD98抗体、抗B7-H3抗体、または抗HER2抗体である、(53)~(72)のいずれか1項に記載の製造方法。
(74)
抗体が、抗TROP2抗体である、(73)に記載の製造方法。
(75)
抗体が、抗CD98抗体である、(73)に記載の製造方法。
(76)
抗体が、抗B7-H3抗体である、(73)に記載の製造方法。
(77)
抗体が、抗HER2抗体である、(73)に記載の製造方法。
本発明に用いられる抗体は、目的抗原、例えば、腫瘍特異抗原(TAA)または腫瘍関連抗原(TSA)に対して産生されうる。かかる抗体は、腫瘍細胞を認識できる特性、かかる腫瘍細胞に結合できる特性、および、かかる腫瘍細胞内に取り込まれて内在化する特性を有する。
本発明に用いられる薬物は、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。薬物は、リンカーの一部また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。薬物は、抗体に特定の構造のリンカー部分を介して結合させるが、本明細書では、この薬物とリンカー部分を含めた薬物リンカーを薬物と称することもある。
本発明に用いられる薬物はリンカーを介して抗体に結合されうる。本発明に用いられるリンカーは、好ましくはN-置換マレイミジル基を有する。リンカーは、開裂型リンカーおよび非開裂型リンカーを挙げることができる。開裂型リンカーとしては、例えば、リソソームプロテアーゼ、エンドソームプロテアーゼなどの細胞内プロテアーゼによって開裂するペプチドリンカーを挙げることができる。
本発明に用いられる薬物リンカー中間体は、上記リンカー化合物のカルボキシル基と抗腫瘍化合物のアミノ基とを縮合剤を用いるなどして反応させることにより製造することができる。
5.抗体-薬物コンジュゲート
抗体-薬物コンジュゲートにおいて、抗体1分子への薬物の結合数は、その有効性、安全性に影響する重要因子である。抗体には4つの鎖間ジスルフィドが存在し、ジスルフィドは2つのチオール基から構成されるため、抗体1分子への薬物の結合数は、2個、4個、6個、または8個となる。
薬物リンカーとしては、好ましくは、
抗体-薬物コンジュゲートの製造は、薬物の結合数が制御されるよう、反応させる原料・試薬の使用量などの反応条件を規定して実施されるが、低分子化合物の化学反応とは異なり、異なる数の薬物が結合した混合物として得られるのが通常である。抗体1分子への薬物の結合数は平均値、すなわち、平均薬物結合数として特定され、表記される。
チオール基を有する抗体は、緩衝液中、-10~10℃で抗体を還元剤と反応させることにより製造することができる。
還元剤としては、例えば、トリス(2-カルボキシエチル)ホスフィンまたはその塩、ジチオトレイトール、2-メルカプトエタノールなどを用いることができ、好ましくはトリス(2-カルボキシエチル)ホスフィンまたはその塩であり、より好ましくはトリス(2-カルボキシエチル)ホスフィン塩酸塩である。
第一工程で得られたチオール基を有する抗体に薬物リンカー中間体を反応させることにより、抗体―薬物コンジュゲート組成物を製造することができる。用いる薬物リンカー中間体の量としては、抗体1分子当たり、2~10モル当量であり、好ましくは4~6モル当量である。
製造した抗体-薬物コンジュゲート組成物は、以下の操作によって濃縮、バッファー交換、精製、抗体濃度および平均薬物結合数の測定を行い、抗体-薬物コンジュゲート組成物の同定を行うことができる。
Amicon Ultra(50,000 MWCO、Millipore Corporation)の容器内に抗体または抗体-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~3800Gにて5~20分間遠心)にて、抗体または抗体-薬物コンジュゲート溶液を濃縮することができる。
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うことができる。その際に、抗体ごとに異なる280nm吸光係数(1.3mLmg-1cm-1~1.8mLmg-1cm-1)を用いる。
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(137mM)およびエチレンジアミン四酢酸(5mM)を含むリン酸緩衝液(10mM、pH6.0;本明細書において「PBS6.0/EDTA」という場合がある。)にて平衡化する。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/EDTA 3.5mLで溶出させた画分(3.5mL)を分取する。この画分を上記(1)と同様の方法で濃縮し、上記(2)と同様の方法で抗体濃度の測定を行ったのちに、PBS6.0/EDTAを用いて抗体濃度を調整することができる。
Sorbitol(5%)を含む酢酸緩衝液(10mM、pH5.5;本明細書において「ABS」という場合がある。)にてNAP-25カラムを平衡化する。このNAP-25カラムに、抗体-薬物コンジュゲート反応水溶液(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2~3回繰り返すことで、未結合の薬物リンカー中間体や低分子化合物(トリス(2-カルボキシエチル)ホスフィン塩酸塩、N-アセチル-L-システインおよび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組成物を得る。
(5-1)HPLC測定方法
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:TSKgel Butyl-NPR(4.6×100mm、2.5μm;東ソー株式会社)
カラム温度:30℃
移動相A:1.5M硫酸アンモニウムを含む、25mMリン酸バッファー(pH7.0)水溶液
移動相B:25mMリン酸バッファー(pH7.0)を75%とイソプロピルアルコールを25%含む混合溶液
グラジエントプログラム:20%-60%(0分-20分)、20%-80%(20-20.1分)、80%-80%(20.1―23分)、80%-20%(23分―23.1分)、20%-20%(23.1分―40分)
サンプル注入量:2μL
本データは、カラムの特性から、薬物結合数の低い抗体-薬物コンジュゲートから順に塩濃度の差により溶出されるため、その各々の面積値を測定する事により、結合数の分布が推定できる。そのピークは、溶出順にD0(薬物リンカーが結合していない抗体)、D2、D4-1、D4-2、D6、D8となり、その分布状況を把握できる。
抗体-薬物コンジュゲート組成物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および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。
A280=AD,280+AA,280=εD,280CD+εA,280CA 式(1)
A370=AD,370+AA,370=εD,370CD+εA,370CA 式(2)
ここで、A280は280nmにおける抗体-薬物コンジュゲート水溶液の吸光度を示し、A370は370nmにおける抗体-薬物コンジュゲート水溶液の吸光度を示し、AA,280は280nmにおける抗体の吸光度を示し、AA,370は370nmにおける抗体の吸光度を示し、AD,280は280nmにおけるコンジュゲート前駆体の吸光度を示し、AD,370は370nmにおけるコンジュゲート前駆体の吸光度を示し、εA,280は280nmにおける抗体のモル吸光係数を示し、εA,370は370nmにおける抗体のモル吸光係数を示し、εD,280は280nmにおけるコンジュゲート前駆体のモル吸光係数を示し、εD,370は370nmにおけるコンジュゲート前駆体のモル吸光係数を示し、CAは抗体-薬物コンジュゲート組成物における抗体濃度を示し、CDは抗体-薬物コンジュゲート組成物における薬物濃度を示す。
抗体-薬物コンジュゲート組成物における抗体一分子あたりの平均薬物結合数は、前述のUV法に加え、以下の逆層クロマトグラフィー(Reversed Phase Chromatography(RPC))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。
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鎖間のジスルフィド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: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%トリフルオロ酢酸(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%(12.5-15分)、42%-29%(15分-15.1分)、29%-29%(15.1分-25分)
サンプル注入量:15μL
(7-3-1)薬物の結合していない抗体の軽鎖(L0)および重鎖(H0)に対して、薬物の結合した軽鎖(薬物が一つ結合した軽鎖:L1)および重鎖(薬物が一つ結合した重鎖: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、L0、L1、H0、H1、H2、H3の順に溶出される。L0および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。
本発明により得られる抗体-薬物コンジュゲート組成物は、腫瘍細胞内に移動した後にリンカー部分が切断され、腫瘍細胞内で薬物が遊離される。
(i)ヒト化抗TROP2抗体重鎖(hTINA1-H1)発現ベクターの構築
配列表の配列番号2に示すヒト化抗TROP2抗体重鎖(hTINA1-H1)のヌクレオチド配列のヌクレオチド番号36~437に示されるヒト化抗TROP2抗体重鎖(hTINA1-H1)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)。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抗TROP2抗体重鎖(hTINA1-H1)の可変領域をコードするDNA配列を含むDNA断片を増幅し、キメラおよびヒト化抗体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化抗TROP2抗体重鎖(hTINA1-H1)発現ベクターを構築した。得られた発現ベクターを「pCMA-G1/hTINA1-H1」と命名した。
プライマーセット
5’-agctcccagatgggtgctgagc-3’(配列番号21:プライマー EG-Inf-F)
5’-gggcccttggtggaggctgagc-3’(配列番号22:プライマー EG1-Inf-R)
配列表の配列番号4に示すヒト化抗TROP2抗体軽鎖(hTINA1-L1)のヌクレオチド配列のヌクレオチド番号38~402に示されるヒト化抗TROP2抗体軽鎖(hTINA1-L1)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)。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抗TROP2抗体軽鎖(hTINA1-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、キメラおよ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抗TROP2抗体軽鎖(hTINA1-L1)発現ベクターを構築した。得られた発現ベクターを「pCMA-LK/hTINA1-L1」と命名した。
プライマーセット
5’-ctgtggatctccggcgcgtacggc-3’(配列番号23:プライマー CM-LKF)
5’-ggagggggcggccaccgtacg-3’(配列番号24:プライマー KCL-Inf-R)
FreeStyle 293F細胞(Invitrogen社)はマニュアルに従い、継代、培養をおこなった。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1.0×106細胞/mLに調製した後に、37℃、8% CO2インキュベーター内で90rpmで一時間振とう培養した。Polyethyleneimine(Polyscience #24765;3.6mg)をOpti-Pro SFM(Invitrogen社;20mL)に溶解し、次にPureLink HiPure Plasmidキット(Invitrogen社)を用いて調製した軽鎖発現ベクター(0.8mg)および重鎖発現ベクター(0.4mg)をOpti-Pro SFM(Invitrogen社;20mL)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)を加えて穏やかに攪拌し、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Disposable Capsule Filter(ADVANTEC #CCS-045-E1H)でろ過した。
(iii)で得られた培養上清から抗体を、rProteinAアフィニティークロマトグラフィー(4-6℃)とセラミックハイドロキシアパタイト(室温)の2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℃で実施した。最初に、培養上清を、PBSで平衡化したMabSelect SuRe(GE Healthcare Bioscience社製、HiTrapカラム)にアプライした。培養上清がカラムに全て入った後、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 CHTType-I 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 Scientific社、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。最後にCentrifugal UF Filter Device VIVASPIN20(分画分子量UF10K,Sartorius社,4℃)にて濃縮し、IgG濃度を20mg/mL以上に調製して精製サンプルとした。
(iv)にて作製したヒト化抗TROP2抗体(hTINA1-H1L1)を、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(137mM)およびエチレンジアミン四酢酸(5mM)を含むリン酸緩衝液(10mM、pH6.0;本明細書において「PBS6.0/EDTA」という場合がある。)にて平衡化した。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/EDTA 3.5mLで溶出させた画分(3.5mL)を分取した。この画分をAmicon Ultra(50,000 MWCO、Millipore Corporation)の容器内に抗体溶液を入れ、遠心機(Allegra X-15R、Beckman Coulter, Inc.)を用いた遠心操作(2000G~3800Gにて5~20分間遠心)にて、抗体溶液を濃縮した。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、280nm吸光係数(1.54mLmg-1cm-1)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21.8mg/mLに抗体濃度を調整した。
(実施例2)薬物リンカー中間体の作製
次式で表される、N-[6-(2,5-ジオキソ-2,5-ジヒドロ-1H-ピロール-1-イル)ヘキサノイル]グリシルグリシル-L-フェニルアラニル-N-[(2-{[(1S,9S)-9-エチル-5-フルオロ-9-ヒドロキシ-4-メチル-10,13-ジオキソ-2,3,9,10,13,15-ヘキサヒドロ-1H,12H-ベンゾ[de]ピラノ[3’,4’:6,7]インドリジノ[1,2-b]キノリン-1-イル]アミノ}-2-オキソエトキシ)メチル]グリシンアミドを、WO2014/057687の実施例58の方法に従って合成した。
(実施例3-1)従来の方法によるヒト化抗TROP2抗体(hTINA1-H1L1) ADC組成物の作製
(i)抗体の還元
ヒト化抗TROP2抗体(hTINA1-H1L1)(15mL:327mg相当、濃度21.8mg/mL;25mM ヒスチジンバッファー)をガラス反応容器に入れ、更に、25mMヒスチジンバッファー(18mL、pH5.0)を加えた。本反応液に、0.5M EDTA水溶液(0.027mL;抗体に対し6当量)を加えたのち、0.1g/mLポリソルベート20水溶液(0.033mL;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.12に調整した。24℃攪拌下に、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩水溶液(1.58mL;抗体一分子に対して2.45当量)を加え、内温を35~36℃になるように3時間加温し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液を冷却し、内温16~17℃にて、攪拌下、実施例2で得た化合物の6.67mg/mL 50%アセトン水溶液(1.71mL;抗体一分子に対して5.2当量)を60分かけて加え、同温度にて20分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン水溶液(0.135mL;抗体一分子に対して3当量)を加え、さらに同温度にて20分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量500mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗TROP2抗体(hTINA1-H1L1)ADC組成物を含有する溶液を17.6mL得た。
[共通操作A]抗体-薬物コンジュゲート組成物における抗体一分子あたりの平均薬物結合数の測定
抗体-薬物コンジュゲート組成物における抗体一分子あたりの平均薬物結合数は、以下の方法を用いる高速液体クロマトグラフィー(HPLC)分析によって求めた。
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)と混合した。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重鎖-軽鎖間および重鎖-重鎖間のジスルフィドを切断したサンプルを、HPLC分析に用いた。
HPLC分析を、下記の測定条件にて行った。
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)
カラム温度:80℃
移動相A:0.05%トリフルオロ酢酸(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%(12.5-15分)、42%-29%(15分―15.1分)、29%-29%(15.1分―25分)
サンプル注入量:15μL
薬物の結合していない抗体の軽鎖(L0)および重鎖(H0)に対して、薬物の結合した軽鎖(薬物が一つ結合した軽鎖:L1)および重鎖(薬物が一つ結合した重鎖: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、L0、L1、H0、H1、H2、H3の順に溶出される。L0および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た。
抗体濃度:16.49mg/mL、抗体収量:290mg(86%)、共通操作A にて測定された抗体一分子あたりの平均薬物結合数(n):4.4であった。各鎖ピーク面積比(%)を表したHPLCクロマトグラフを図5に示す。
1.HPLC測定方法
HPLC分析を、下記の測定条件にて行った。
検出器:紫外吸光度計(測定波長:280nm)
カラム:TSKgel Butyl-NPR(4.6×100mm、2.5μm;東ソー株式会社)
カラム温度:30℃
移動相A:1.5M硫酸アンモニウムを含む、25mMリン酸バッファー(pH7.0)水溶液
移動相B:25mMリン酸バッファー(pH7.0)を75%とイソプロピルアルコールを25%含む混合溶液
グラジエントプログラム:20%-60%(0分-20分)、20%-80%(20-20.1分)、80%-80%(20.1―23分)、80%-20%(23分―23.1分)、20%-20%(23.1分―40分)
サンプル注入量:2μL
本データは、カラムの特性から、薬物結合数の低い抗体-薬物コンジュゲートから順に塩濃度の差により溶出されるため、その各々の面積値を測定する事により、薬物結合数の分布を推定した。そのピークは、溶出順にD0(薬物リンカーが結合していないもの)、D2、D4-1、D4-2、D6、D8となり、その分布状況は、D0:4.8%、D2:16.8%、D4-1:24.6%、D4-2:13.1%、D6:24.8%、D8:12.6%であった(図6)。
(i)抗体の還元
ヒト化抗TROP2抗体(hTINA1-H1L1)(22.9mL:500mg相当、濃度21.8mg/mL;25mMヒスチジンバッファー)を、ガラス反応容器に入れ、更に、25mM ヒスチジンバッファー(22mL、pH5.0)を加えた。本反応液に、0.5M EDTA水溶液(0.0344mL;抗体に対し5当量)を加えたのち、0.1g/mLポリソルベート20水溶液(0.050ml;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.12に調整し、冷却した。攪拌下、内温0~1℃にて、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩水溶液(2.54mL;抗体一分子に対して2.58当量)を加え、内温0~1℃にて6時間攪拌加し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液に、内温0~2℃にて、攪拌下、実施例2で得た化合物の6.03mg/mL 50%アセトン水溶液(2.99mL;抗体一分子に対して5.1当量)を10分かけて加え、同温度にて、40分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン水溶液(0.206 mL;抗体一分子に対して3当量)を加え、さらに同温度にて10分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量700mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗TROP2抗体(hTINA1-H1L1)ADC組成物を含有する溶液を23.6mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗TROP2抗体(hTINA1-H1L1)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として27640を、重鎖のモル吸光係数として83810を推定値として用いた。
従来の方法により作製されたヒト化抗TROP2抗体ADC組成物(実施例3-1)の平均薬物結合数は4.4であり、D4-1の含有率は24.6%であった。一方、本発明の方法により作製されたヒト化抗TROP2抗体ADC組成物(実施例3-2)の平均薬物結合数は4.1であり、D4-1の含有率は53.3%であった。
(i)ヒト化抗CD98抗体重鎖(hM23-H1)発現ベクターの構築
配列番号11に示すヒト化抗CD98抗体重鎖(hM23-H1)のヌクレオチド配列のヌクレオチド番号58~405に示されるヒト化抗CD98抗体重鎖(hM23-H1)の可変領域をコードするDNA配列を含むDNA断片(ヌクレオチド番号36~422)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hM23-H1の可変領域をコードするDNA配列を含むDNA断片を増幅し、キメラおよびヒト化抗体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化抗CD98抗体重鎖(hM23-H1)発現ベクターを構築した。得られた発現ベクターを「pCMA-G1/hM23-H1」と命名した。
プライマーセット
5’-AGCTCCCAGATGGGTGCTGAGC-3’(配列番号21:プライマー EG-Inf-F)
5’-GGGCCCTTGGTGGAGGCTGAGC-3’(配列番号22:プライマー EG1-Inf-R)
配列番号13に示すヒト化抗CD98抗体軽鎖(hM23-L1)のヌクレオチド配列のヌクレオチド番号61~405に示されるヒト化抗CD98抗体軽鎖(hM23-L1)の可変領域をコードするDNA配列を含むDNA断片(ヌクレオチド番号38~420)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抗CD98抗体軽鎖(hM23-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、キメラおよ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抗CD98抗体軽鎖(hM23-L1)発現ベクターを構築した。得られた発現ベクターを「pCMA-LK/hM23-L1」と命名した。
プライマーセット
5’-CTGTGGATCTCCGGCGCGTACGGC-3’(配列番号23:プライマー CM-LKF)
5’-GGAGGGGGCGGCCACCGTACG-3’(配列番号24:プライマー KCL-Inf-R)
ヒト化抗CD98抗体を実施例1の(iii)と同様の方法で生産した。pCMA-G1/hM23-H1とpCMA-LK/hM23-L1との組合せによって取得されたヒト化抗CD98抗体を「hM23-H1L1」と命名した。
(iii)で得られた培養上清から抗体を実施例1の(iv)と同様の方法で精製した。
(iv)にて精製したヒト化抗CD98抗体(hM23-H1L1)を実施例1の(v)と同様の方法でバッファー交換および濃度調整した。その際に、280nm吸光係数(1.65mLmg-1cm-1)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40mg/mLに抗体濃度を調整した。
(実施例5-1)従来の方法によるヒト化抗CD98抗体(hM23-H1L1)ADC組成物の作製
(i)抗体の還元
ヒト化抗CD98抗体(hM23-H1L1)(12mL:480mg相当、濃度40mg/mL;25mMヒスチジンバッファー)をガラス反応容器に入れ、更に、25mMヒスチジンバッファー(36mL、pH5.0)を加えた。本反応液に、0.5M EDTA水溶液(CALBIOCHEM;0.0394mL;抗体に対し6当量)を加えたのち、0.1g/mLポリソルベート20(日油株式会社)水溶液(0.048mL;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.10に調整した。21℃攪拌下に、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩(Nacalai Tesque, Inc.)水溶液(2.17mL;抗体一分子に対して2.31当量)を加え、内温を35~36℃になるように3時間加温し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液を冷却し、内温17~18℃にて、攪拌下、実施例2で得た化合物の6.20mg/mL 50%アセトン水溶液(2.74mL;抗体一分子に対して5.0当量)を7分かけて加え、同温度にて、40分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン(キシダ化学)水溶液(0.197mL;抗体一分子に対して3当量)を加え、さらに同温度にて30分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量600mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗CD98抗体(hM23-H1L1)ADC組成物を含有する溶液を21.6mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗CD98抗体(hM23-H1L1)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として41370を、重鎖のモル吸光係数として77810を推定値として用いた。
(i)抗体の還元
ヒト化抗CD98抗体(hM23-H1L1)(12.5mL:500mg相当、濃度40mg/mL;25mMヒスチジンバッファー)をガラス反応容器に入れ、更に、25mMヒスチジンバッファー(27.5mL、pH5.0)を加えた。本反応液に、0.5M EDTA水溶液(CALBIOCHEM;0.041mL;抗体に対し6当量)を加えたのち、0.1g/mLポリソルベート20(日油株式会社)水溶液(0.050mL;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.10に調整した。反応液を冷却し、内温0~1℃攪拌下に、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩(Nacalai Tesque, Inc.)水溶液(2.75mL;抗体一分子に対して2.80当量)を加え、内温を0~1℃になるように6時間攪拌し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液を内温0.7-1.2℃にて、攪拌下、実施例2で得た化合物の6.08mg/mL 50%アセトン水溶液(3.14mL;抗体一分子に対して5.4当量)を10分かけて加え、同温度にて、50分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン(キシダ化学)水溶液(0.205mL;抗体一分子に対して3当量)を加え、さらに同温度にて30分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量600mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗CD98抗体(hM23-H1L1)ADC組成物を含有する溶液を23.6mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗CD98抗体(hM23-H1L1)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として41370を、重鎖のモル吸光係数として77810を推定値として用いた。
従来の方法により作製されたヒト化抗CD98抗体ADC組成物(実施例5-1)の平均薬物結合数は4.0であり、D4-1の含有率は27.8%であった。一方、本発明の方法により作製されたヒト化抗CD98抗体ADC組成物(実施例5-2)の平均薬物結合数は4.1であり、D4-1の含有率は51.0%であった。
(実施例6)ヒト化抗B7-H3抗体ADC組成物の作製
(実施例6-1)従来の方法によるヒト化抗B7-H3抗体(M30-H1-L4)ADC組成物の作製
(i)抗体の還元
ヒト化抗B7-H3抗体(M30-H1-L4)(WO2014/057687の参考例1の方法に従って作製、12.4mL:250mg相当、濃度20.1mg/mL;25mMクエン酸バッファー)をガラス反応容器に入れ、更に、25mMヒスチジンバッファー(18mL、pH7.5)を加えた。本反応液に、0.5M EDTA水溶液(CALBIOCHEM;0.018mL;抗体に対し5当量)を加えたのち、0.1g/mLポリソルベート80(日油株式会社)水溶液(0.013mL;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.02に調整した。35℃攪拌下に、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩(Nacalai Tesque, Inc.)水溶液(1.05mL;抗体一分子に対して2.15当量)を加え、内温を35~36℃になるように2時間加温し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液を冷却し、内温15~16℃にて、攪拌下、実施例2で得た化合物の6.22mg/mL 50%アセトン水溶液(1.36mL;抗体一分子に対して4.8当量)を4分かけて加え、同温度にて、20分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン(キシダ化学)水溶液(0.102mL;抗体一分子に対して3当量)を加え、さらに同温度にて20分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量300mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗B7-H3抗体(M30-H1-L4)ADC組成物を含有する溶液を13.1mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗B7-H3抗体(M30-H1-L4)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として30160を、重鎖のモル吸光係数として87250を推定値として用いた。
(i)抗体の還元
ヒト化抗B7-H3抗体(M30-H1-L4)(WO2014/057687の参考例1の方法に従って作製、27.1mL:500mg相当、濃度18.5mg/mL;10mMヒスチジンバッファー)をガラス反応容器に入れ、更に、10mMヒスチジン水溶液(25mL)を加えた。本反応液に、精製白糖(MERCK;1.25g)と0.5M EDTA水溶液(CALBIOCHEM;0.041mL;抗体に対し6当量)を加えたのち、0.1g/mLポリソルベート80(日油株式会社)水溶液(0.050mL;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.08に調整した。反応液を冷却し、内温0~1℃攪拌下に、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩(Nacalai Tesque, Inc.)水溶液(2.08mL;抗体一分子に対して2.13当量)を加え、内温を0~1℃になるように5.5時間攪拌し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液を内温0~1℃にて、攪拌下、実施例2で得た化合物の6.04mg/mL 50%アセトン水溶液(2.82mL;抗体一分子に対して4.8当量)を20分かけて加え、同温度にて、20分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン(キシダ化学)水溶液(0.205mL;抗体一分子に対して3当量)を加え、さらに同温度にて20分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量800mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗B7-H3抗体(M30-H1-L4)ADC組成物を含有する溶液を23.6mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗B7-H3抗体(M30-H1-L4)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として30160を、重鎖のモル吸光係数として87250を推定値として用いた。
従来の方法により作製されたヒト化抗B7-H3抗体ADC組成物(実施例6-1)の平均薬物結合数は3.8であり、D4-1の含有率は27.9%であった。一方、本発明の方法により作製されたヒト化抗B7-H3抗体ADC組成物(実施例6-2)の平均薬物結合数は4.1であり、D4-1の含有率は58.4%であった。
(i)抗体の還元
ヒト化抗HER2抗体(トラスツズマブ;米国特許第5821337号)(22.3mL:500mg相当、濃度22.4mg/mL;25mM ヒスチジンバッファー)をガラス反応容器に入れ、更に、25mMヒスチジンバッファー(27mL、pH5.0)を加えた。本反応液に、0.5M EDTA水溶液(0.034mL;抗体に対し5当量)を加えたのち、0.1g/mLポリソルベート20水溶液(0.050mL;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.12に調整した。22℃攪拌下に、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩水溶液(2.12mL;抗体一分子に対して2.15当量)を加え、内温を22~25℃になるように3時間攪拌し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液を冷却し、内温11~13℃にて、攪拌下、実施例2で得た化合物の6.15mg/mL 50%アセトン水溶液(2.77mL;抗体一分子に対して4.8当量)を20分かけて加え、同温度にて20分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン水溶液(0.206mL;抗体一分子に対して3当量)を加え、さらに同温度にて20分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量600mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗HER2抗体ADC組成物を含有する溶液を22.7mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗HER2抗体(トラスツズマブ)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として26150を、重鎖のモル吸光係数として81290を推定値として用いた。
(実施例7-2)本発明の方法によるヒト化抗HER2抗体ADC組成物の作製
ヒト化抗HER2抗体(トラスツズマブ;米国特許第5821337号)(22.3mL:500mg相当、濃度22.4mg/mL;25mMヒスチジンバッファー)を、ガラス反応容器に入れ、更に、25mM ヒスチジンバッファー(25mL、pH5.0)を加えた。本反応液に、0.5M EDTA水溶液(0.034mL;抗体に対し5当量)を加えたのち、0.1g/mLポリソルベート20水溶液(0.050ml;抗体に対し0.01%)を添加した後、0.3Mリン酸水素二ナトリウム水溶液を加え、pH7.13に調整し、冷却した。攪拌下、内温0~1℃にて、1.00mg/mL トリス(2-カルボキシエチル)ホスフィン塩酸塩水溶液(2.37mL;抗体一分子に対して2.40当量)を加え、内温0~1℃にて6時間攪拌加し、抗体の鎖間ジスルフィドを還元した。
上記(i)で得られた溶液に、内温0~2℃にて、攪拌下、実施例2で得た化合物の6.14mg/mL 50%アセトン水溶液(2.84mL;抗体一分子に対して4.9当量)を10分かけて加え、同温度にて、40分間撹拌し、薬物リンカー中間体を抗体へ結合させた。次に、50mM N-アセチルシステイン水溶液(0.206 mL;抗体一分子に対して3当量)を加え、さらに同温度にて50分間撹拌し、薬物リンカー中間体の反応を停止させたのち、10%酢酸水溶液を用いて、pH5.0に調整した。
上記(ii)で得られた溶液を、Pellicon XL(日本ミリポア株式会社、50cm2)を用い、ローラーポンプにて、10mMヒスチジンバッファー(pH5.0)を加えながら循環させ、排水量600mLになるまで洗浄作業を行い、低分子を除去した。その後、濃縮を行い、ヒト化抗HER2抗体ADC組成物を含有する溶液を21.7mL得た。
実施例3-1の(iv)に記載の共通操作Aと同様に平均薬物結合数を測定した。ヒト化抗HER2抗体(トラスツズマブ)の場合、そのアミノ酸配列に従って、軽鎖のモル吸光係数として26150を、重鎖のモル吸光係数として81290を推定値として用いた。
従来の方法により作製されたヒト化抗HER2抗体ADC組成物(実施例7-1)の平均薬物結合数は3.9であり、D4-1の含有率は34.1%であった。一方、本発明の方法により作製されたヒト化抗HER2抗体ADC組成物(実施例7-2)の平均薬物結合数は4.0であり、D4-1の含有率は55.2%であった。
実施例3、4、6、および7の結果から、平均薬物結合数は、従来の製造方法により製造された抗体-薬物コンジュゲート組成物および本発明の製造方法により製造された抗体-薬物コンジュゲート組成物ともに、3.5~4.5であった。一方、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率は、従来の製造方法により製造された抗体-薬物コンジュゲート組成物では35%以下であるのに対し、本発明の製造方法により製造された抗体-薬物コンジュゲート組成物では50%以上であった。このように、本発明の製造方法を用いることにより、平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である抗体-薬物コンジュゲート組成物を選択的に製造できることが示された。
試験例2の結果から、本発明の製造方法により製造された抗体-薬物コンジュゲート組成物は、従来の製造方法により製造された抗体-薬物コンジュゲート組成物よりも優れた安全性を有することが示された。
以上により、本発明の抗体-薬物コンジュゲート組成物(平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である抗体-薬物コンジュゲート組成物)は、従来の製造方法により製造された抗体-薬物コンジュゲート組成物(平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が35%以下である抗体-薬物コンジュゲート組成物)よりも優れた安全性を有することが示された。
配列番号2:ヒト化抗TROP2抗体重鎖(hTINA1-H1)のアミノ酸配列
配列番号3:ヒト化抗TROP2抗体軽鎖(hTINA1-L1)のヌクレオチド配列
配列番号4:ヒト化抗TROP2抗体軽鎖(hTINA1-L1)のアミノ酸配列
配列番号5:抗TROP2抗体(TINA1)のCDRH1のアミノ酸配列
配列番号6:抗TROP2抗体(TINA1)のCDRH2のアミノ酸配列
配列番号7:抗TROP2抗体(TINA1)のCDRH3のアミノ酸配列
配列番号8:抗TROP2抗体(TINA1)のCDRL1のアミノ酸配列
配列番号9:抗TROP2抗体(TINA1)のCDRL2のアミノ酸配列
配列番号10:抗TROP2抗体(TINA1)のCDRL3のアミノ酸配列
配列番号11:ヒト化抗CD98抗体重鎖(hM23-H1)のヌクレオチド配列
配列番号12:ヒト化抗CD98抗体重鎖(hM23-H1)のアミノ酸配列
配列番号13:ヒト化抗CD98抗体軽鎖(hM23-L1)のヌクレオチド配列
配列番号14:ヒト化抗CD98抗体軽鎖(hM23-L1)のアミノ酸配列
配列番号15:抗CD98抗体(M23)のCDRH1のアミノ酸配列
配列番号16:抗CD98抗体(M23)のCDRH2のアミノ酸配列
配列番号17:抗CD98抗体(M23)のCDRH3のアミノ酸配列
配列番号18:抗CD98抗体(M23)のCDRL1のアミノ酸配列
配列番号19:抗CD98抗体(M23)のCDRL2のアミノ酸配列
配列番号20:抗CD98抗体(M23)のCDRL3のアミノ酸配列
配列番号21:プライマーEG-Inf-Fのヌクレオチド配列
配列番号22:プライマーEG1-Inf-Rのヌクレオチド配列
配列番号23:プライマーCM-LKFのヌクレオチド配列
配列番号24:プライマーKCL-Inf-Rのヌクレオチド配列
配列番号25:ヒト化抗B7-H3抗体重鎖(M30-H1)のアミノ酸配列
配列番号26:ヒト化抗B7-H3抗体軽鎖(M30-L4)のアミノ酸配列
配列番号27:抗B7-H3抗体(M30)のCDRH1のアミノ酸配列
配列番号28:抗B7-H3抗体(M30)のCDRH2のアミノ酸配列
配列番号29:抗B7-H3抗体(M30)のCDRH3のアミノ酸配列
配列番号30:抗B7-H3抗体(M30)のCDRL1のアミノ酸配列
配列番号31:抗B7-H3抗体(M30)のCDRL2のアミノ酸配列
配列番号32:抗B7-H3抗体(M30)のCDRL3のアミノ酸配列
配列番号33:ヒト化抗HER2抗体重鎖のアミノ酸配列
配列番号34:ヒト化抗HER2抗体軽鎖のアミノ酸配列
Claims (25)
- (i)緩衝液中、抗体を還元剤と反応させ、鎖間ジスルフィドを還元する工程;および、
(ii)(i)で得られたチオール基を有する抗体に対して、薬物リンカー中間体を反応させる工程;
を含む、抗体-薬物コンジュゲート組成物の製造方法であって、
(i)の工程の反応温度が、-10~10℃であり、
製造される抗体-薬物コンジュゲート組成物の、平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である、
ことを特徴とする、製造方法。 - 製造される抗体-薬物コンジュゲート組成物の、平均薬物結合数が4.0~4.1である、請求項1に記載の製造方法。
- (i)の工程の反応温度が、-5~5℃である、請求項1または2に記載の製造方法。
- (i)の工程の反応温度が、-3~3℃である、請求項3に記載の製造方法。
- (i)の工程の反応温度が、0~2℃である、請求項4に記載の製造方法。
- (i)の工程の反応温度が、0~1℃である、請求項5に記載の製造方法。
- 還元剤が抗体一分子あたり2~3モル当量で用いられる、請求項1~6のいずれか1項に記載の製造方法。
- 還元剤がトリス(2-カルボキシエチル)ホスフィンまたはその塩である、請求項1~7のいずれか1項に記載の製造方法。
- トリス(2-カルボキシエチル)ホスフィンの塩がトリス(2-カルボキシエチル)ホスフィン塩酸塩である、請求項8に記載の製造方法。
- 緩衝液が、ヒスチジンバッファーである、請求項1~9のいずれか1項に記載の製造方法。
- 緩衝液が、キレート剤を含む、請求項1~10のいずれか1項に記載の製造方法。
- キレート剤がエチレンジアミン四酢酸である、請求項11に記載の製造方法。
- 抗体が、抗TROP2抗体、抗CD98抗体、抗B7-H3抗体、または抗HER2抗体である、請求項1~12のいずれか1項に記載の製造方法。
- 薬物リンカー中間体が、N-置換マレイミジル基を有する、請求項1~13のいずれか1項に記載の製造方法。
- 請求項1~15のいずれか1項に記載の製造方法によって製造される、抗体-薬物コンジュゲート組成物。
- 平均薬物結合数が3.5~4.5であり、重鎖-軽鎖間チオールに薬物リンカーが4個結合した抗体-薬物コンジュゲートの含有率が50%以上である、抗体-薬物コンジュゲート組成物。
- 平均薬物結合数が4.0~4.1である、請求項17に記載の抗体-薬物コンジュゲート組成物。
- 抗体が、抗TROP2抗体、抗CD98抗体、抗B7-H3抗体、または抗HER2抗体である、請求項17または18に記載の抗体-薬物コンジュゲート組成物。
- 請求項16~20のいずれか1項に記載の抗体-薬物コンジュゲート組成物を含有する、医薬組成物。
- 腫瘍および/または癌の治療のための、請求項21に記載の医薬組成物。
- 肺癌、腎癌、尿路上皮癌、大腸癌、前立腺癌、多型神経膠芽腫、卵巣癌、膵癌、乳癌、メラノーマ、肝癌、膀胱癌、胃癌、子宮頸癌、子宮体癌、頭頸部癌、食道癌、胆道癌、甲状腺癌、リンパ腫、急性骨髄性白血病、急性リンパ性白血病、慢性骨髄性白血病、および/または多発性骨髄腫の治療のための、請求項22に記載の医薬組成物。
- 請求項16~20のいずれか1項に記載の抗体-薬物コンジュゲート組成物を投与することを特徴とする、腫瘍および/または癌の治療方法。
- 肺癌、腎癌、尿路上皮癌、大腸癌、前立腺癌、多型神経膠芽腫、卵巣癌、膵癌、乳癌、メラノーマ、肝癌、膀胱癌、胃癌、子宮頸癌、子宮体癌、頭頸部癌、食道癌、胆道癌、甲状腺癌、リンパ腫、急性骨髄性白血病、急性リンパ性白血病、慢性骨髄性白血病、および/または多発性骨髄腫の治療方法である、請求項24に記載の治療方法。
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TW201705982A (zh) | 2017-02-16 |
ES2938186T3 (es) | 2023-04-05 |
JP6787890B2 (ja) | 2020-11-18 |
TWI827921B (zh) | 2024-01-01 |
JPWO2017002776A1 (ja) | 2018-05-31 |
US20210386865A1 (en) | 2021-12-16 |
EP3315512B1 (en) | 2022-11-23 |
KR20180021723A (ko) | 2018-03-05 |
TW202130368A (zh) | 2021-08-16 |
EP4180455A1 (en) | 2023-05-17 |
EP3315512A4 (en) | 2019-02-13 |
IL256558A (en) | 2018-02-28 |
IL256558B (en) | 2022-04-01 |
HUE061408T2 (hu) | 2023-06-28 |
JP7118117B2 (ja) | 2022-08-15 |
AU2016286898A1 (en) | 2018-01-04 |
US20180147292A1 (en) | 2018-05-31 |
US11173213B2 (en) | 2021-11-16 |
CN107922477A (zh) | 2018-04-17 |
BR112017027690A2 (pt) | 2018-10-09 |
CA2990572C (en) | 2022-07-26 |
CN107922477B (zh) | 2022-11-01 |
AU2016286898B2 (en) | 2022-12-08 |
CN116059395A (zh) | 2023-05-05 |
IL290959B (en) | 2022-12-01 |
IL290959B2 (en) | 2023-04-01 |
TWI725037B (zh) | 2021-04-21 |
CA2990572A1 (en) | 2017-01-05 |
EP3315512A1 (en) | 2018-05-02 |
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IL290959A (en) | 2022-04-01 |
HK1247211A1 (zh) | 2018-09-21 |
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