TW201117814A - New maytansinoids and the use of said maytansinoids to prepare conjugates with an antibody - Google Patents
New maytansinoids and the use of said maytansinoids to prepare conjugates with an antibody Download PDFInfo
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- TW201117814A TW201117814A TW099133090A TW99133090A TW201117814A TW 201117814 A TW201117814 A TW 201117814A TW 099133090 A TW099133090 A TW 099133090A TW 99133090 A TW99133090 A TW 99133090A TW 201117814 A TW201117814 A TW 201117814A
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- GRVDJDISBSALJP-UHFFFAOYSA-N methyloxidanyl Chemical group [O]C GRVDJDISBSALJP-UHFFFAOYSA-N 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- BDRTVPCFKSUHCJ-UHFFFAOYSA-N molecular hydrogen;potassium Chemical compound [K].[H][H] BDRTVPCFKSUHCJ-UHFFFAOYSA-N 0.000 description 1
- 229940125645 monoclonal antibody drug Drugs 0.000 description 1
- 125000002950 monocyclic group Chemical group 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 125000004108 n-butyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000000740 n-pentyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000004123 n-propyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 125000001971 neopentyl group Chemical group [H]C([*])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000000269 nucleophilic effect Effects 0.000 description 1
- 125000002347 octyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 125000003538 pentan-3-yl group Chemical group [H]C([H])([H])C([H])([H])C([H])(*)C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 150000004968 peroxymonosulfuric acids Chemical class 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 125000004437 phosphorous atom Chemical group 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 229940047920 potassium 75 mg Drugs 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 239000001120 potassium sulphate Substances 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010079317 prolyl-tyrosine Proteins 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000003586 protic polar solvent Substances 0.000 description 1
- 125000004076 pyridyl group Chemical group 0.000 description 1
- 125000000714 pyrimidinyl group Chemical group 0.000 description 1
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 231100000272 reduced body weight Toxicity 0.000 description 1
- RWWYLEGWBNMMLJ-YSOARWBDSA-N remdesivir Chemical compound NC1=NC=NN2C1=CC=C2[C@]1([C@@H]([C@@H]([C@H](O1)CO[P@](=O)(OC1=CC=CC=C1)N[C@H](C(=O)OCC(CC)CC)C)O)O)C#N RWWYLEGWBNMMLJ-YSOARWBDSA-N 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 229930182490 saponin Natural products 0.000 description 1
- 150000007949 saponins Chemical class 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N serine Chemical compound OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 150000003385 sodium Chemical class 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007614 solvation Methods 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 125000005346 substituted cycloalkyl group Chemical group 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 125000000472 sulfonyl group Chemical group *S(*)(=O)=O 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 125000004434 sulfur atom Chemical group 0.000 description 1
- 210000004233 talus Anatomy 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 108010071097 threonyl-lysyl-proline Proteins 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- ITMCEJHCFYSIIV-UHFFFAOYSA-M triflate Chemical compound [O-]S(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-M 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000004565 tumor cell growth Effects 0.000 description 1
- 108010079202 tyrosyl-alanyl-cysteine Proteins 0.000 description 1
- 108010071635 tyrosyl-prolyl-arginine Proteins 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
- 108010052774 valyl-lysyl-glycyl-phenylalanyl-tyrosine Proteins 0.000 description 1
- 108010021199 valyl-valyl-valine Proteins 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
- 229910052727 yttrium Inorganic materials 0.000 description 1
- VWQVUPCCIRVNHF-UHFFFAOYSA-N yttrium atom Chemical compound [Y] VWQVUPCCIRVNHF-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D498/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms
- C07D498/12—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms in which the condensed system contains three hetero rings
- C07D498/18—Bridged systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/5375—1,4-Oxazines, e.g. morpholine
- A61K31/5383—1,4-Oxazines, e.g. morpholine ortho- or peri-condensed with heterocyclic ring systems
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/68033—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a maytansine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
- A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pharmacology & Pharmacy (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nitrogen And Oxygen Or Sulfur-Condensed Heterocyclic Ring Systems (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicinal Preparation (AREA)
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
201117814 六、發明說明: 【發明所屬之技術領域】 本發明係關於新穎類美登素及該類美登素用於製備與抗 體結合的結合物之用途。本發明亦關於包含該類美登素與 該等結合物之組合物。 【先前技術】 已出現許多製品嘗試以單株抗體-藥物結合物特異性靶 向腫瘤細胞(Sela等人,於/wwwnocortyMgflies 189-216 (C. Vogel 編,1987)中;Ghose 等人’於 rargeied 1-22 (E. Goldberg 編,1983)中;Diener 等人,於 dwiAo办 1-23 (J. Rodwell編 ’ 1988)中; 專/^,於 Antibody mediated delivery systems 25-53 (J. Rodwell 編,1988)中;Bumol 等人,於 办 we山βγίβ/ηβ 55-79 (J. Rodwell 編,1988) 中)。亦參見:Monneret C·等人,Cawcer 2000, 87(11),829-38 ; Ricart A.D.等人,C7/m.ca/ /Vaci/ce 2007,245-255 ; Singh R•及 Rickson H.K·, Therapeutic Antibodies: Methods and Protocols, 2009, 525, 445-467。已使用不同類之細胞毒性劑,諸如紫杉烧衍生 物(taxane derivative)(WO 06061258)、 來普黴素衍生物 (leptomycine derivative)(W〇 07144709)、CC-1065及類似 物(WO 2007102069)或諸如甲胺嗓吟(meth〇trexate)、道諾 黴素(daunorubicin) ' 阿黴素(doxorubicin)、長春新鹼 (vincristine)、長春鹼(vinblastine)、美法侖(melphalan)、 150937.doc 201117814 絲裂黴素C(mitomycin C)、苯丁酸氮茶(chlorambucil)與抗 體結合。 使用對腫瘤細胞具有親和力的靶向抗體使得細胞毒性劑 直接在腫瘤細胞附近傳遞或直接在腫瘤細胞中傳遞成為可 能,從而增加細胞毒性劑之功效,同時將通常與細胞毒性 劑有關之副作用減至最低。
類美登素為來源於美登素(maytansin)之細胞毒性劑,美 登素為分離自東非灌木齒葉美登木serraia)之天 然產物(US 3896111)。已製備許多類美登素:參見US 4151042 ; J.Med.Chem. 1978, 21, 3 1-37 ; Nature 1977, 270, 721-722, Chem.Pharm.Bull. 1984, 3441-345 1 ; US 4248870 ; US 4137230 ; Chem.Bull. 1984, 3441。 US 5,208,020 > US 5,416,064^ R.V.J. Chari, 31 Advanced Drwg De//ver_y 89-104 (1998)描述了 類美登素結合 物,諸如 L-DMl(A)或 L-DM4(A,):
(糾〇 類美登素之結合物描述於EP 0425235及WO 2004/103272 中。在EP 0425235中描述了以下式(Bl)、(B2)或(B3)之類 150937.doc 201117814 美登素:
0 丫” 1 CHa (B1)—
(B3) 其中Z〇、Z,4Z2表示Η或SR。 在WO 2004/103272中描述了式(C)之類美登素:
其中Υ'表示 (CR7CR8)1(CR9=CR1〇)pC = CqAr(CR5CR6)mDu(CR11=CR12)r (C三ChBtCCRsCRJuCHSZ,其中A、B及D表示視情況經 取代之環烷基、環烯基、雜芳基或雜環烷基。 在WO 03/068144中,描述式(D)之化合物:
其中Z為細胞毒性劑且Q為R2COO、R2R3NCOO、 150937.doc 201117814 R2OCOO、R20、R2CONR3、R2R3N、R2OCONR3 或 S。尺2為 SCR4R5R6。z可為選自以下各者之類美登素衍生物:
更特定言之,揭示以下化合物(D):
化合物(D)因此在聚乙二醇化連接基團中含有内部二硫 鍵。 2008年12月8日,Immunogen Inc.亦在曰内瓦舉行之歐洲 抗體會議(European Antibody Congress in Geneva)中揭示式 (E)之結合物:
Ο mAb-N^^o^0'〇 〜〇*ν Π (Ε), 150937.doc 201117814 且已在2008年10月在曰内瓦舉行之第二十屆e〇rtcnci_ AACR論壇會中揭示式(E,)之結合物:
【發明内容】 定義 •「烧基」意謂可為鏈中具有1至20個碳原子的直鏈或分 支鏈脂族烴基或具有3至10個碳原子的環狀脂族烴基。較 佳烷基在鏈中具有I至12個碳原子。例示性烷基包括甲 基、乙基、正丙基、異丙基、2,2-二甲基丙基、正丁基、 第二丁基、正戊基、3-戊基、辛基、壬基、癸基; •「環烷基」意謂具有3至1〇個碳原子的環狀脂族烴基。 較佳環烷基在環狀鏈中具有3至8個碳原子。例示性環烷基 包括環丙基、環丁基、環戊基及環己基; •「芳基」意謂ό至14個碳原子,較佳6至丨〇個碳原子之 芳族單環或多環烴環系統。例示性芳基包括苯基或萘基。 •「雜芳基」意謂3員至14員,較佳5員至1〇員不飽和穩 定單環、雙環或多環中環中至少一成員為雜原子。雜 原子通常為(但不限於)氧、氮、硫、硒或磷原子。雜原子 較佳為氧、氮或硫原子。例示性雜芳基包括吡啶基、吼咯 150937.doc 201117814 基、噻吩基、呋喃基、嘧啶基及三唑基; 「雜環院基」意謂含有至少一個雜原子之環院基,其 中環中至少一成員為雜原子; 所定 •「烷氧基」意謂-〇-烷基,其中烷基係如上所定義 •「烧氧基」意s胃-0-C0-烧基’其中坑基係如上 義; •「伸烷基」意謂由直鏈或分支鏈烷移除兩個氫原子所 形成之通式-CmH2m-之览基。例示性伸烧基包括亞甲基 (-CH2-)、伸乙基(-CR2CH2-)、伸丙基(_CH2CH2CH2-)、伸 丁基(-CH2CH2CH2CH2-)、伸異丁基()、伸己基 (-CH2CH2CH2CH2CH2CH2-)。直鏈伸院基可由式 特 定表示,其中m為1至20之整數; •「EphA2受體」係指一種路胺酸激酶,屬於Eph受體家 族(回顧於 Pasquale,E. B.等人,2005, CW/ 6,462-475 中),且包含 Genbank 寄存編號 NM_004431(人類 EphA2)、NM_010139(鼠類 EphA2)或 NXM—345596(大鼠EphA2)中之胺基序列。人類EphA2為較 佳EphA2受體。本文所用之術語「EphA2配位體」係指結 合於EphA2受體且視情況活化(例如刺激自體磷酸 化)EphA2受體之蛋白質。本文中之較佳EphA2配位體為 「ephrinA 1」,其結合於EphA2受體且包含Genbank寄存編 號NM_004428(人類ephrinAl)中之胺基序列; •「多株抗體」為在一或多種其他不同抗體中或存在下 產生的抗體。多株抗體一般係在幾種其他產生不同抗體之 150937.doc 201117814 B-淋巴細胞存在下由一種B淋巴細胞所產生。多株抗體通 常直接獲自經免疫之動物; •「單株抗體」為獲自實質上同源抗體群之抗體,亦即 形成該群體之抗體除可少量存在之可能天然產生之突變外 基本上相同。該等抗體係針對單一抗原決定基且因此具有 高度特異性; •「裸抗體」為不與類美登素結合之抗體; •「抗原決定基」為抗原上供抗體結合之位點。其可由 鄰接殘基形成或由非鄰接殘基因抗原蛋白質摺疊而緊密接 近來形成。鄰接胺基酸所形成之抗原決定基在暴露於變性 溶劑時通常保持’而非鄰接胺基酸所形成之抗原決定基在 此暴露下通常失去。 【實施方式】 新穎類美登素 本發明係關於一種式⑴化合物:
其中 ALKJ^(Ci-c6)伸烷基; 1及Xz各獨立地為以下基團之一 :-CH=CH-、-CO-、 150937.doc 201117814 -CONR-,-NRCO- > -COO- ^ -OCO- > -OCONR- ^ -NRCOO-、_NRCONR,…_NR_、_s(〇)n(n=〇、【或 2) 或-O-; • R及R,獨立地為H或((VC6)烷基; • I為1至40之整數,較佳為1至2〇之整數,且更佳為上至 10之整數; •當為-CH=CH-時,j為對應於i之整數且當χ2不 為-CH=CH-時,j為對應於2之整數; • Zb為一簡單鍵、-〇-或-NH-且Rb為Η或(Cl-C6)烷基' (c^c:7)環烷基、芳基、雜芳基或(c3_C7)雜環烷基;或 Z b為早鍵且R卜為H a 1。 更特疋έ之’ X2為-CH = CH-或-CONR-,CO基連接於 -Xi-ALK-基且R為η或(CVCe)烧基。更特定言之,_X|_ALK_ 為-8-(^2-。更特定言之,1為3、4、5、6、7、8、9或10。 本發明之特徵可為式(II)化合物:
式(Π)更確切地涵蓋以下化合物: 150937.doc -II - 201117814
150937.doc -12· 201117814 該等化合物可以鹼或鹽形式存在或以該鹼或該鹽之溶劑 合物或水合物形式存在。 本發明之化合物包含反應性化學基團-C(=0)ZbRb(GCRl), 其對存在於抗體上之反應性化學基團(GCR2)具有反應性。 GCR1與GCR2之間的反應使得經由共價鍵將細胞毒性劑連 接於抗體上成為可能。因此,該化合物易於與抗體結合。 更特定言之,Zb為Ο ;在此情況下,GCR1為羧酸官能基 (Rb=H)或酯官能基。更特定言之,-C( = 0)ZbRb為-COOH、 -COOCCi-CJ 烷基,諸如-COOCH3 或-COOCH2CH=CH2。在 酯官能基中,對抗體胺基(諸如離胺酸基團)呈現出良好反 應性的「經活化」者為較佳。舉例而言,經活化之目旨可為
,SO,WI M=H或陽離子
-其中GI表示至少一種誘導基團,諸如-N〇2或鹵 基(例如-F)。該等經活化之酯的實例為以下者:-
GCR2可為例如位於抗體表面離胺酸殘基側由離胺酸攜 帶之ε-胺基、鉸鏈區之醣基或鏈内S-S鍵還原後半胱胺酸 之硫醇基(Garnett VLC.專尺,Advanced Drug Delivery 2001,5义171-216)。最近,新穎方法已針對藉由 突變引入半胱胺酸(Junutula J.R·等人,TVaiwre 价okc/mo/og少 2008, 26,925-932 ; WO 09026274)或引入非 天然胺基酸,從而使得開發新類型蛋白質化學物質成為可 150937.doc -13- 201117814
能(de Graaf A.J.等人,C/zem. 2009,2009年 2 月 3 日(Review) ; DOI: 10.102 l/bc800294a ; WO 2006/069246 以及 Chin J.W.等人,丄4CS 2002,724, 9026-9027 (technology ReCode®))。 本發明之化合物可用於製備共價連接有至少一個下式之 類美登素片段的結合物:
因此,式(I)化合物可用於製備一種結合物,其中該類美 登素片段共價連接於抗體。 製備式(I)化合物之一般流程 可根據流程1製備式(I)化合物:
流程1 + RGj—ALK-X;-(CH办OCHjCHjVCbOJ-ZA-- (I)
中間體P!含有RG!反應性基團,其能與連接於含有中間 體P2之PEG的反應性基團RG2&應形成X】。舉例而言,可 經由在諸如DIEA之鹼存在下藉由親核取代使具有RGf-SH 150937.doc -14- 201117814 之Pi與具有RG2=-Br之P2&應形成X丨=S。該反應之實例提 供於實例1、2中。 具有RGp-SH之P〗的實例為L-DM1及L-DM4以及EP 1313738之化合物 11a、c、d、g: L-DM1 : ALK-SH=-CH2CH2-SH ; L-DM4 : ALK-SH=-CH2CH2CMe2-SH ; 11a : ALK-SH=-CH2-SH ; 11c : ALK-SH=-CH2CH2CH2-SH ; lid : ALK-SH=-CH2CH2CH2CH2-SH ; llg : ALK-SH=-CHMe-CH2-SH。 其他反應實例提供於表I中。
表I 條目 Χι χ2 p2 RG, rg2 反應條件 1 -S- -CO- 鹵基-乙 醯基連接 基團 -SH 鹵基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG>=-SH2pi(例 如L-DM1或L-DM4)經函基-乙醯基連 接基團親核取代 2 -S- -CONR- 鹵基-SI 胺連接基 團 -SH 鹵基 在中性或鹼性條件(有機鹼' 無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RGiz-SHiP^例 如L-DM1或L-DM4)經齒基-醯胺連接 基團親核取代 2' -S- -CONR- 磺酸酯· 醯胺連接 基團 -SH 經活化 之磺酸 酯 在中性或驗性條件(有機驗、無機驗 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RGi=-SH之Pi(例 如L-DM1或L-DM4)經活化磺酸醋-醯 胺連接基團(例如甲續醯基、甲苯石黃 酿基、三氟甲磺酸酯)親核取代 150937.doc -15- 201117814 3 -S(0)n-η=1 或2 -CONR- 鹵基-酿 胺連接基 困 -SH 鹵基 在輕度氧化條件(過硫酸氫鉀、過酸) 下’在非質子性極性溶劑中,可控 氧化條目1所得之產物 4 -S- -NRCO- 齒基-酿 胺連接基 團 -SH 鹵基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG〗=-SH之Pi(例 如L-DM1或L-DM4)經函基-醯胺連接 基團親核取代 5 -S- -COO- 函基-S旨 連接基團 -SH 鹵基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG丨=-SH之Pi(例 如L-DM1或L-DM4)經鹵基-醋連接基 團親核取代 6 -S- -OCO- 鹵基-碳 酸酯連接 基團 -SH 鹵基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG丨=-SH之Pi(例 如L-DM1或L-DM4)經鹵基-碳酸酯連 接基團親核取代 7 -S· OCONR- 或- NRCOO- 鹵基- 胺基甲酸 酯連接基 團 -SH 鹵基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RGi=-SH之Pi(例 如L-DM1或L-DM4)經_基-胺基甲酸 酯連接基團親核取代 8 -s- RCONR,- 鹵基-服 連接基團 -SH 齒基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG丨=-SH2P!(例 如L-DM1或L-DM4)經鹵基-脲連接基 團親核取代 9 -s- -NR- 鹵基-胺 連接基團 -SH 卤基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RGi^SHiPi(例 如L-DM1或L-DM4)經鹵基-胺連接基 團親核取代 10 -S(0)„-n=0、1 或2 -S(0)„-n=0 ' 1 或2 鹵基-硫 基連接基 團 -SH 齒基 在中性或鹼性條件(有機鹼、無機鹼 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG丨=-SH之Pi(例 如L-DM1或L-DM4)經函基-硫基連接 基團親核取代,最後在輕度氧化條 件(過硫酸氫鉀、過酸)下,在非質子 性極性溶劑中,可控氧化由此獲得 之產物 150937.doc -16· 201117814 在中性或鹼性條件(有機鹼、無機鹼 11 -S- -0- _基-謎 連接基團 -SH 鹵基 或負載鹼)下,在質子性或非質子性 極性溶劑中,具有RG产-SH2P1(例 如L-DM1或L-DM4)經鹵基-醚連接基 團親核取代 對於一些式(I)化合物而言,可在P!與P2反應後、另 一 -ZbRb基團之至少一次轉化後獲得最終所需的-ZbRb基 團。實例提供於流程1’中,其中-ZbRb=-0-烯丙基轉化 0
—〇—N 為-ZbRb= 0 2. fit
流程Γ 同樣,亦可在卩1與卩2之間反應前,對攜帶-ZbRb基圑之中 間體使用至少一次轉化。 另一實例為-ZbRb=-OH轉化為-ZbRb=Hal,其需要諸如 SOCI〗之醯化劑。 製備P,
Pi可根據流程2、以美登醇為起始物質製備:
流程2 150937.doc -17- 201117814 美登醇藉由酯化反應與含有反應性醯基-COOZ之中間體 P3反應,其中Z為Η或鹵素原子。該反應描述於WO 2004/103272 之圖 3a-d 以及 WO 2007/021674 中。當 Ζ 為 Η 時,酯化可藉由增強酸官能基反應性之偶合劑進行。 製備Ρ2 製備ρ2之起始物質為可市面上購得之PEG化合物或可用 該市售PEG化合物經由熟習此項技術者已知之至少一個化 學反應來製備。PEG化合物可在市面上購得,例如購自 JenKem Technology USA Inc. 2033 W. McDermott Dr. Suite 320 #188, Allen, TX 75013-4675, USA。 舉例而言,使用市售化合物HOOCCH2CH2(OCH2CH2)r OCH2CH2NH2 製備其中 X2=-CONR-且 RG2=Hal 之 P2(P2=Hal-ALK-CONR-CH2CH2(OCH2CH2)i-COZbRb)描述如
R=H
HaN Br 或 I 步驟(i):形成醯胺鍵且活化酸基團;在諸如DCM之極性 非質子性溶劑中分別進行兩個步驟:使胺基與鹵基烷酸N-羥基丁二醯亞胺酯(例如li基乙酸酯)反應,接著當場添加 諸如DIC之偶合劑。 R关Η 150937.doc •18* 201117814
步驟(Π):羧酸以酯形式保護且胺以三氟乙醯胺形式保 護;在諸如DCM之極性非質子性溶劑中分別進行兩個步 驟:藉由三甲基矽烷基重氮甲烷及曱醇處理來保護酸,接 著藉由添加TFAA及TEA保護胺; 步驟(iii):胺之烷基化及酯之鹼性水解;在諸如THF之 極性非質子性溶劑中以兩個獨立步驟進行反應:在攜帶諸 如鹵烧RHal之離去基的反應物存在下,藉由NaH處理來使 胺炫基化,且添加LiOH水溶液; 步驟(i):在步驟(iii)之後,進行步驟(i)(其中R=H)的反 應。 同樣,使用市售化合物Hal-CH2CH=CHCH2(OCH2CH2)r COOtBu 製備 X2 = -CH=CH-且 RG2=鹵基之 P2(P2=Hal-CH2-CH=CH-CH2(OCH2CH2)i-COZbRb)描述如下:
負載型DCC
結合物之製備方法 結合物可由包含以下步驟之方法獲得: (i) 使視情況緩衝之抗體水溶液與式(I)化合物之溶液接 觸; (ii) 隨後視情況使⑴中形成之結合物與可能存在於溶液 150937.doc -19- 201117814 中之未反應5式劑及任何聚集物分離。 細胞結合劑之水溶液可用至少一種緩衝劑緩衝,諸如鱗 酸鉀或N-2-羥乙基哌嗪·Ν,·2_乙磺酸(Hepes緩衝劑)或緩衝 劑混合物(諸如以下實例中所揭示之緩衝劑A)。緩衝劑視 抗體性質而定。式(I)化合物溶解於有機極性溶劑(或極性 溶劑混合物)中,例如DMSO或DMA。 反應溫度通常在20至40°C之間變化。反應時間可在1至 24小時之間變化。抗體與細胞毒性劑之間的反應可使用折 射測定計及/或UV偵測器藉由尺寸排阻層析法來監 測。若結合物產量太低’則可延長反應時間及/或可添加 式⑴化合物。 為進行步驟(ii)之分離,熟習此項技術者可使用許多不 同層析方法:結合物可藉由例如SEC、吸附層析(諸如離子 父換層析,1EC)、疏水性相互作用層析(HIC)、親和性層 析、混合載體層析(諸如羥基磷灰石層析)或高效液相層析 (HPLC)來純化。亦可使用透析或透據純化。 可使用之方法的實例描述於實例I中。 如本文中所使用之術語「聚集物」意謂可在兩個或兩個 以上抗體之間所形成之結合物,該等抗體已因結合而改變 或未改變°聚集物可在大量參數影響下形成,諸如溶液中 抗體之河/農度、溶液pH、高剪切力、鍵結二聚體數目及其 疏 Kit ,皿度(參見 Wang & Gosh, 2008, J. Mew卜ane ^ 311 316,及其中引用之參考文獻);應注意,一些此 等 > 數之相對影響並未清楚確立。就蛋白質及抗體而言, 150937.doc 201117814 熟習此項技術者應參考Cromwel丨等人(2006, ^ 8(3): E572-E5 79)。可使用熟習此項技術者所熟知技術(諸 如SEC)測定聚集物之含量(參見Walter等人,1993, Biochem., 212(2): 469-480) ° 步驟⑴或⑼後’可對含有結合物之溶液進行超遽及/或 透濾之額外步驟(in)。 在此等步驟結束時以水溶液形式回收結合物。 抗體 術語「抗體」係、α最廣泛含義用於本文中且特定涵蓋諸 如IgG、IgM、IgA、IgD及IgE之任何同型單株抗體(包括全 長單株抗體)、多株抗體、多特異性抗體、嵌合抗體及抗 體片段。對特異性抗原具有反應性之抗體可藉由重組方法 產生,諸如選擇噬菌體或類似載體中之重組抗體庫,或用 抗原或編碼抗原之核酸使動物免疫。 典型抗體包含由二硫鍵連接之兩個相同重鏈及兩個相同 輕鏈。各重鏈及輕鏈含有恆定區及可變區。如本文所用, 「VH」或「VH」係指抗體免疫球蛋白重鏈(包括卜、 scFv、dsFv' Fab、Fab,或F(ab,)2片段重鏈)之可變區。提 及「VL」或「VL」係指抗體免疫球蛋白輕鏈(包括Fv、 scFv ' dsFv、Fab、Fa1y或F(aly)2片段輕鏈)之可變區。各 可變區含有三個稱為「互補決定區」(「CDR」)或「高變 區」之區段,其主要負責結合抗原之抗原決定基。自^^端 依次編號,其通常稱為CDR1、CDR2& CDR3。可變區之 較高度保守部分稱為「構架區」(「FR」)。天然重鏈及輕 150937.doc 21 201117814 鏈之可變域各包含四個FR區,該等Fr區廣泛採用β片構型 且經三個形成環的CDR連接,該等環連接β片結構且有時 候形成β片結構之一部分。各鏈中之CDR藉由FR區緊密結 合在一起且與另一鏈之CDR—起促進抗體之抗原結合位點 形成(參見 Kabat 4 人,Sequences of Proteins of Immunological Interest,第 5 版,National Institute 〇f Health, Bethesda, MD, 1991) 〇 抗體(詳見 Janeway 等人,《immun〇bi〇l〇gy»,第 5 版, 2001,Garland Publishing,New York)可選自例如 w〇 04043344、WO 08010101 . w〇 08047242 或 \y〇 05009369(抗CA6)中提及者。 亦可考慮識別A類Eph受體家族成員(諸如EphA2受體, 較佳人類受體)且用作該受體之拮抗劑的抗體或其片段。 該抗體缺乏任何促效劑活性。該抗體或其抗原決定基結合 片段可為申請專利範圍第12-15項中所述者。 人類化抗體或其抗原決定基結合片段較佳具有另外抑制 表現EphA2受體之癌細胞生長的能力。人類化抗體或其抗 原決定基結合片段較佳具有另外抑制表現Eph A2受體之轉 移性癌細胞遷移的能力。 人類化抗體可為人類化2H11R35R74抗體或其抗原決定 基結合片段。人類化抗體可由編碼hu53.2Hll之聚核^:酸 序列之定點突變誘發獲得(WO 2008/010101)。較佳提供 2H11R3 5R74抗體之表面重塑(resurfaced)或人類化形式, 其中該抗體或其片段之輕鏈與重鏈中之表面暴露殘基經i 150937.doc •22· 201117814 換而與已知人類抗體表面更密切相似。人類化2H11R35R74 抗體或其抗原決定基結合片段具有改良之特性。舉例而 言,人類化2H11R35R74抗體或其抗原決定基結合片段特 異性識別EphA2受體。人類化2H11R35R74抗體或其抗原決 定基結合片段更佳具有另外抑制EphA2受體表現細胞生長 的能力。 2H11R3 5R74抗體之人類化形式亦在本文中就以下充分 表徵:其輕鏈與重鏈可變區之相應胺基酸序列、輕鏈及重 鏈可變區基因之DNA序列、CDRs鑑定、其表面胺基酸鑑 定、及其以重組形式表現之方法之揭示。然而,範疇不限 於包含該等序列之抗體及片段。反而,亦考慮特異性結合 EphA2受體之所有抗體及片段。特異性結合EphA2受體之 抗體及片段較佳拮抗該受體之生物活性。該等抗體另外更 佳實質上缺少促效劑活性。因此,抗體及抗原決定基結合 抗體片段可在骨架、CDRs及/或輕鏈及重鏈之胺基酸序列 上不同於2H11R35R74抗體或其人類化衍生物,且仍屬於 本發明之範疇。 2H11R3 5R74抗體之CDR係藉由模型化來鑑定且已預測 其分子結構。此外,雖然CDR對於抗原決定基識別而言具 有重要作用,但其並非本發明之抗體及片段所必需的。因 此,提供具有經改良特性之抗體及片段,該等經改良特性 藉由例如本發明抗體之親和力成熟所產生。 53.2H11可能來源之小鼠輕鏈IgVK及JK生殖系基因及重 鏈IgVh及Jh生殖系基因已經鑑定且揭示於WO 2008/010101 150937.doc •23· 201117814 中。該等生殖系序列之寄存編號分別為MMU23 1196及 AF303833。該等生殖系基因序列適用於鑑定抗體中之體細 胞突變,包括CDR中之體細胞突變。 2H11R3 5R74抗體重鏈及輕鏈可變區序列及其CDR序列 列示於本案中。該資訊可用於產生2H11R35R74抗體之人 類化形式。亦可藉由hu53.2Hll之定點突變誘發獲得本發 明之人類化2H11R35R74抗體。該等人類化抗EphA2抗體或 其衍生物亦可用作本發明結合物之細胞結合劑。 因此,在一實施例中,本發明提供包含一或多個CDR的 人類化抗體或其抗原決定基結合片段,該等CDR具有選自 由SEQ ID NO: 1、2、3、4、5、6組成之群的胺基酸序 列。在一較佳實施例中,本發明之人類化抗體包含至少一 條重鏈及至少一條輕鏈,其中該重鏈包含三個具有由SEQ ID NO: 1、2及3表示之胺基酸序列的連續CDR,且其中該 輕鏈包含三個具有由SEQ ID NO: 4、5及6表示之胺基酸序 列的連續CDR。 人類化2H11R35R74抗體或其片段較佳包含具有由SEQ ID NO. 12組成之胺基酸序列的VH。包含具有由SEQ ID NO. 14組成之胺基酸序列之的人類化2H11R35R74抗體 或其片段亦較佳。人類化2H11R35R74抗體較佳包含至少 一條重鏈及至少一條輕鏈,其中該重鏈包含三個具有由 SEQ ID NO: 1、2及3表示之胺基酸序列的連續CDR,其中 該輕鏈包含三個具有由SEQ ID NO: 4、5及6表示之胺基酸 序列的連續CDR,其中該重鏈具有由SEQ ID NO. 12組成 150937.doc •24· 201117814 ID NO. 14組成 之胺基酸序列’且其中該輕鏈具有由SEq 之胺基酸序列。 結合物 體之類美登素分
結合物通常包含1至1 〇個共價連接於抗 子(所謂「藥物與抗j 體及所用類美登素3 類美登素/抗體比率 質)而變化。因此, 物,其包含:若干由於藥物與抗體比率不同而彼此不同之 結合物;視情況含有之裸抗體;視情況含有之聚集物。所 測定之DAR因此為平均值。 本發明因此亦關於一種結合物,其包含—或多個共價連 接於抗體之如申請專利範圍第1-8項中任_項之化合物。 該連接較佳係經由醯胺鍵。該抗體較佳如申請專利範圍第 12-15項中任一項所定義。 本文所用測定DAR之方法在於以分光光度法量測實質上 經純化之結合物的溶液在25 2 nm及2 80 nm下之吸光度之比 率(此在步驟(ii)之後)。特定而言,該DAR可使用分別在 280 nm及252 nm下所量測之抗體的消光係數以分光光度法 來測疋.Sa28〇=224,000 Μ 丨 cm 1 及 εΑ252=82,880 M-1 cm·1 ;假 定抗體平均分子量為160,000,且對於類美登素而言, ε〇28〇=5,180 M-km·、] sD252=26,159 M-icnT1。計算方法來源 於 Antony S. Dimitrov(編),LLC,2009,Therapeutic Antibodies and Protocols > 第 525卷,445,Springer Science 150937.doc •25· 201117814 且更詳細描述如下: 經由SEC分析單體峰(可計算「DAR(SEC)」參數)或者使 用典型分光光度計設備(可計算「dar(uv)」參數)量測結 合物在252 nm(A252)及280 nm(A28Q)下之吸光度。吸光度可 表示如下: 八252 = X£D252Xc;A χεΑ252) Α280 = (CD X£D28〇)+(Ca XSA28〇) 其中: • CD及CA分別為類美登素及抗體之溶液之濃度, • £[>252及6〇28()分別為類美登素在252 nm及280 nm下之莫 耳消光係數, • εΑ252及εΑ28〇分別為抗體在252 nm及280 nm下之莫耳消 光係數。 解析該等具有兩個未知數之兩個方程式產生下列方程 式: CD = [(εΑ28〇χ Α252)- (εΑ252 X Α280)]/[(εϋ252 χ εΑ280)- (εΑ252 χ ε〇280)]
Ca - [Α28〇 - (CD χ εϋ28())]/εΑ28。 隨後由藥物濃度與抗體濃度之比率計算平均DAR : DAR=cd/ca。 使用UV分光光度計量測之平均dAR(DAR(UV))更特定言 之超過4,更特定言之在4與1〇之間,甚至更特定言之在4 與7之間。 s玄結合物以及式(I)化合物可用作抗癌劑。有利的是,該 抗體可使藥劑選擇性靶向腫瘤細胞,從而使類美登素乾向 該等細胞之鄰近處或直接靶向該等細胞内(參見<<Antib〇dy_ I50937.doc -26- 201117814 drug conjugates for cancer therapy» Carter P.J.等人, Cancer J. 2008, 14, 154-169 ; «Targeted cancer therapy: conferring specificity to cytotoxic drugs» Chari R., Ace. C/zew. Λβί. 2008,岑7,98-107)。可治療實體或液體腫瘤。 結合物可調配為緩衝水溶液形式,濃度較佳介於1 mg/ml與1 0 mg/ml之間。該溶液可原樣投與或其可經稀釋 而形成用於灌注之溶液。 [實例1 方法A:高壓液相層析-質譜分析(LCMS) 已在Waters UPLC-SQD系統上,在正離子及/或負離子電 喷模式(ES+/-)中獲得質譜。層析條件如下:管柱: ACQUITY BEH C18,1.7 μηι-2·1χ30 mm ;溶劑:A : Η2Ο(0· 1% 曱酸)B : CH3CN(0· 1% 甲酸);管柱溫度:45°C ; 流速·· 0.6 mL/min ;梯度(2分鐘):1分鐘:5°/。至50%之B ; 1.3分鐘:100%之B ; 1.45分鐘:100%之B ; 1.75分鐘:5% 之B。 方法B:高壓液相層析·質譜分析(LCMS) 已在Waters ZQ系統上,在正離子及/或負離子電喷模式 (ES+/-)中獲得質譜。層析條件如下:管柱:xBridge cig 2.5 μηι-3χ50 mm ;溶劑:a : h2〇(0.1% 甲酸)B : Ch3CN (o.i%甲酸);管柱溫度:70。〇;流速:〇·9 mL/min ;梯度 (7分鐘)· 5.3分鐘:5%至1〇〇%之b ; 5.5分鐘:1〇〇%之b ; 6.3分鐘:5%之丑。 方法C :質譜分析(MS) 150937.doc •27- 201117814 已在Waters UPLC-SQD系統上,在正離子及/或負離子電 噴模式(ES+/-)中獲得質譜。層析條件如下··管柱··
ACQUITY BEH C18 1.7 μιη-2·1χ50 mm ;溶劑:a : H2O (0.1%甲酸)B : CH3CN(0.1%甲酸);管柱溫度:5〇。匸;流 速:1 mL/min ;梯度(2分鐘):0.8分鐘:5%至50%之B ; 1.2 分鐘:100%之 B ; 1.85 分鐘:1〇〇%之 B ; 195 分鐘:5% 之B。 方法D:高壓液相層析-質譜分析(LcmS) 已在Waters UPLC-SQD系統上,在正離子及/或負離子電 喷模式(ES+/-)中獲得質譜。層析條件如下:管柱: ACQUITY BEH C18 1_7 μηι-2·1χ50 mm ;溶劑:A : H20 (0.1% 甲酸)B : CH3CN(0.1〇/o 曱酸);管柱溫度:5(rc ;流 速.1 mL/min ’梯度(2分鐘):0.8分鐘:5%至50%之B ; 1.2分鐘.100%之B ; 1.85分鐘:1〇〇%之b ; 1.95分鐘:50/〇 之B。 方法G:結合物之脫糖基化及高解析度質譜分析(HRMS) 脫糖基化為一種藉助於醣苷酶之酶促消化技術。脫糖基 化由 500 μΐ結合物 +1〇〇 μι Tris緩衝液(50 mM HC1)+10 μΐ寡 醣酶F酶(100個單位冷凍乾燥酶/1〇〇…水)構成。使介質渦 旋且在37 C下維持一夜。隨後準備脫糖基化樣品以用 HRMS分析。在Waters Q_Tof_2系統上,在電喷正離子模式 中(ES + )獲得質譜。層析條件如下:管柱:4 μηι BioSuite 250 URH SEC 4.6x300 mm(Waters);溶劑:A :甲酸敍 25 mM+l0/〇甲酸’ B . CH3CN ;管柱溫度:3〇。匸;流速:〇_4 150937.doc 〇〇 201117814 mL/min ;等度溶離·· 70°/。A+30°/。B(15 分鐘)。 方法Η:分析型尺寸排阻層析(SEC) > 管柱:TSKgel G3000 SWXL 5 μηι管柱,7.8 mmx30 cm, TOSOH BIOSCIENCE, LLC Part #08541 > 移動相:KC1(0.2 M)、KH2P〇4(0.052 M)、K2HP04(0.107 M)、iPrOH(20體積 %) >分析條件:在0.5 mL/min下等度溶離30分鐘
Elite HPLC 系統(Merck)上使用 L2455 DAD分 光光度計積測器進行分析。 緩衝液内含物 >緩衝液A(pH 6.5) : NaCl(50 mM)、磷酸鉀緩衝液(50 mM) ' EDTA(2 mM) >緩衝液HGS(pH 5.5):組胺酸(10 mM)、甘胺酸(130 mM)、蔗糖 5%(w/v)、HC1(8 mM) 所用縮寫
AcOEt :乙酸乙酯;ALK : ((:!-(:12)伸烷基,尤其(CVC6) 伸烷基;DAR :藥物抗體比率;DBU : 1,8-二氮雜雙環 [5.4.0]十一碳-7-烯;DCC : Ν,Ν’-二環己基碳化二亞胺; DCM :二氣曱烷;DEAD :偶氮二羧酸二乙酯;DIC : Ν,Ν1-二異丙基碳化二醯亞胺;DIPEA : Ν,Ν-二異丙基乙基 胺;DMA :二曱基乙醯胺;DMAP : 4-二甲基胺基吡啶; DME :二曱氧基乙烷;DMF :二曱基甲醯胺;DMSO :二 曱亞砜;ε :莫耳消光係數;EEDQ : 2-乙氧基-1-乙氧羰 基-1,2-二氫喹啉;EDCI : Ν-(3-二曱基胺基丙基)-Ν’-乙基 150937.doc •29· 201117814 碳化二亞胺;EDTA :伸乙基-二胺-四乙酸;Fmoc :苐基 曱氧基羰基;Hal :鹵原子;HOBt : 1-羥基苯并三唑; HEPES : 4-(2-羥乙基)-1-哌嗪-乙磺酸;HRMS :高解析度 質譜分析;NHS : N-羥基丁二醯亞胺;iPrOH :異丙醇; NMP : N-甲基吡咯啶酮;Rf :滯留因子;RP :減壓; RT :室溫;SEC :尺寸排阻層析;TBDMS :第三丁基二曱 基矽烷基;TEA :三乙胺;TFA :三氟乙酸;TFAA :三氟 乙酸酐;TFF :切向流過濾;THF :四氫呋喃;TIPS :三 異丙基矽烷基;TLC :薄層層析;tR :滯留時間。 實例中所用之抗體 使用兩種抗體製備結合物: > hu2Hll :(在 WO 2008010101 中亦稱為 hu53 2H11): 該抗體係由按照布達佩斯條約(Budapest Treaty)寄存於 美國菌種保存中心(American Type Culture Collection), 寄存編號為PTA-7662之融合瘤產生且描述於PCT申請案 WO 2008/010101 中; > hu2HllR35R74 :該人類化抗體結合於EphA2受體且 藉由 hu53 2H11(由 SEQ ID NO: 18 之重鏈及 SEQ NO: 16 之輕鏈組成)之定點突變誘發來獲得。 實例1 150937.doc •30· 201117814 3-[2<2-[2<2-漢-乙醢胺基 >乙氧基]-乙氡基]-乙氡基)
1.1. 製備結合物 hu2HllR35R74-PEG4_NHAc-DM4 在室溫磁力攪拌下,添加9 mL hu2HllR35R74(14.36 mg/ml於緩衝液A中),隨後添加16.85 mL緩衝液A、3.23 mL 1 M HEPES、1.59 mL DMA,接著添加 1.64 mL 10 mM L-DM4-AcNH-PEG4-CONHS活化酯之DMA溶液。在室溫下 1小時30分鐘後,再添加 0.085 mL 10 mM L-DM4-AcNH-PEG4-CONHS活化酯之DMA溶液。在室溫下1小時45分鐘 後,粗結合介質以60 mL HGS緩衝液稀釋且在Pellicon 3匣 上藉由TFF純化。使樣品相對於約10個樣品體積之HGS緩 衝液透濾且隨後收集。以額外10 mL HGS緩衝液洗滌TFF 槽及管線。混合兩種溶液,經由0.22 μπι PVDF過濾滅菌, 在Amicon 15上濃縮且經由0.22 μηι PVDF過渡滅菌。由此 獲得 17 mL hu2HllR35R74-PEG4-NHAc-DM4 結合物 (c=5.76 mg/ml)。隨後分析結合物之最終載藥率及單體純 度。SEC 分析(方法 H) : DAR(SEC) = 5.4 ; RT=16.757 分鐘; 單體純度=99.5% ; HRMS資料:參見圖1。 1.2. 製備 L-DM4-AcNH-PEG4-CONHS活化酯 150937.doc •31 - 201117814 在室溫磁力攪拌下,將154.3 mg L-DM4(根據w〇 04/103272製備-參見化合物4b)引入玻璃瓶中。隨後添加90 mg 3-[2-(2-{2-[2-(2-溴-乙醯胺基)-乙氧基]-乙氧基卜乙氧 基)-乙氧基]-丙酸2,5-二側氧基-吡咯啶-1-基酯於0.94 mL DMA中之溶液’隨後添加36 μΐ DIEA。在室溫下23小時 後’反應介質以5 mL AcOEt稀釋且以7 mL水洗蘇。以5 mL AcOEt萃取水相。合併之有機相經MgS04乾燥,在減壓 下濃縮至乾。獲得228 mg淺黃色黏性油狀物,該產物以最 低量DMA稀釋且在30 g C18接枝矽膠上藉由急驟層析純化 (溶離梯度為水:乙腈95:5至5:95(體積比))。在減壓下濃縮 溶離份2及3後,獲得無色黏性油狀物,該產物以最低量 DMA稀釋且在30 g C18接枝矽膠上藉由急驟層析純化(溶 離梯度為水:乙腈95:5至5:95(體積比))。在減壓下濃縮溶離 份33至35後’獲得41 mg呈白色酥皮狀產物形式之l_dm4-
AcNH-PEG4-CONHS 活化酯。質譜(b) : rt=4.06 分鐘; [M+H-H20] + : m/z 1164; [M+H] + : m/z 1182; [M-H+HC02H]-: m/z 1226 ; 'H NMR (500 MHz,δ (ppm),氣仿·d): 0.80 (s, 3H); 1.21 (s,3H); 1.22 (s,3H); 1.25 (m,1H); 1.29 (d, /=6,7 Hz, 6H); 1.46 (m, 1H); 1.57 (d, J=13.4 Hz, 1H); 1.64 (s,3 H ); 1.76至 183 (m,1H); 1.88至 1.96 (m,1H); 2.18 (dd, /=2.5及 14.3 Hz,1H); 2.36 (m,1H); 2.53 (m, 1H); 2.61 (dd, «/=12.5及14.3取111);2.82至2.92(111,101^);2.98(€1, /=16.7 Hz, 1H); 3.03 (d, J=9.6 Hz, 1H); 3.15 (d, 7=12.9 Hz, 1H); 3.22 (s,3H); 3.32 (寬單峰,1H); 3 36 (s,3H); 3 42 150937.doc -32· 201117814 (m, 2H); 3.50 (d, J=9.1 Hz, 1H); 3.53 (t, J=5.2 Hz, 2H); 3.58至 3.67 (m, 13H); 3.84 (t,J=6.4 Hz, 2H); 3.99 (s,3H); 4.27 (m,lH); 4.77 (dd,《7=2.9及 11.9 Hz,1H); 5.42 (q,/=6.7 Hz,1H); 5.66 (dd, J=9.1 及 15.4 Hz,1 H ); 6.23 (s,lH); 6·43 (dd,J=11.3 及 15.4 Hz,1H); 6.64 (d,*7=1.1 Hz,1H); 6.74 (d, J=11.3 Hz, 1H); 6.85 (d, 7=1.1 Hz, 1H); 7.08 (t, 7=5.2 Hz, 1H)。 1.3.製備3-[2-(2_{2-[2-(2-漠-乙醯胺基)-乙氧基】-乙氧基}-乙氧基)-乙氧基】-丙酸2,5-二側氧基-吡咯啶-1-基酯 在室溫磁力攪拌下,將671.4 1^3-(2-{2-[2-(2-胺基-乙 氧基)-乙氧基]-乙氧基}_乙氧基)-丙酸(CA(PEG)4, Pierce) 引入玻璃瓶中。隨後添加597.4 mg溴-乙酸2,5-二側氧基-吡 咯啶-1-基酯於14 mLDCM中之溶液。在室溫下15分鐘後, 添加0.396 mL DIC。1小時30分鐘後,在燒結玻璃上過濾 粗反應介質’且在1〇〇 g CN接枝矽膠上藉由急驟層析純化 濾液(溶離梯度為正庚烷/iPrOH/AcOEt,其中iPrOH部分遞 增)。在減壓下濃縮溶離份30至45後,獲得呈無色油狀之 3-2-(2-{2-[2-(2-溴-乙醯胺基)-乙氧基]-乙氧基卜乙氧基)_乙 氧基]-丙酸2,5-二側氧基-吼咯啶-1-基酯。質譜(A): RT=0_74 min; [M+H]+: m/z 483/485(由於 Br之兩個同位素 而出現兩個峰);[M_H+HC02H]-: m/z 527/529(由於Br之兩 個同位素而出現兩個峰)。 可根據公開方案製備溴-乙酸2,5-二側氧基-吼α各啶-1 _基 酯(Biochemistry 1974, 481)。 150937.doc -33- 201117814 實例2
2_1·製備結合物 hu2HllR35R74-PEG4-NMeAc_DM4 在室溫磁力攪拌下,添加4 mL hu2HllR35R74(14.36 mg/ml於缓衝液A中),隨後添加7.5 mL緩衝液A、1.45 1 Μ mL HEPES、1.05 mL DMA,接著添加 0.39 mL 10 mM L-M4-AcNMe-PEG4_CONHS活化酯之DMA溶液。在室溫下 30分鐘後,再添加0.19 mL 10 mM L-DM4-AcNMe-PEG4-CONHS活化酯之DMA溶液。在室溫下3小時後,粗結合介 質以65 mL HGS緩衝液稀釋且在Pellicon 3匣上藉由TFF純 化。使樣品相對於約個樣品體積之HGS緩衝液透濾且隨 後收集。以額外1 〇 mL HGS緩衝液洗蘇TFF槽及管線。混 合兩種溶液,在Ami con 15上濃縮且經由0.22 μιη PVDF過 濾、滅菌。由此獲得8-5 mL hu2HllR35R74-PEG4-NMeAc-DM4結合物(c=6.01 mg/ml)。隨後分析結合物之最終載藥 率及單體純度。SEC 分析(H) : DAR(SEC)=5.5 ; RT=16.7 分 鐘;單體純度=99.4。/。; HRMS資料:參見圖2。 2.2.製備 L-DM4-AcNMe_PEG4-CONHS活化醋 在室溫磁力攪拌下,將133.4 mg L_DM4引入玻璃瓶中。 150937.doc • 34· 201117814 隨後添加85 mg 3-{2-[2-(2-{2-[(2-溴-乙醯基)-甲基-胺基]-乙氧基}-乙氧基)-乙氧基]-乙氧基卜丙酸2,5_二側氧基_。比咯 咬-卜基酯於0.2 mL DMA中之溶液,隨後添加32.9 μΐ DIEA。在室溫下!小時後,在3〇 g CIS接枝矽膠上藉由急 驟層析純化反應介質(溶離梯度為水:乙腈95:5至5:95(體積 比))。在減壓下濃縮含有所需產物之溶離份後,獲得71.3 mg呈無色玻璃狀產物形式之L_DM4-AcNMe-PEG4-CONHS 活化酯。質譜(D) : RT=0_98分鐘;[M+H-H20] + : m/z 1178 (主信號);[M+Na] + : m/z 1218; [M-H+HC02H]-: m/z 1240 ; 4 NMR (500 MHz,δ (ppm),氣仿-d): 0.81 (s,3H); 1.20 至 1.33 (m, 13H); 1.42至 1.52 (m, 1H); 1.56至 1.61 (m,1H); 1.65 (s, 3H); 1.73 至 1.83 (m,1H); 1.96至 2.04 (m,1H); 2.19 (£1〇1,>/=2.8及14.4 1^,111);2.29至2.41(111,111);2.55至2.66 (m,2H); 2·83 至 2.93 (m,12H); 3_04 (d,/=9.8 Hz,1H); 3.12 ((1,1/=12.7 1^,11"1);3.18至3.25(〇,511);3.37(5,311);3.47 至3·54(m,3H);3.57至3·68(m,15H);3·85(t,>/=6·6Hz, 2H); 3.99 (s,3H); 4.29 (m,1H); 4.79 (dd, *7=2.8及 12.2 Hz, 1H); 5.41 (q, /=6.7 Hz,1H); 5.68 (dd, J=9.3及 15.2 Hz, 1H); 6.23 (s,1H); 6.43 (dd,/=11.0及 15.2 Hz,1H); 6.66 (s, 1H); 6.74 (d,/=11.0 Hz,lH);6.83(s,1H)。 2.3.製備3-{2-[2_(2-{2-[(2_溴·乙醯基)-甲基-胺基】-乙氧 基)-氧基)-乙氧基]-乙氡基卜丙酸2,5-二側氧基-«^比咯啶-l-基酯 150937.doc -35- 201117814
在室溫磁力撥拌下,將115.1 mg 3-(2-{2-[2-(2-曱基胺 基-乙氧基)-乙氧基]-乙氧基乙氧基)·丙酸、1 5 mL DCM、97.3 mg溴-乙酸2,5-二侧氧基-吡咯啶_ι_基酯依次引 入圓底燒瓶中。2小時後,添加72 μΐ DIEA,且在室溫下 再經1小時後’添加70·2 μΐ DIC。粗反應介質在室溫下保 持4小時’在_20°C下保持16小時,且隨後在30 g矽膠上藉 由急驟層析純化(溶離梯度為DCM:甲醇〇:1〇〇至3:97(體積 比))^在減壓下濃縮含有所需產物之溶離份後,獲得8 5.8 mg呈白色固體形式之3·{2-[2-(2-{2-[(2-溴-乙醯基)-曱基_ 胺基]-乙氧基}-乙氧基)-乙氧基]-乙氧基卜丙酸2,5_二側氧 基比咯啶-1-基酯。質譜(A) : RT=0.84分鐘;[M+H] + : m/z 497/499 〇 2·4.製備3-(2-{2-[2-(2-甲基胺基-乙氧基)-乙氧基]-乙氧基}_ 乙氧基)-丙酸
在氬氣之惰性氛圍下,在磁力攪拌下,將120.1 mg 3-[2-(2-{2-[2-(2,2,2-三氟-乙醯胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-丙酸曱酯、1 mL無水THF及59.8 μΐ CH3I依次引入 圓底燒瓶中。使用約o°c之冰/水浴冷卻反應介質,且小份 緩慢添加16.1 mg NaH(50%純度於油中)。在〇°C下15分鐘 150937.doc •36· 201117814 及在室溫下1小時後,粗反應介質在減壓下濃縮至乾且 以0.5 mL THF及0.8 mL水稀釋。在室溫下,隨後添加3〇 6 mg LiOH至反應介質中。粗反應介質在室溫下保持2小 時’在-20°C下保持16小時’且隨後在30 g ci8接枝碎膠上 藉由急驟層析純化(溶離梯度為水:乙腈95:5至5:95(體積 比))。在減壓下濃縮含有所需產物之溶離份後,獲得115 3 mg呈黃色油狀之3-(2-{2-[2-(2-曱基胺基-乙氧基)_乙氧基]_ 乙氧基}-乙氧基)-丙酸。 2.5.製備3-[2-(2-{2 - [2-(2,2,2-三氟-乙酿胺基)_乙氧基卜乙氧 基}_乙氧基)-乙氧基】-丙酸甲醋
在氬氣之惰性氛圍下,在磁力攪拌下,將230 mg 3-(2-{2-[2-(2-胺基-乙氧基)·乙氧基]-乙氧基乙氧基丙酸 (CA(PEG)4, Pierce)、2 mL DCM及1 mL甲醇依次引入圓底 燒瓶中。在室溫下,緩慢添加1 mL三曱基石夕烧基重氮甲烧 (於己院中之2 Μ溶液)至反應介質中。在室溫下2小時後, 藉由添加乙酸中和過量三甲基矽烷基重氮甲烷。粗物質隨 後在減壓下蒸乾。所得殘餘物以2 mL DCM稀釋,使用水-冰浴冷卻至(TC,隨後依次添加363 μι TEa及300 μΐ TFAA。在室溫下2小時3〇分鐘及在-⑼·^下19小時後,依 次添加363 μΐ TEA及300 μΐ TFAA。在室溫下4小時30分鐘 後’將粗物質儲備於_2〇°C且隨後在30 g C18接枝矽膠上藉 150937.doc -37- 201117814 由急驟層析純化(溶離梯度為水:乙腈9 5:5至5:9 5 (體積 比))。在減壓下濃縮含有所需產物之溶離份後,獲得123 mg呈淺黃色油狀之3-[2-(2-{2-[2-(2,2,2-三氟·乙醯胺基)-乙 氧基]-乙氧基}-乙氧基)-乙氧基]-丙酸甲酯。質譜(A): RT=0.90分鐘;[M+H] + : m/z 376; [M-H]-: m/z 374。 實例3
3.1.製備結合物 hu2HllR35R74-PEG8-NHAc-DM4 在室溫磁力攪拌下,添加4 mL hu2HllR35R74(14.36 mg/ml於緩衝液A中),隨後添加7.5 mL緩衝液A、1.45 mL 1 M HEPES、1.05 mL DMA,接著添加 0,405 mL 10 mM L-DM4-AcNMe-PEG8-CONHS活化酯之DMA溶液。在室溫下 30分鐘後,再添加0.1 mL 10 mM L-DM4-AcNMe-PEG8-CONHS活化酯之DMA溶液。在室溫下1小時45分鐘後,粗 結合介質以60 mL HGS緩衝液稀釋且在Pellicon 3匣上藉由 TFF純化。使樣品相對於約10個樣品體積之HGS緩衝液進 行透濾且隨後收集。以額外10 mL HGS緩衝液洗滌TFF槽 及管線。混合兩種溶液,在Amicon 1 5上濃縮且經由0.22 150937.doc 38 - 201117814 μηι PVDF過濾滅菌。由此獲得 7 mL lu^HllRJSR^-PEGS-
AcNMe-DlVH結合物 (c=6.95 mg/ml)。 隨後分析結合物之最 終載藥率及單體純度。SEC分析(H) : DAR(SEC)=5.0 ; RT=16.5 93分鐘;單體純度=99.5% ; HRMS資料:參見圖 3 ° 3.2·製備 L-DM4-AcNH-PEG8-CONHS活化酉旨 在室溫磁力攪拌下,將65 mg 3-丨2-[2-(2-丨2-[2-(2_丨2_[;2_ (3-溴-丙醯胺基)-乙氧基]-乙氧基}•乙氧基)_乙氧基]•乙氧 基}-乙氧基)-乙氧基]-乙氧基}-丙酸2,5-二側氧基· 比洛 啶-1-基酯引入玻璃瓶中,隨後引入67.7 mg L-DM4於0.85 mL DMA及16.5 μΐ DIEA中之溶液。在室溫下48小時後, 在1 0 g石夕膠上藉由急驟層析純化反應介質(溶離梯度為 DCM:MeOH 100:0至90:10(體積比))。在減壓下濃縮溶離份 18至26後,獲得17 mg呈無色玻璃狀之l-DM4-AcNH-PEG8-CONHS活化酯。質譜(b) : RT=4.08分鐘;[M+H-H20] + : m/z 1340 (主信號);[M+Na] + : m/z 1380; [M-H+HC02H]-: m/z 1402 ; *11 NMR (400 MHz, δ (ppm) » 氣仿-d): 0.81 (s,3H); 1.22 (s,3H); 1.23 (s,3H); 1.26 (m, 1H); 1.30 (d,J=6.8 Hz,6H); 1.41 至 1.52 (m, 1H); 1.65 (s, 3H); 1_80 (m,1H); 1.89至 1.99 (m,1H); 2.19 (m,1H); 2.37 (m,1H); 2.47至 2·67 (m,2H); 2.81 至 2.93 (m,10H); 2.99 (d, J=16.6 Hz,1H); 3.04 (d,/=9.8 Hz,1H); 3.16 (寬雙峰, J=13.7 Hz,1H); 3.23 (s,3H); 3.32 (寬單峰,1H); 3.37 (s, 3H); 3.44 (m, 2H); 3.51 (d, J=9.1 Hz, 1H); 3.54 (t, 7=5.4 150937.doc -39· 201117814 1^,211);3.59至3.73(111,2姐);3.86(1,/=6_6 112,211);4.00 (5,311);4.22至4.33(111,1印;4.78(£1(1,</=2.9及12.2 112, lH);5.43(q,J=6,8Hz,lH);5.67(dd,J=9.0&15.2Hz, 1H); 6.23 (s,1H); 6.44 (dd,J=11.2及 15·2 Hz, 1H); 6.65 (d, J=1.5 Hz, 1H); 6.75 (d, J=n.2 Hz, 1H); 6.86 (d, J=1.5 Hz, 111);7.02至7.13(111,11^)。
3.3.製備 3-{2·[2-(2-{2-[2-(2-{2-[2-(3-溴-丙醯胺基)-乙氧 基]-乙氧基卜乙氧基)-乙氧基]-乙氧基}-乙氧基)-乙氧基卜乙 氧基}-丙酸2,5-二側氧基_吡咯啶_;!_基酯 BrJL
在室溫磁力攪拌下,將100 mg 3-(2-{2-[2-(2-胺基-乙氧 基)-乙氧基]-乙氧基}-乙氧基)·丙酸(CA(PEG)4,Pierce)、2 1^〇0]^及53.5 11^溴_乙酸2,5-二側氧基-吡咯啶_1_基酯依 次引入玻璃瓶中。在室溫下1小時後,添加35.1 μΐ DIC。1 小時後’粗反應介質在燒結玻璃上過濾,在減壓下濃縮至 乾,以10 mL AcOEt稀釋,在燒結玻璃上過濾且在減壓下 濃縮至乾。獲得76.5 mg呈無色油狀之3-{2-[2-(2-丨 {2-[2-(3-溴-丙醯胺基)-乙氧基乙氧基}_乙氧基)_乙氧基]_ 乙氧基}•乙氧基)-乙氧基]-乙氧基}-丙酸2,5 -二側氧基_„比嘻 啶-1-基酯。質譜(A) : RT=0.80 分鐘;[M+H] + : m/z 659/661; [M-H+HC02H]-: m/z 703/705。 實例4 150937.doc -40- 201117814
4.1. 製備結合物 hu2HllR35R74-PEG4-烯丙基-DM4 在室溫磁力授拌下’添加4 mL hu2Hl 1R35R74(14·36 mg/ml於緩衝液Α中)’隨後添加7.5 mL緩衝液A、1.45 mL 1 M HEPES、1.14 mL DMA,接著添加 0.3 mL 10 mM L-DM4-烯丙基-PEG4-CONHS活化酯之DMA溶液。在室溫 下30分鐘後,再添加0.125 mL 10 mM L-DM4-烯丙 基-PEG4-CONHS活化酯之DMA溶液。在室溫下1小時25分 鐘後,粗結合介質以65 mL HGS緩衝液稀釋且在Pellicon 3 匣上藉由TFF純化。樣品相對於約10個樣品體積之HGS緩 衝液透滤·且隨後收集。以額外1 0 mL HGS緩衝液洗務TFF 槽及管線。混合兩種溶液,在Amicon 1 5上濃縮且經由 0.22 μιη PVDF過濾滅菌。獲得8.0 mL hu2Hll R35R74-PEG4-稀丙基-DM4結合物(c=5·22 mg/ml)。隨後分析結合 物之最終載藥率及單體純度。SEC分析(H) : DAR(SEC) = 5.3 ; RT=16.767分鐘;單體純度=99.4% ; HRMS資料:參 見圖4。 4.2. 製備L-DM4-烯丙基-PEG4-CONHS活化醋 在室溫磁力攪拌下,將70 mg L-DM4、45 mg 3-(2-{2- 150937.doc • 41 · 201117814 [2-(4-溴-丁- 2-烯氧基)-乙氧基]-乙氧基}-乙氧基)-丙酸2,5-一側氧基-0比B各0定_ 1 -基酉曰(7臭-稀丙基-PEG4_CONHS)、0.5 mL DM A及23.5 μΐ DIE A依次引入玻璃瓶中。在室溫下2小 時及在-20°C下17小時後,添加50 μΐ DIEA。在室溫下24小 時後,在30 g C1 8接枝矽膠上藉由急驟層析純化反應介質 (溶離梯度為水:乙腈95:5至5:95(體積比))^在減壓下濃縮 含有預期產物之溶離份後,獲得47.1 mg呈白色固體形式 之L-DM4-烯丙基-PEG4-CONHS活化酯。質譜(D): RT=1.06分鐘;[M+Na] + : m/z 1173 ; 1H NMR (500 ΜΗζ,δ (PPm),氣仿-d): 0.81 (s,3H); 1.18至 1.39 (m,13H); 1.42至 1.52 (m,1H); 1.58 (d,/=13.4 Hz,1H); 1.65 (s,3H); 1.73 至 1.82 (m,1H); 1.86至 1.95 (m,1H); 2.19 (d,·7=14·3 Hz,1H); 2.40(111,11"1);2.51至2.65(111,2印;2.82至2.95(111,911);2.98 至 3.07 (m,2H); 3.12 (d,《7=12.6 Hz,1H); 3.18至 3.27 (m, 1H); 3.23 (s, 3H);3.36 (s, 3H); 3.51 (d, /=9.1 Hz, 1H); 3.54 至3.82(111,13:9);3.86(1</=6.4^12,2印;3.91至3.95(111, 2H); 3.99 (s, 3H); 4.28 (t, J=11.0 Hz, 1H); 4.78 (dd5 J=2.6 及 11.9 Hz,1H); 5.44 (q,*7=6.7 Hz,1H); 5.49至 5.63 (m, 2H); 5.68 (dd,7=9.1 及 15.0 Hz,1H); 6.24 (s,1H); 6.43 (dd, 及 15.0 Hz,1H); 6.66 (s,1H); 6.77 (d,《7=11.1 Hz,lH); 6.83 (s, 1H)。 4.3.裝備3-(2-{2-[2-(4-溴-丁 -2-烯氧基)乙氧基]-乙氧基}- 乙氧基)-丙酸2,S-二側氧基-吡咯啶-1-基酯 150937.doc •42· 201117814
在室溫下,將200 mg 3-(2-{2-[2-(4-溴-丁 _2_烯氧基)_乙 氧基]-乙氧基}-乙氧基)-丙酸、4 mL DCM及232.3 mg負載 型DCC(2當量)依次引入玻璃瓶中。在室溫下1小時後,添 加64.8 mg NHS。在室溫下5小時後,在燒結玻璃上過遽粗 物質,以DCM洗蘇固體,且合併之壚液在減壓下濃縮至 乾。在15 g石夕膠上藉由急驟層析純化(溶離梯度為 MeOH:DCM 0:100至10:90(體積比)),且在減壓下濃縮含有 預期產物之溶離份,獲得46 mg呈淺黃色油狀之3·(2_{2_[2_ (4-漠-丁-2-烯氧基)-乙氧基]-乙氧基乙氧基)_丙酸2,5_二 側氧基-°比π各°定-1 -基醋( >臭-稀丙基-PEG4-CONHS)。質譜 (A) . RT=1.02分鐘;[M+H] + : m/z 454/456; [M+Na] + : m/z 476/478; [M-H+HC02H]-: m/z 498/500。 4.4.製備3-(2-{2-[2·(4-溴-丁-2-烯氧基)-乙氧基]_乙氧基}· 乙氧基)-丙酸
〜^〇H 在室溫下,丨g 3_(2_{2_[2-(4_溴_ 丁 _2_烯氧基)_乙氧基]_ 乙氧基卜乙氧基)-丙酸第三丁酯(市售)、6 mL TFA及3 mL DCM之溶液攪拌3小時,且隨後在減壓下濃縮至乾。油狀 殘餘物以曱笨稀釋且在減壓下濃縮至乾,產生853 mg呈標 色油狀之3-(2-{2_[2_(4_溴-丁 _2_烯氧基)_乙氧基]_乙氧基}_ 150937.doc -43· 201117814 乙氧基)-丙酸。 實例5 5.1.製備結合物 hu2Hll-PEG4-NHAc-DM4 結合物hu2Hll-PEG4-NHAc-DM4可以類似於實例1的方 式來製備:在室溫攪拌下,添加1 mL hu2Hll(8.52 mg/ml 於緩衝液A中),隨後添加0.7 mL緩衝液A、0.213 mL 1 Μ HEPES、0.7 mL DMA,隨後添加 L-DM4-AcNH-PEG4-CONHS活化酯經0.128 mL DMA稀釋之10 mM DMA溶液 0.085 mL。在室溫下2小時後,粗介質在Amicon 4上以 7000 G濃縮,在Nap-10管柱上與HGS緩衝液緩衝性交換, 且最後在5 mL Zeba管柱上純化。由此獲得1.15 mL hu2Hll-PEG4-NHAc-DM4結合物(c=3.78 mg/ml)。分析結 合物之最終載藥率及單體純度。SEC分析(方法H) : DAR (UV)=6.6 ; DAR(SEC)=5.6 ; RT=15.387 分鐘;單體純度 =99.7% ; HRMS資料:參見圖5。 同樣,製備包括hu2Hll且描述於實例6-8中之其他結合 物(參見表Ila)。 實例9 :抑制MDA-MB231腫瘤細胞(來自ECAAC參考# 92020424)生長 用胰蛋白酶處理指數生長期細胞且使其再懸浮於適當培 養基中(DMEM/F12 Gibco #21331 ; 10% SVF Gibco # 10500-056 ; 2 nM麩醯胺酸Gibco # 25030)。將含有血清之 完全培養基中的細胞懸浮液以5000個細胞/孔之密度分佈 於 96 孔 Cytostar 培養板(GE Healthcare Europe,#RPNQ0163) 150937.doc •44- 201117814 中。塗佈4小時後,將結合物之連續稀釋液以1〇_7與1〇_12 Μ 之間範圍之濃度一式三份添加至孔中。在37°C /5% C02 下,在結合物存在下,培養細胞3天。第4天,添加1 0 μΐ 14C-胸苷溶液(0_1 μ(:ί/孔(Perkin Elmer # NEC56825000))至 各孔申。在實驗開始後96小時用microbeta放射性計數器 (Perkin Elmer)量測14C-胸苷之吸收量。自測試孔讀數減去 無細胞試劑空白讀數且以存活分率形式對該等數據作圖, 存活分率係藉由結合物處理細胞之讀數除以媒劑處理細胞 之對照孔平均讀數所獲得。在該等實驗中,在實驗開始時 將裸抗體(hu2Hll或hu2HllR35R74)以1 μΜ之濃度添加至 各孔中且如先前所述量測增殖之抑制。 表Ila及lib中所報導之結果表明,根據在裸hU2Hll或 hu2HllR35R74存在下進行之競爭研究,該等結合物對 MDA-MB23 1細胞顯示強活體外增殖抑制特性且經由結合 於抗原起作用。 表Ila 結合物 DAR (UV) ICs〇 [pM] 單獨結合物 +裸1 hu2Hll hu2H 11 -PEG4-NHAc-DM4 (參見實例1.1) 6.6 895 75223 hu2H 11 -PEG8-NHAc-DM4 5.6 1007 20726 hu2Hl 1-PEG4-烯丙基-DM4 5.1 437 25907 hu2H 11 -PEG4-NMeAc-DM4 4.1 1400 85379 比率 " 一 84 ~· 一 21 59 150937.doc • 45- 61 201117814 表lib IC5〇 [pM] 結合物 DAR (UV) 單獨結合物 +裸 hu2HllR35R74 比率 hu2Hl 1R35R74-PEG4-NHAC-DM4 5.9 147 29731 202 hu2H 11R35R74-PEG8-NHAC-DM4 4.9 400 24955 62 hu2Hl 1R35R74-PEG4-烯丙基-DM4 5.3 161 7820 49 hu2Hll R35R74-PEG4-NMeAc-DM4 5.4 217 36400 168 實例 10 : L-DM4-AcNH-PEG4-COOMe
10.1.製備無藥物之L-DM4-AcNH-PEG4-COOMe
在室溫磁力攪拌下,在Ar之惰性氛圍下,將57.4 mg 3-[2-(2-{2-[2-(2-溴-乙醯胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-丙酸甲酯、80 mg L-DM4於0.44 mL DMA中之溶 -46 * 150937.doc 201117814 液及最後19.6 μΐ DIPEA依次引入玻璃瓶中。在室溫下1 §小 時後,粗物質以10 mL水稀釋,以3x7 ml AcOEt萃取。收 集有機相,經MgSCU乾燥、過濾且在減壓下濃縮至乾。獲 得124 mg無色油狀物,該產物以最低量DMA稀釋且在C18 接枝石夕膠上藉由急驟層析純化(Merck,C18, 5 g, 25-40 μιη, 18 ml/min,溶離梯度為水:乙腈1〇〇:〇至5:95(體積比))。在 減壓下濃縮含有預期化合物之溶離份後,獲得12.8 mg呈 無色膜形式之曱酯L-DM4-AcNH-PEG4-COOMe。質譜 (C) : tR=0.97 分鐘;[M+H] + : m/z 1099; [M-H]-: m/z 1097 ; ]H NMR (500 MHz, δ (ppm),氣仿-d): 0.80 (s, 3H); 1.21 (s,3H); 1.22 (s,3H); 1_24至 1.35 (m,7H); 1.46 (td,J=6.4及 10.2 Hz,1H); 1.57 (d, /=13.4 Hz,1H); 1.64 (s, 3H);1.80(ddd,J=4.9&11.5&14.6Hz,lH);1.92(m,iH); 2.18(04,</=2.3及14.7 1^,111);2.36((1(1(1,>/=4.8及11.4及 16.2 Hz,1H); 2.52 (ddd,/=5.1 及 11.3及 16.3 Hz,1H); 2.59 (t, J=6.4 Hz, 2H); 2.63 (m, 1H); 2.86 (s, 3H); 2.88 (m, lH); 2.98 (d,《7=16.7 Hz, 1H); 3.03 (d,《7=9.6 Hz, 1H); 3.15 (山 /=12.6 Hz, 1H); 3.23 (s, 3H); 3.36 (s, 3H); 3.42 (m, 2H); 3.50 (d, /=9.1 Hz, 1H); 3.54 (m, 2H); 3.63 (m, 13H); (s, 3H); 3.75 (t, J=6.4 Hz, 2H); 3.99 (s, 3H); 4.27 (t, J=ll-3 Hz,1H); 4.78 (dd, J-2.2及 12.1 Hz, 1H); 5.42 (q,《7=6.9 Hz, 1H); 5.66 (dd,/=9.1 及 15,4 Hz, 1H); 6,21 (s,1H); 6,43 (dd, «7=11.0及 15_4 Hz, 1H); 6.64 (s,1H); 6.74 (d,·7=11,〇 Hz, 1H); 6.85 (s, 1H); 7.05 (t,/=5.5 Hz,1H)。 150937.doc -47· 201117814 10.2.製備3-丨2-(2-{2-[2-(2-溴-乙醯胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-丙酸曱酯
分子量=400.27 分子式=C14H26BrN07 在室溫磁力攪拌下,將100 mg 3-(2-{2-[2-(2-胺基-乙氧 基)-乙氧基]-乙氧基}-乙氧基)-丙酸(CA(PEG)4,Pierce)、 89 mg溴-乙酸2,5-二侧氧基-吡咯啶-1-基酯及2 mL DCM依 次引入玻璃瓶中。在室溫下1小時後,添加0.7 mL MeOH 及0.3 8 mL於己烷中之2 Μ三.曱基矽烷基重氮曱烷溶液。在 室溫下1小時後,粗反應混合物在減壓下濃縮至乾,隨後 以最低量DMA稀釋且在C1 8接枝矽膠上藉由急驟層析純化 (Merck,C18,5 g, 25-40 μηι,18 ml/min,溶離梯度為水:乙 腈100:0至5 :95(體積比))。在減壓下濃縮含有預期化合物之 溶離份後,獲得58 mg呈無色油狀之3-[2-(2-{2-[2-(2-溴-乙 醯胺基)-乙氧基]-乙氧基}-乙氧基)-乙氧基]-丙酸曱酯。質 譜(A) : tR=0.75 分鐘;[M+H] + : m/z 400/402。 可按照公開方案(Biochemistry 1974,481)製備溴-乙酸 2,5 -二側氧基-D比洛°定-1 -基醋。 實例 11 : L-DM4-AcNMe-PEG4-COOMe
I50937.doc 48- 201117814 工1.1 製備無藥物之 L-DMtAcNMe-PEGd-COOMe
在室溫磁力攪拌下,將30 mg L-DM4、20.8 mg 3-{2-[2-(2-{2-[(2-溴-乙醯基)_甲基_胺基]_乙氧基}•乙氧基)_乙氧 基]-乙氧基}-丙酸曱酯於0.3 mL DMA中之溶液及7.4 μΐ DIPEΑ依次引入玻璃瓶中。在室溫下18小時後,反應介質 以7 mL AcOEt稀釋且以5 mL水洗滌兩次。有機相以鹽水洗 滌’經MgSCU乾燥’過濾且在減壓下濃縮至乾。獲得39 mg無色玻璃狀物’該產物以最低量dmA/MeOH混合物稀 釋且在C18接枝石夕膠上藉由層析純化(xTerra⑧C18,5 μπι, 50x30 mm,30 ml/min,溶離梯度為水:乙腈95:5至5:95(體 積比))。在減壓下濃縮含有預期化合物之溶離份後,獲得 7.8 mg呈無色固體形式之甲酯L-DM4-AcNMe-PEG4-COOMe。質譜(C) : tR=1.00分鐘;[m+H-H20] + : m/z 1095; [M+Na+] + : m/z 1135; [M-H+HC02H]-: m/z 1157; [M-H]-: m/z 1111 ; 'H NMR (500 MHz, δ (ppm), DMSO-d6): 0.78 (s. 3H); 1.12 (d, ./=6.6Hz, 3H); 1.16 (m,9H); 1.26 (m, 1H); 1.40至 1.51 (m, 2H); 1.59 (s,3H); 1.62 (m,1H); 1.87 (m, 111);2.04(111,111);2.28(111,111);2.47至2.58(111部分遮罩, 4H); 2.73 (s, 3H); 2.76 (s, 1H); 2.80 (d, J=9.6 Hz, 1H); I50937.doc •49· 201117814 2.95 (s,2H); 3.10 (s,3H); 3.16至3.48 (部分遮蔽之多重 峰,21H); 3.25 (S,3Η); 3·59 (S,3H); 3.63 (t,4 Hz, 2H); 3.93 (s’ 3H); 4.08 (m,1H); 4.53 (dd,>2.9至 11.9 HZ, 1H); 5.89 (s, 1H); 6.49^6.58 (m5 2H); 6.62 (m, 1H); 6.84 (s,1H); 7.19 (s,1H) 〇 ll·2.製備3-{2-[2·(2-{2·【(2-溴-乙醢基)_甲基_胺基]乙氧 基}_乙氧基)-乙氧基】-乙氧基}-丙酸曱酯 Β
分子量=414.30 分子式=C15H28BrN〇7 在室溫磁力攪拌下,在Ar之惰性氛圍下,依次添加i27 mg 3-(2-{2-[2-(2·甲基胺基-乙氧基卜乙氧基]_乙氧基卜乙氧 基)-丙酸甲酯、102.2 mg溴-乙酸2,5-二側氧基-吡咯啶_:丨_基 酯及1·2 mL DCM。45分鐘後,粗物質在減壓下濃縮且在5 g CN接枝矽膠上藉由急驟層析純化(溶離梯度為正庚烷 /iPrOH/AcOEt,其中iPr0H部分遞增)。在減壓下濃縮含有 預期化合物之溶離份後,獲得122 mg呈無色油狀之3_{2_ [2-(2-{2-[(2-溴·乙醯基)·甲基-胺基]_乙氧基丨·乙氧基)乙氧 基]-乙氧基}-丙酸甲酯。質譜(A) : tR=0.84分鐘;[M+H] + : m/z 414/416。 可按照公開方案(Biochemistry 1974,481)製備演_乙酸 2,5 -二側氧基_ n比洛咬_ 1 _基酯。 11.3.製備3-(2-{2-丨2-(2-甲基胺基-乙氧基)_乙氧基】·乙氧 基}-乙氧基)-丙酸甲酯 150937.doc -50· 201117814
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分子量=293.36 分子式=C13H27N00 在室溫磁力攪拌下,在Ar之惰性氛圍下,將27 1.7 mg 3-[2-(2-{2-[2-(第三丁氧幾基-甲基-胺基乙氧基卜乙氧 基}_乙氧基)-乙氧基]-丙酸曱醋溶解於2 mL於二β惡烧中之4 Ν鹽酸溶液中。1 8小時後,粗反應介質在減壓下濃縮,溶 解於4 mL MeOH中且通過3 g SCX SPE管柱(以10 mL MeOH調節’以lOmLMeOH洗滌且以2N氨水之MeOH溶液 溶離)。在減壓下濃縮溶離份後,獲得146 mg無色油狀 物。將該油狀物溶解於AcOEt中,經MgS04乾燥,過濾、且 在減壓下蒸發。獲得127 mg呈無色油狀之3-(2-{2-[2-(2-曱 基胺基-乙氧基)-乙氧基]-乙氧基}-乙氧基)_丙酸曱酯。質 譜(A) : tR=0.40 分鐘;[M+H] + : m/z 294。 II.4·製備3-[2-(2-(2-[2-(第三丁氧羰基-曱基-胺基)_乙氧 基】-乙氧基}-乙氧基)-乙氧基】-丙酸甲醋
分子量=393.48 分子式=C18H35N08 在室溫磁力攪拌下’在Ar之惰性氛圍下,用水-冰-氣化 鈉將227 mg 3-[2-(2-{2-[2-(第三丁氧羰基-甲基-胺基兴乙氧 基]-乙氧基}-乙氧基)-乙氧基]-丙酸於0.644 mL DCM且 0.644 mL MeOH中之溶液冷卻至約〇°c。添加0.449 mL 2 Μ 三曱基矽烷基重氮曱烷之己烷溶液,且在室溫下丨7小時 後’添加0.5 Μ乙酸之MeOH溶液以獲得5與6之間之pH值。 150937.doc •51· 201117814 粗物質以AcOEt(30 mL)稀釋,以水(2x15 mL)及鹽水(15 mL)洗滌’且有機相經MgS〇4乾燥,過濾且在減壓下蒸 發°獲得209.7 1^呈無色油狀之3-[2-(2-{2-[2-(第三丁氧羰 基-曱基-胺基)-乙氧基]-乙氧基乙氧基)-乙氧基]-丙酸甲 酯。質譜(A) : tR=l.i8分鐘;[M+H] + : m/z 294, 338, 394。 11.5.製備3-[2-(2-{2-【2-(第三丁氧羰基-甲基-胺基)-乙氧 基]-乙氧基卜乙氧基)-乙氧基】-丙酸
十I 分子量=379.45 〇 〇 分子式=C17H33N08 在室溫磁力攪拌下,在Ar之惰性氛圍下,用水-冰-氯化 納將330 mg市售3-(2-{2-[2-(2-第三丁氧基羰基胺基-乙氧 基)-乙氧基]-乙氧基}-乙氧基)-丙酸於4mL THF中之溶液冷 卻至約0°C。緩慢添加72.2 mg NaH(70%於油中),且在25 分鐘後添加Mel。移除冷卻浴且在室溫下3小時後,添加 120 mg NaH及0.2 mL Mel。在室溫下1.5小時後,添加經稀 釋之乙酸水溶液以獲得5與6之間的pH值。粗反應混合物以
AcOEt(30 mL)稀釋,以水(2x20 mL)及鹽水(1〇 mL)洗滌, 且有機相經MgS04乾燥,過濾且在減壓下蒸發。獲得227 mg呈無色油狀之3-[2-(2-{2-[2-(第三丁氧羰基·甲基-胺基)_ 乙氧基]-乙氧基}-乙氧基)-乙氧基]-丙酸。質譜(A): tR=1.02分鐘;[M+H] + : m/z 280,380。 實例 12 : L-DM4-烯丙基-PEG^COOMe 150937.doc -52- 201117814
TFA,CH,CL
12.1·製備無藥物之L-DM4-烯丙基-PEG4-COOMe
在室溫磁力攪拌下,將36.7 mg L-DM4、20.8 mg 3_(;2_ {2-[2-(4-溴-丁-2-烯氧基)-乙氡基]-乙氧基卜乙氧基)_丙酸 甲酯於0.37 mL DMA中之溶液及最後9.4 μΐ DIPEA依次引 入玻璃瓶中。在室溫下1.5小時及在-1 8 下1 8小時後,在 C18 接枝矽膠(Merck, C18, 5 g,25-40 μηι,18 ml/min,溶離 梯度為水··乙腈100··0至5..95(體積比))上藉由急驟層析純化 反應介質。在減壓下濃縮含有預期化合物之溶離份後,獲 得18.2 mg白色固體且在CN接枝矽膠上藉由急驟層析純化 (溶離梯度為正庚烧/iPrOH/AcOEt,其中Ac〇Et部分遞 增)。獲得4.02 mg呈白色固體形式之曱酯^七河肛烯丙 1·〇9分鐘;[m_h+hc〇〇H]-: 。H NMR (500 ΜΗζ,δ 基-PEG4-COOMe。質譜(〇:。=1· m/z 1112; [M+Na] + : m/z 1090 〇 (ppm) ’ 氣仿-d): 0.80 (s,3H); 1.18至1.26 (m, 7H); 127至 150937.doc ·53· 201117814 1.31 (m,6H); 1·40至 1.50 (m,1H); 1.57 (d,J=13.7 Hz, 3H); 1.64(8,3印;1.77((1(1(1,</=4.8及11.7及14.5 1^,111);1.91 (ddd,t/=4.8&11.7&14.5Hz,lH);2.18(dd,</=2.5&14.3 Hz, 1H); 2.38 (m, 1H); 2.57 (m, 4H); 2.86 (s5 2H); 2.89 (m, 1H); 3.00 (m5 1H); 3.04 (d, 7=9.9 Hz, 1H); 3.11 (d, /=12.3 Hz, 1H); 3.23 (s, 3H); 3.35 (s, 3H); 3.50 (d, 7=9.1 Hz, 1H); 3.55 (m, 2H); 3.63 (m, 10H); 3.69 (s, 3H); 3.75 (t, J=6.4 Hz, 2H); 3.92 (d, /=5.2 Hz, 2H); 3.98 (s, 3H); 4.27 (ddd, •7=1.6及 10.4及 12.3 Hz,1H); 4.7 8 (dd,/=3.0及 11.8 Hz,1H); 5,43(9,>/=6.8 1^,111);5.48至5,61(111,2^1);5.67(<1(1,1/=9.2 及 15.2 Hz,1H); 6.20 (d, «7=0.5 Hz,1H); 6.42 (dd,<7=11.3及 15.4 Hz, 1H); 6.65 (d, J=\.9 Hz, 1H); 6.77 (d, J=11.3 Hz, 1H); 6.82 (d,J=l.l Hz,1H)。 12.2.製備3-(2-{2-【2-(4-溴-丁 -2-烯氧基)-乙氧基]-乙氧基}-乙氧基)-丙酸甲酯
分子量=369.26 分子式=C14H25Br06 在磁力攪拌下,向50 mg 3-(2-{2-[2-(4-溴-丁-2-烯氧基)-乙氧基]-乙氧基}_乙氧基)-丙酸於1 mL DCM及0.25 mL MeOH中之冷卻溶液(水_冰浴)中添加0.11 mL 2 Μ於己烷中 之三曱基矽烷基重氮甲烷溶液。移除水-冰浴後,粗反應 混合物在室溫下攪拌1.5小時,以2滴乙酸中和且在減壓下 濃縮至乾。與曱苯共沸蒸發後’獲得47 mg呈琥珀色油狀 之3-(2-{2-[2-(4-溴-丁-2-烯氧基)-乙氧基]-乙氧基卜乙氧 150937.doc • 54· 201117814 tR=1.12 分鐘;[M+H] + : m/z 基)-丙酸甲酯。質譜: 369/371 ° 3·〇{2·[2-(4-溴-丁-2-烯氧基)-乙氧基乙氧基卜乙氧 基)-丙酸之製備已描述於實例4中。 實例 13: L-DlVU-AcNH-PEGs-COOMe
13.1.製備無藥物之L-DM4-AcNH-PEG8-COOMe
在室溫磁力授拌下’將6〇 mg L-DM4、11.4 mg碳酸鉀及 75 mg 3-{2-[2-(2-{2-[2-(2-{2-[2-(2-溴-乙醯胺基)-乙氧基]_ 乙氧基}-乙氧基)-乙氧基]-乙氧基丨-乙氧基乙氧基乙氧 基}-丙酸甲酯於0.7 mL DMA中之溶液依次引入玻璃瓶中。 在室溫下22小時後’反應介質以12 mL水稀釋,以2xl〇 mL AcOEt萃取。收集有機相,經MgS〇4乾燥,過濾且在減壓 150937.doc -55· 201117814 下農縮至乾。獲得50 mg無色油狀物’該產物以最低量 DCM稀釋且在5 g CN接枝矽膠上藉由急驟層析純化(溶離 梯度為正庚烧/iPrOH/AcOEt,其中iPrOH部分遞增)。在,咸 壓下濃縮含有預期化合物之溶離份後,殘餘物以最低量 DMA 稀釋且在 <:18接枝矽膠(Merck,C18, 5 g, 25-40 μιη,12 ml/min,溶離梯度為水:乙腈1〇〇:〇至5:95(體積比))上藉由 急驟層析純化。在減壓下濃縮含 '有預期化合物之溶離份 後,獲得3.3 mg呈無色膜形式之曱酯L-DM4-AcNH-PEG8-COOMe » 質譜(C) : tR=0.97分鐘;[M-H]-+ HCOOH: m/z 1319。j NMR (400 ΜΗζ,δ (ppm),氣仿-d): 0.73 (s. 3H): 1.10至 1·19 (m,7H); 1.21 (s, 3H); 1.23 (s,3H); 1.39 (td,/=6.1 及 10.1 Hz, 1H); 1.50 (d,《/=13.2Hz,1H); 1.57 (s, 3H); 1‘71 (m,1H); 1.85 (m,1H);2.11 (dd,J=3.4及 14.2 Hz, 1H); 2.29 (ddd,/=5.1 及 11.1 及 16.〇 Hz,1H); 2·45 (ddd, •/=5.4及11.2及16.11^,111);2.52((,</=6.6 1^,311);2.79(8, 3H); 2.82 (m, 1H); 2.90 (m, lH); 2.96 (d, J=9.3 Hz, 1H); 3.07 (d,/=13.2 Hz,1H); 3.15 (s,3H); 3.28 (s,3H); 3.35 (m, 2H); 3.44 (m, 3H); 3.56 (s, 29H); 3.61 (s, 3H); 3.68 (t, J=6.6 Hz, 2H); 3.91 (s, 3H); 4.20 (t, J=12.0 Hz, 1H); 4.70 (dd,J=3.2及 12.0 Hz,1H);· 5.35 (q,·7=7,〇 Hz,1H); 5.59 (dd, «/=9.0及 15.4 Hz, 1H); 6.13 (s,1H); 6.35 (dd,*7-11.0及 15.4 Hz, 1H); 6.57 (d, 7=1.5 Hz, lH); 6.67 (d, /=11.2 Hz, 1H); 6.78 (d,《7=1.5 Hz,1H); 6.96 (t,*/=5.ό Hz,1H)。 13.2.製備 3-{2-[2-(2-{2-[2-(2-{2-【2-(2-溴-乙醯胺基)-乙氧 150937.doc ·56· 201117814 基]-乙氧基}-乙氧基-乙氧基】-乙氧基卜乙氧基乙氧基]-乙 氧基}-丙酸甲酯 Η 分子量=576.48 ° 分子式=C22H42BrN011 在室溫磁力攪拌下,在Ar之惰性氛圍下,將200 mg 3-[2-(2-{2-[2-(2-{2-[2-(2-胺基-乙氧基)-乙氧基]-乙氧基}_ 乙氧基)-乙氧基]-乙氧基丨-乙氡基兴乙氧基]_丙酸 (CA(PEG)8,Pierce)、1.7 mL DCM、0.9 mL MeOH 及 0.34 mL2M於己烧中之三曱基矽烷基重氮曱烷溶液依次引入玻 璃瓶中。在室溫下3 0分鐘後’添加〇 · 1 mL 2 Μ於己烧中之 三曱基矽烷基重氮甲烷溶液。在室溫下25分鐘後,藉由添 加幾滴乙酸中和反應混合物,在減壓下濃縮至乾,與甲苯 共彿。由此獲得之無色油狀物以106.9 mg溴-乙酸2,5-二側 氧基比咯啶-1-基酯於〇.7 mL DCM中之溶液稀釋。在室溫 下30分鐘及在4。(:下16小時後,在20 g CN接枝矽膠上藉由 急驟層析純化粗物質(溶離梯度為正庚烷/iPr〇H/Ae〇Et, 其中iPrOH部分遞增)。在減壓下濃縮含有預期化合物之溶 離份後,獲得175 mg呈無色油狀之3-{2-[2-(2-{2-[2-(2-{2_ [2-(2-溴-乙醯胺基)_乙氧基]_乙氧基}•乙氧基_乙氧基]-乙氧 基}-乙氧基)-乙氧基]-乙氧基}-丙酸甲酯。質譜(B) ·· tR=2.79分鐘;[M+H] + : m/z 576/578。 了按照么開方案製備》臭-乙酸2,5 -二側氧基-π比π各。定_ 1 _美 酯(Biochemistry,1974, 481)。 150937.doc •57- 201117814
14.1.製備無藥物之L-DM4-Mal-PEG4-COOMe
在室溫磁力攪拌下,依次添加160 mg L-DM4、115·8 mg 3-{2-[2-(2-{2-[3-(2,5-二側氧基-2,5-二氫-°比咯-1-基)-丙醯 胺基]-乙氧基}-乙氧基)-乙氧基]-乙氧基}•丙酸2,5-二側氧 基-吡咯啶-1-基酯(市售,SM(PEG)4,Pierce)、0.6 mL DMA、55.1 mg 負載型 DIPEA(3.72 mmol/g)、0·3 mL額外 DMA及6 μΐ DIPEA。在室溫下1小時及在-20°C下16小時 後,過濾粗反應混合物,以DCM洗滌且在20 g矽膠上藉由 急驟層析純化(溶離梯度為DCM:MeOH,其中MeOH比重遞 增)。在減壓下濃縮含有預期產物之溶離份後,獲得110 mg無色膜狀物且在10 g石夕膠上純化(溶離梯度為 DCM:MeOH,其中MeOH比重遞增)。在減壓下濃縮含有預 期產物之溶離份後,獲得19.6 mg呈無色玻璃形式之曱酯 L-DM4-Mal-PEG4-COOMe。質譜(A): tR=1.31/1.32 分鐘(2 150937.doc -58- 201117814 種非對映異構體);m/z 1208。NMR (500 MHz, δ (ppm) ’ 氣仿-d): 0.73 (s,3H); 1·22 (m,13H); 1.39 (m, 1H); 1.52 (d, J=13.7 Hz, 1H); 1.57 (s, 3H); 1.78 (m, 1H); 1.99 (m, 1H); 2.11 (ddd,/=1.8及 1.9及 14.1 Hz,1H); 2.24 (ddd,J=4.7及 11_5及 15.9 Hz, 1H); 2.38 (m,3H); 2.46 (m, 1H); 2.53 (m, 3H); 2.69 (s, 1H); 2.82 (s, 3H); 2.94 (dd, •/=4,7&9.6Hz,lH);3.08(m,3H);3.14(s,3H);3.29(s, 3H); 3.34 (q, /=5.3 Hz, 2H); 3.43 (d, /=9.1 Hz, 1H); 3.48 (td,J=1.8及 5.1 Hz, 2H); 3.58 (s, 13H); 3.67 (m,7H); 3.91 (s, 3H); 4.22 (t, /=11.3 Hz, 1H); 4.70 (m, 1H); 5.29 (m, 1H); 5.59 (m,1H); 6_30 (大單峰,1H); 6.35 (dd,J=ll.〇及 15.4 Hz,1H); 6.61 (m, 2H); 6.76 (s, 1H)。 實例15 :抑制MDA-MB-231及HCT116腫瘤細胞生長 將指數生長期細胞用胰蛋白酶處理且再懸浮於其相應培 養基中(DMEM/F12 Gibco #21331; 10% SVF Gibco # 10500-056; 2 nM麩醯胺酸 Gibco # 25030 用於 MDA-MB231 及MDA-A1細胞;DMEM (Gibco # 11960) 10% SVF Gibco # 10500-056; 2 nM 麩醯胺酸 Gibco # 25030 用於 HCT116 細 胞)。將含有血清之完全培養基中的細胞懸浮液以5000個 細胞/孔之密度分佈於96孔Cytostar培養板(GE Healthcare Europe,# RPNQ0163)中(MDA-MB231,HCT116)。塗佈 4小 時後,將連續稀釋液一式三份添加至孔中。在藥物存在 下,在37°C/5% C02下將細胞培養3天。第4天,添加1〇 μι 14C 胸苷溶液(0.1 μ(:ί/孔(Perkin Elmer # NEC56825000))至 150937.doc -59- 201117814 各孔中。在實驗開始後96小時,用microbeta放射性計數器 (Perkin Elmer)量測14C胸苷之吸收量。自測試孔讀數減去 無細胞試劑空白讀數且以存活分率形式對該等數據作圖, 存活分率係藉由結合物處理細胞之讀數除以媒劑處理細胞 之對照孔平均讀數所獲得。 表III:細胞增殖抑制(IC5〇(nM)) 實例 無藥物 MDA-MB231 HCT116 10 L-DM4-AcNH-PEG4-COOMe 15.3 13 11 L-DM4-AcNMe-PEG4-COOMe 28.3 18.5 12 L-DM4-烯丙基-PEG4-COOMe 1.5 0.8 14 L-DM4-Mal-PEG4-COOMe 31.7 33.6 由此可見’以醋-COOMe形式測試之產物對兩種不同細 胞株呈現強活體外增殖抑制性。 實例16 : hu2Hll及hu2HllR35R74之結合物的活體内評估 為評估結合物之抗腫瘤活性,動物每日稱重且每週2次 藉由測徑規量測腫瘤。使用公式質量(mg)=[長度(mm) X寬 度(mm)2]/2計算腫瘤重量。在最大無毒性劑量(HNTD)下評 估抗腫瘤活性。 在最低點(組平均值)造成20%體重損失(bwl)或10°/〇或 10%以上藥物死亡之劑量視為過度毒性劑量《動物體重包 括腫瘤重量。主要療效終點為ΔΤ/AC、中值消退百分數、 部分及完全消退(PR及CR)以及無腫瘤倖存者(TFS)。 各治療組(T)及對照組(C)之腫瘤體積變化係如下計算: 對於各腫瘤而言,自指定觀測日之腫瘤體積減去首次治療 150937.doc -60· 201117814 曰(分期日)之腫瘤體積。對於治療組而言計算中值△ τ且對 於對照組而言計算中值△(:。隨後計算比率ΔΤ/Δ(::且以百分 _ ^M(Tt-TO) 數表示:% AT/AC= ^^"(Cf_CQ)xl〇〇 ° 當ΔΤ/Δ(2低於40%時,認為該劑量在治療上有效且當 △ TMC低於10%時,認為該劑量非常有效。若at/ac低於 0,則認為該劑量高度有效且註明消退百分比(參考丨): 腫瘤消退% :定義為治療組在指定觀測日之腫瘤體積減 幅與首次治療第一天之腫瘤體積相比的。/(^計算各動物在 特定時間點的消退% 〇 體積t()-體積t %(在t時): 隨後計算各組之中值消退% xlOO。 消退 部分消退(PR):若腫瘤體積減小至開始治療時腫瘤體積 之50%,則消退定義為部分消退。 完全消退(CR):當腫瘤體積=〇 mm3時實現完全消退(當 不能記錄腫瘤體積時視為CR)。 TFS :無腫瘤定義為在研究結束時腫瘤不可偵測之動 物。 hu2Hll結合物與hu2HllR35R74結合物之間的比較
150937.doc -61 - 201117814
評估hu2hll結合物及hu2hllR35R74結合物在2種劑量下 針對皮下植入雌性SCID小鼠中之強烈表現標靶之可測量原 發性結腸腫瘤CR-LRB-004P的抗腫瘤活性。對照組不予處 理。劑量係以毫克/公斤蛋白質表示。第1 5天藉由靜脈内 (IV)快速注射投與 40 mg/kg 及 10 mg/kg 之 hu2hllR35R74 結 合物。為給予等效劑量之DM4,投與44 mg/kg及11 mg/kg 之hu2hll結合物。結果提供於表IV中。 對CR-LRB-004P腫瘤使用單一投藥進度時, hu2hl 1 R35R74 結合物在 40 mg/kg 及 1 0 mg/kg 下有效, △ TMC分別為28°/。及39°/。,而hu2hll結合物僅在40 mg/kg下 有效,ATMC為26%。在10 mg/kg下,hu2hll結合物在該 模型中無效。由該等結果可知,hu2hllR35R74結合物在較 低劑量下顯示出優於hu2h 11結合物之活性。 結合物優化、選擇最佳藥物抗體比率DAR-DAR對 hu2HllR35R74結合物抗SCID雌性小鼠前列腺癌PC-3之抗 腫瘤活性的影響 DAR對hu2HllR3 5R74結合物抗腫瘤活性的影響係藉由 比較兩種有效低劑量在六種不同藥物抗體比率(DAR)下對 皮下植入雌性SCID中之前列腺PC-3腫瘤的影響來評估。 對照組不予處理。劑量係以毫克/公斤蛋白質表示。藉由 150937.doc -62- 201117814 UV方法測定DAR。第16天,分別以3.4、4.4、5.9、6.2、 7.4及8.4之DAR、藉由靜脈内(IV)快速注射投與10 mg/kg及 5 mg/kg hu2HllR3 5R74結合物。結果提供於表V中。 150937.doc 63- 201117814 猶A^w^w龠垅凝Ύ«^Ί 七sitalus^荽伞垅Tf卜llsoiuqznq^#^皱 IlJaznq 雄街-ΛΙ^ 註解 有效 有效 有效 無效 第21曰 之生物 統計口值3 0.011 0.0174 0.0008 00 第30曰 之無腫瘤 倖存者 Ο 〇 〇 v〇 〇 00 ο 消退 PR PR 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/6 0/8 0/8 若<0, 第21曰 中值 ATMC°/。 (消退。/〇) 1 m? S 鱗< S I筚 # ΰ m s -14.7(25) -18.2(25) -13別25) -15別25) -14.7 (27) i I i 1 00 ο 概 ^ ΌΙ S 茶(φί Q # S δ n< s 0 Φ4 δ 5 ^ 杂茶$ ^ iii c # 40(1.6) 10(0.4) 44(1.6) 11 (0.4) I 52 J2 I IV 16 mL/kg > 16 mL/kg 1 藥劑 hu2hllR35R74- 結合物 DAR(UV)=5.9 hu2hll-結合物 DAR(UV)=5.3 對照 •64- 150937.doc 201117814 W 鑪,f'sl'talus ε-ud 璨铎瓦柘戡 Ύ 琛智^i-*>IvaliI:K-^#♦tTrSSHUKznq^^-A^ 。((Λα)Ηνα 嫦 Ηνα 辉)趄))$骤趔^ 註解 無效 無效 有效 無效 南效 南效 高效 南效 1¾效 高效 1¾效 而效 惡病質 生物統計 piia Γ- CN T3 mm 0.0020 NS <0.0001 0.0013 <0.0001 0.0043 <0.0001 <0.0001 <0.0001 0.0014 第34日之 無腫瘤 倖存者 00 00 Ο 〇 〇〇 ^ Ο 〇 〇〇 ^ Ο 〇 00 oo o o oo oo o o CO oo o o 0/10 消退 CR 完全 〇〇 00 Ο 〇 00 〇〇 Ο 〇 〇〇 〇〇 Ο '—1 00 oo o o OO 00 o o oo oo (N O 0/10 PR 部分 〇〇 ^ Ο 〇 〇〇 ^ ^ 〇 〇〇 〇〇 oo oo CN (N oo oo oo oo ?5 0/10 第27曰 中值 消退% 1 42.8 13.4 66 35.5 59.6 0 1 '< 第27曰 中值 ΔΤ/Δ<:(%) Ο (N (N m ? ? ? 2 o o V V 穸00 0 1 < 每隻小鼠在 最低點之 ^ S f擊 -3.4(23) -11.9(30) -4.4(17) -4.5(25) /—V /—Ν 卜寸 —ΓΛ mm uo as -5.4(17) -8.0(34) /—N /—N 卜卜 o m \ό -6.3(17) -15.6(34) -19.6(27) 荽 灘 死亡 00 00 Ο 〇 〇〇 ^ Ο 〇 00 oo o o 00 00 〇 〇 00 00 o o 00 oo o o 0/10 每次注射 劑量 mg/kg (總劑量) 2 ^ 2 ^ 2 ^ 2 ^ 2 ^ 2 ^ 進度(天數) VO 投藥途徑/ 每次注射 齊J量 (ml/kg) IV 16 mL/kg IV 16 mL/kg IV 16 mL/kg IV 16 mL/kg IV 16 mL/kg IV 16 mL/kg 藥劑 DAR=3.4 DAR=4.4 DAR=5.9 DAR=6.2 DAR=7.4 DAR=8.4 對照 龚<0贺 w^案备,^¾¾ να4* 150937.doc •65 · 201117814 使用單一投藥進度’ hu2HllR35R74結合物在l〇 mg/kg 下展示活性的DAR為4.4至8.4。hu2HllR35R74結合物在5 mg/kg下展示活性的DAR為5.9至4。總之,DAR對 hu2HllR3 5R74結合物之腫瘤活性有影響。由該等關於特 定腫瘤之結果可知,DAR(UV)應大於4。最佳DAR應至少 等於5.9。
實例17 :藉由hu2HllR35R74結合物之PK參數評估DAR 在單次靜脈内(IV)投與雄性CD-I小鼠20 mg/kg結合物 後,評估hu2HllR35R74結合物在不同藥物抗體比率(DAR) 下之藥物動力學特性。量測結合物之血漿含量以在標準條 件下確立基礎單次劑量藥物動力學參數。將?1^參數與裸親 本抗體之彼等參數相比較。藉由特定ELISA技術量測結合 物及其抗體組分(總抗體,結合抗體與任何去結合抗體之 總和)之血漿濃度。結果提供於圖7上。 結果展示DAR值與總抗體組分暴露量之間的逆相關性’ 對於0、3.4、4.3、5.9、6.6及7.4之0人11,人1;(:0-〇〇值分別 為 83,000,000、61,000,000、48,000,000、46,000,000、 41,000,000及 27,000,000 ng.h/mL。 類似地,DAR值與結合物暴露量之間存在逆相關性,對 於 3.4、4.3、5.9、6.6 及 7.4 之 DAR,AUCO-oo 值分別為
39.000. 000 、30,000,000 ' 27,000,000 ' 29,000,000 A 20.000. 000 ng'h/mL 〇 DAR值與抗體組分清除率之間完全相關,對於DAR 0、 3.4、4.3、5_9、6.6及 7.4,CI值分別為 0.00024、0.0003 3、 150937.doc •66· 201117814 0.00042、0.00043、0.00049及 0.00074 L/h/kg ° 類似地,DAR值與結合物清除率之間幾乎完全相關,對 於0入113.4、4.3、5_9、6.6及7.4,(:1值分別為 0.0005 1、 0.00066、0.00075、0.00069、0.00099 L/h/kg ° 總之,DAR對PK參數有影響,當DAR增加時,暴露量減 小且清除率增加。根據功效及PK評估之結果,最佳DAR應 包括在5.9與7.4之間。 實例18 :評估hu2HllR35R74結合物在SCID雌性小鼠中抗 前列腺癌PC-3之作用 在8種劑量下評估具有DAR=5.9之抗體藥物結合物 hu2HllR35R74結合物針對皮下植入雌性SCID小鼠中之強 烈表現標靶之可測量前列腺PC-3腫瘤的抗腫瘤效應。對照 組不予處理。劑量係以毫克/公斤蛋白質表示。在第1 7曰 藉由靜脈内(IV)快速注射投與160、120、80、40、20、 10、5及2.5 mg/kg下之該等結合物。結果提供於表VI中。 使用單一投藥進程,發現所測試結合物之最大劑量(160 mg/kg)具有毒性,誘發體重損失及藥物相關死亡。在 HNTD( 1 20 mg/kg)及其他最低劑量下,化合物高度有效。 對於除2.5 11^/1^外之所有劑量而言,11112111111351174結合 物誘發部分消退且對於120、80及20 mg/kg而言,其誘發 完全消退。另外,該腫瘤模型為惡病質,且化合物在最低 點投與時降低體重損失(與對照相比)。總之, hu2HllR3 5R74結合物對前列腺PC-3腫瘤模型展示高活性 及良好劑量效應。 I50937.doc •67· 201117814 寒昶聲«^w Ί-rtifalus £:0<1璨锘«:桓黩<«^^荽^激>卜尨5£尨111^3||擊^-1>^ 150937.doc αΐΝΗ s 1¾ 9¾ (如祝)(令命) (碱¥龊绰鹉) (®Ηί敏絮) sPHlw S 泛。/0蜮诨 %3爹妄牛(BiJs ¥ί敬 (Υ莫) Β6寸Mr δ ϊ 5、¥# 蘅灘 (3Με)
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Lr.z ο.ς 001 OOCN 00寸 008 p bo:a/luI9I ΛΙ -68- 8/0 8/0 001 (sF—(·6(ν_ 00/0 201117814 【圖式簡單說明】 圖1 :實例1之脫糖基化結合物在去卷積後之HRMS質 譜; 圖2 :實例2之脫糖基化結合物在去卷積後之HRMS質 譜; 圖3 :實例3之脫糖基化結合物在去卷積後之HRMS質 譜; 圖4 :實例4之脫糖基化結合物在去卷積後之HRMS質 譜; 圖5 :實例5之脫糖基化結合物在去卷積後之HRMS質 譜; 圖6A-6C :序列; 圖7 :各種DAR下hu2HllR3 5R74結合物之PK參數:條形 圖表示在單次劑量靜脈内投與雄性CD-1小鼠(n=4)20 mg/kg含於HGS中之結合物後,若干結合物之暴露量 (AUC(O-inf);左)及清除率(CI ;右)與DAR的關係。在圖 上,2H11-DM4(底部)係指hu2HllR35R74結合物。 該等圖展示,每種結合物均存在攜帶0至10個類美登素 之結合物的分佈(D〇 :無類美登素;Dx : X個類美登素)。 150937.doc -69- 201117814 序列表 <110>法商赛諾菲-安萬特公司 <120>新穎類美登素(MAYTANSINOIDS)及該類美登素用於製備與抗體結合的結合劑之用途 <130> FR2009-104 <140〉 099133090 <141> 2010-09-29 <150> 09305939.2 ' <151> 2009-10-02 <160> 18 <170> Patentln version 3.3 <210> 1 <211> 5 <212> PRT <213> 智人 <400> 1
Ala Tyr Tyr Met His <210> 2 <211> 17 <212> PRT <213>小家鼠 <400> 2
Leu val Asn Pro Tyr Asn Gly Phe Ser Ser Tyr Asn Gin Lys Phe Gin 15 10 15
Gly <210> 3 <211> 10 <212> PRT <213> 小家鼠 <400> 3 Glu Phe Tyr Gly Tyr Arg Tyr Phe Asp val 1 5 10 <210> 4 <211> 16 <212> PRT <213> 小家鼠 <400> 4
Lys Ser Ser Gin ser Leu lie His Ser
Asp Gly Arg Thr Tyr Leu Asn 10 15 <210> 5 <211> 7 <212> PRT <213>小家鼠 <400> 5
Leu Val Ser Arg Leu Asp Ser 150937-序列表.doc 201117814 <210> 6 <211> 9 <212> PRT <213>小家鼠 <400> 6
Trp Gin Gly Ser His Phe Pro Arg Thr <210> 7 <211> 357 <212> DNA <213>小家鼠 <220> <221> CDS <222> (1)..(357) <400> 7 gag Glu 1 cag Gin ctg Leu caa Gin 5 cag Gin tet Ser gga Gly cct Pro 9?g Glu 10 ctg Leu aag Lys cct Pro ggg Gly 15 get Ala 48 tea Ser aag Lys att lie 20 tcc ser tgc Cys aag Lys K tet Ser 25 gat tac Gly Tyr tea Ser ttc Phe act Thr 30 gcc Ala tac Tyr 96 tac Tyr atg Met cac tgg gtg His Trp val 35 aag Lys caa Gin agt Ser 40 cat His gta aag agt val Lys Ser ett Leu 45 gag tgg Glu Trp att lie 144 gga Gly ett Leu 50 aat Asn cct tac Pro Tyr aat Asn 55 ggt Gly ttt Phe agt Ser age Ser tac Tyr 60 aac Asn cag Gin aat Asn ttc Phe 192 gag Glu 65 gac Asp aag gcc Lys Ala age Ser ttg Leu 70 act Thr gta Val gat Asp aga Arg ttc Phe 75 tcc Ser age Ser acc Thr gcc Ala tac Tyr 80 240 atg Met gaa Glu etc Leu cac His age Ser 85 ctg Leu aca Thr tet Ser gag Glu gac Asp 90 tet Ser gca Ala 9tc Val tat tac tgt Tyr Tyr Cys 95 288 gca Ala aga Arg gaa Glu ttc Phe 100 tac Tyr ggc Gly tac Tyr egg Arg tac Tyr 105 ttc Phe gat Asp gt〒 val tgg Trp ggc Gly 110 gca Ala 336 acc Thr geg gtc Ala val 115 acc Thr tcc Ser tea Ser 357 <210> 8 <211> 119 <212> PRT <213>小家鼠 <400> 8
Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu val Lys Pro Gly Ala 15 10 15 150937·序列表.doc 201117814
Ser val Lys lie Ser cys Lys Ala Ser Gly Tyr Ser Phe Thr Ala Tyr 20 25 30
Tyr Met His Trp Val Lys Gin Ser His Val Lys Ser Leu Glu Trp lie 35 40 45
Gly Leu val Asn Pro Tyr Asn Gly Phe Ser Ser Tyr Asn Gin Asn Phe 50 55 60
Glu Asp Lys Ala Ser Leu Thr Val Asp Arg Phe Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala val Tyr Tyr Cys 85 90 95
Ala Arg Glu Phe Tyr Gly Tyr Arg Tyr Phe Asp val Trp Gly Ala Gly 100 105 110
Thr Ala val Thr val Ser ser 115 <210> 9 <211> B39 <212> DNA <213> 小家鼠 <220> <221> CDS <222> (1)..(339)
ατ o 9G1 t y QT 9G c e th t p 9Γ ch a T 99 g厂 c A too c ΓΟ CPI t e th t p t s ai c H aΓ 9 9 c e 9Γ t s c A
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Ly <400> 9 gat Asp 1 atg Met acc Thr 5 cag Gin act Thr cca Pro etc Leu act Thr 10 ttg Leu teg Ser acc Thr att lie 15 gga Gly 48 caa Gin cca Pro gec Ala tcc Ser 20 ate lie tet Ser tgc cys aag Lys tea Ser 25 agt Ser cag Gin age Ser etc Leu ata lie 30 cat His agt Ser 96 gat Asp gga Gly aga Arg 35 aca tat Thr Tyr ttg Leu aat tgg Asn Trp 40 ttg Leu tta Leu cag Gin agg Arg cca Pro 45 ggc GTy cag Gin tet Ser 144 cca Pro aag Lys 50 ege Arg eta Leu att lie tat Tyr ctg Leu 55 fa? tet Ser aga Arg ctg Leu gac Asp 60 tet Ser gga gtc Gly Val cct Pro 192 gac Asp 65 agg Arg ttc Phe act Thr ggc Gly agt Ser 70 gga Gly tea Ser ggg Gly aca Thr gat Asp 75 ttc Phe aca Thr ctg Leu aaa Lys ate lie 80 240 age Ser aga Arg gtg gag get Val Glu Ala gag Glu gat Asp ttg Leu gga Gly fai tat Tyr tat Tyr tgc Cys tgg Trp caa Gin ggt Gly 288 85 95 150937-序列表.doc 201117814 <210> 10 <211> 113 <212> PRT <213> 小家鼠 <400> 10
Asp val val Met Thr Gin Thr Pro Leu Thr Leu Ser'Val Thr lie Gly 15 10 15
Gin Pro Ala Ser He Ser Cys Lys Ser Ser Gin Ser Leu lie His ser 20 25 30
Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gin A「g Pro Gly G]n Se「 35 40 45
Pro Lys Arg Leu lie Tyr Leu val Ser Arg Leu Asp Ser Gly val Pro 50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 8〇
Ser Arg val Glu Ala Glu Asp Leu Gly val Tyr Tyr Cys Trp Gin Gly 85 90 95
Ser His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 105 110
Arg <210> 11 <211> 357 <212> DNA <213> 智人 <220> <221> CDS <222> (1)..(357) <400> 11 cag gtg caa ctg gtg caa tcc ggt gcc gag gtc gtc aaa ccc gga gca
Gin val Gin Leu val Gin Ser Gly Ala Glu Val val Lys Pro Gly Ala 15 10 15 tct gtg aag ata tcc tgt aag gcc tcc ggc tac act ttt aca gcc tac
Ser val Lys lie Ser cys Lys Ala ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30 tat atg cat tgg gtt aaa cag agt ccc gtg cag tcc ctg gaa tgg ate
Tyr Met His Trp val Lys Gin Ser Pro val Gin Ser Leu Glu Trp lie 35 40 45 ggc ttg gtg aac cct tat aac gga ttc tea agt tac aat caa aag ttt
Gly Leu val Asn Pro Tyr Asn Gly Phe Ser Ser Tyr Asn Gin Lys Phe 50 55 60 4· 150937-序列表.doc 201117814 cag Gin 65 ggc Gly aag Lys tcc Ser ctg Leu 70 act Thr fa? gac Asp aga Arg tet Ser 75 agt tcc Ser Ser aca Thr tac Tyr 80 240 atg Met gag Glu etc Leu cat His tea Ser 85 ctg Leu aca Thr tea Ser gaa Glu gac ASp 90 age Ser gcc gta Ala val tac Tyr tat Tyr 95 tgc cys 288 gca cgt Ala Arg gag Glu ttc Phe 100 tac Tyr ggc Gly tat Tyr aga Arg tac Tyr 105 ttt Phe gac Asp gtc tgg val Trp ggc Gly 110 caa Gin ggc Gly 336 aca Thr gtc val 115 aca Thr gtg Val age Ser tet Ser 357 <210> 12 <211> 119 <212> PRT <213> 智人 <400> 12
Gin val Gin Leu val Gin Ser Gly Ala Glu val Val Lys Pro Gly Ala 1 5 10 15
Ser val Lys lie Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25 30
Tyr Met His Trp Val Lys Gin Ser Pro val Gin ser Leu Glu Trp lie 35 40 45
Gly Leu Val Asn Pro Tyr Asn Gly Phe Ser Ser Tyr Asn Gin Lys Phe 50 55 60
Gin Gly Lys Ala ser Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Glu Leu His ser Leu Thr Ser Glu Asp Ser Ala val Tyr Tyr cys 85 90 95
Ala Arg Glu Phe Tyr Gly Tyr Arg Tyr Phe Asp Val Trp Gly Gin Gly 100 105 110
Thr Ala Val Thr Val Ser Ser 115 <210> 13 <211> 336 <212> DNA <213> 智人 <220> <221> CDS <222> (1)..(336) <400> 13 gac gtc gtg atg aca caa acc cct ctg tcc ctg age gtc act ctg gga 48
Asp val val Met Thr Gin Thr Pro Leu Ser Leu Ser val Thr Leu Gly 150937-序列表.doc 96 201117814 15 10 15 caa ccc get tee att age tgc aaa tea tea caa tet etc ate cac tea
Gin Pro Ala Ser lie Ser cys Lys Ser ser Gin ser Leu lie His Ser 20 25 30 144 192 240 288 336 gac ggc cgt aeg tac etc aat tgg ctg ctg cag aga cca gga cag tee
Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gin Arg Pro Gly Gin Ser 35 40 45 cct aaa agg ett ate tac ctg gtc tet cgt ttg gac tet ggt gta cca
Pro Lys Arg Leu lie Tyr Leu Val Ser Arg Leu Asp Ser Gly Val Pro 50 55 60 gac egg ttt act ggt tee ggg gee gga acc gat ttc act ctg aag att
Asp Arg Phe Thr Gly ser Gly Ala Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80 tee agg gtg gaa get gaa gat etc gga gtg tat tat tgc tgg cag ggc
Ser Arg Val Glu Ala Glu Asp Leu Gly val Tyr Tyr Cys Trp Gin Gly 85 90 95 age cat ttc ccc cgt act ttt ggt gag ggt acc aaa ttg gaa att aag Ser His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 105 110 <210> 14 <211> 112 <212> PRT <213> 智人 <400> 14
Asp val val Met Thr Gin Thr Pro Leu Ser Leu Ser val Thr Leu Gly 1 5 10 15
Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin ser Leu lie His Ser 20 25 30
Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gin Arg Pro Gly Gin Ser 35 40 45
Pro Lys Arg Leu lie Tyr Leu val Ser Arg Leu Asp ser Gly val Pro 50 55 60
Asp Arg Phe Thr Gly Ser Gly Ala Gly Thr Asp Phe Thr Leu Lys lie 65 70 75 80
Ser Arg val Glu Ala Glu Asp Leu Gly val Tyr Tyr Cys Trp Gin Gly 85 90 95
Ser His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu lie Lys 100 l〇5 HO <210> 15 <211> 717 <212> DNA <213> 智人 <220> 150937·序列表.doc 48201117814 <221> CDS <222> (1)..(717) <400> 15 atg Met 1 gga Gly tgg Trp tet Ser tgc cys 5 ate lie ate lie ctg Leu ttt phe etc Leu 10 act Thr acc Thr 15 gga Gly gtg Val cac His agt Ser gac Asp 20 gt〒 val gtg val atg Met aca Thr caa Gin 25 acc Thr cct Pro ctg Leu tcc Ser ctg Leu 30 age Ser act Thr ctg Leu gga Gly 35 caa Gin ccc Pro tcc Ser att lie 40 age Ser tgc cys aaa Lys tea ser tea Ser 45 caa Gin tet Ser etc Leu ate lie cac His 50 tea Ser gac Asp ggc Gly cgt Arg aeg Thr 55 tac Tyr etc Leu aat Asn tgg Trp ctg Leu 60 ctg Leu cag Gin aga Arg cca Pro gga Gly 65 cag Gin tcc ser cct Pro aaa Lys agg Arg 70 ett Leu ate lie tac Tyr ctg Leu 75 tet Ser cgt Arg ttg Leu gac Asp tet Ser 80 ggt Gly cca Pro gac Asp egg Arg 85 ttt Phe act Thr ggt Gly tcc Ser 999 Gly 90 gee Ala gga Gly acc Thr gat Asp ttc Phe 95 act Thr ctg Leu aag Lys att lie tcc Ser 100 agg Arg gtg val gaa Glu 9« Ala gaa Glu 105 gat Asp etc Leu gga gtg Gly val tat Tyr 110 tat Tyr tgc Cys tgg Trp cag ggc Gin Gly 115 age Ser cat His ttc Phe ccc Pro cgt Arg 120 act Thr ttt Phe ggt ggg gat Gly Gly Gly 125 acc Thr aaa Lys ttg Leu gaa Glu att lie 130 aag Lys cgt Arg aeg gtg get gca Thr val Ala Ala 135 cca Pro tet Ser gt〒 val ttc Phe 140 ate lie ttc Phe ccg Pro cca Pro tet Ser 145 gat A$p gag cag Glu Gin ttg Leu aaa Lys 150 tet Ser gga Gly act Thr tet Ser 155 tgc cys ctg Leu ctg Leu 160 aat Asn aac Asn ttc Phe tat Tyr ccc Pro 165 aga Arg gag Glu aaa Lys gta cag val Gin 170 tgg Trp aag Lys fa? gat Asp 175 aac Asn etc Leu caa Gin teg Ser 180 ggt Gly aac Asn tcc Ser cag Gin gag Glu 185 agt Ser aca Thr gag cag GIG Gin 190 gac Asp age Ser aag Lys gac Asp age Ser 195 acc Thr tac Tyr age Ser etc Leu age Ser 200 age Ser acc Thr ctg Leu aeg Thr ctg Leu 205 age Ser aaa Lys gca Ala gac tac Asp Tyr 210 gag Glu aaa Lys cac His aaa Lys gtc val 215 tac Tyr 9cc Ala tgc cys gaa Glu 9tc val 220 acc Thr cat His cag ggc Gin Gly ctg Leu 225 age Ser teg Ser ccc Pro 9tc val aca Thr 230 aag Lys age Ser ttc Phe aac Asn agg gga Gly gag Glu tgt cys tag 96 144 192 240 288 336 384 432 480 528 576 624 672 <210> 16 <211> 238 <212> PRT <213> 智人 150937·序列表.doc 717 201117814 <400> 16 Met Gly Trp
Ser Cys lie lie Leu Phe Leu val Ala Thr Ala Thr Gly 5 10 15 val His Ser
Asp val val Met Thr Gin Thr Pro Leu Ser Leu Ser val 20 25 30
Thr Leu Gly 35
Gin Pro Ala Ser lie Ser Cys Lys Ser Ser Gin ser Leu 40 45 lie His Ser 50
Asp Gly Arg Thr Tyr Leu Asn Trp Leu Leu Gin Arg Pro 55 60
Gly Gin Ser 65 pro Lys Arg Leu lie Tyr Leu val Ser Arg Leu Asp Ser 70 75 80
Gly val Pro
Asp Arg Phe Thr Gly Ser Gly Ala Gly Thr Asp Phe Thr 85 90 95
Leu Lys lie
Ser Arg Val Glu Ala Glu Asp Leu Gly val Tyr Tyr Cys 100 105 110
Trp Gin Gly 115
Ser His Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu 120 125
Glu lie Lys 130
Arg Thr val Ala Ala Pro Ser val Phe lie Phe pro pro 135 140
Ser Asp Glu 145
Gin Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu 150 155 160
Asn Asn Phe
Tyr Pro Arg Glu Ala Lys Val Gin Trp Lys Val Asp Asn 165 170 175
Ala Leu Gin ser Gly Asn Ser Gin Glu ser val Thr Glu Gin Asp Ser 180 185 190
Lys Asp ser 195
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu ser Lys Ala 200 205
Asp Tyr Glu 210
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly 215 220
Leu Ser ser 225
Pro val Thr Lys Ser Phe Asn Arg dy Glu cys <210> 17 <211> 1404 <212> DNA <213> 智人 150937-序列表.doc 201117814 <220> <221> CDS <222> (1)..(1404) <400> 17 atg Met 1 gga tgg Gly Trp tee Ser tgt Cys 5 att lie att lie ctg Leu ttt Phe etc Leu 10 aca Thr aca Thr 15 ggc Gly 48 fa? cat His agt Ser cag gtg Gin val 20 caa Gin ctg Leu Ώ caa Gin 25 tee Ser ggt Gly gee gag gtc Ala Glu val 30 gtc val aaa Lys 96 ccc Pro gga gca Gly Ala 35 tet Ser gtg val aag Lys ata lie tee Ser 40 tgt Cys aag Lys tee Ser gac tac Gly Tyr 45 act Thr ttt Phe 144 aca Thr 50 tac Tyr tat Tyr atg Met cat His tgg Trp 55 fa? aaa Lys cag Gin agt Ser ccc Pro 60 cag Gin tee ser ctg Leu 192 gaa Glu 65 tgg Trp ate lie ggc Gly ttg Leu gtg Val 70 aac Asn cct pro tat aac Tyr Asn gga Gly 75 ttc Phe tea Ser agt Ser tac Tyr aat Asn 80 240 caa Gin aag Lys ttt Phe cag ggc Gin Gly 85 aag Lys tee Ser ctg Leu act Thr 90 gac Asp aga Arg tet Ser agt Ser 95 tee Ser 288 aca Thr tac Tyr atg Met 100 etc Leu cat His tea Ser ctg Leu 105 aca Thr tea Ser gaa Glu gac Asp age Ser 110 336 tac tat Tyr Tyr tgc Cys 115 gca cgt Ala Arg gag Glu ttc Phe tac Tyr 120 ggc tat aga tac Gly Tyr Arg Tyr ttt Phe 125 gac Asp gt〒 Val tgg Trp 384 ggc Gly caa Gin 130 ggc Gly aca Thr 9cc Ala aca Thr 135 gtg Val age Ser tet Ser 9« Ala tee Ser 140 act Thr aag Lys ggc Gly cca Pro 432 teg Ser 145 val ttc Phe ccc Pro ctg Leu gca Ala 150 ccc pro tee Ser tee Ser aag Lys age Ser 155 acc Thr tet Ser ggg ggc aca Gly Gly Thr 160 480 geg gee Ala Ala ctg Leu ggc tgc Gly Cys 165 ctg gtc Leu val aag Lys gac tac Asp Tyr 170 ttc Phe ccc Pro gaa Glu ccg Pro fa? 175 aeg Thr 528 teg Ser tgg Trp aac Asn 180 tea Ser ggc Gly Ala ctg Leu acc Thr 185 age Ser ggc Gly 沿 cac His acc Thr 190 ttc phe ccg Pro 576 get gtc Ala Val eta Leu 195 cag Gin tee Ser tea Ser gga Gly etc Leu 200 tac Tyr tee Ser etc Leu age Ser age Ser 205 緦sy acc Thr 624 39 ccc Pro 210 tee Ser age Ser age Ser ttg Leu ggc Gly 215 acc Thr cag Gin acc tac Thr Tyr ate lie 220 tgc Cys aac Asn gtg Val aat Asn 672 cac His 225 aag Lys ccc Pro age Ser aac Asn acc Thr 230 aag Lys 渤 gac Asp aag Lys aaa Lys 235 gtt gag Val Glu ccc Pro aaa Lys tet Ser 240 720 tgt cys gac ASP aaa Lys act Thr cac His 245 aca Thr tgc cys cca pro ccg Pro tgc cys 250 cca Pro gca Ala cct Pro gaa Glu etc Leu 255 ctg Leu 768 150937-序列表.doc 201117814
ggg Gly gga Gly ccg pro tea Ser 260 ttc Phe etc Leu ttc Phe ccc Pro 265 cca pro aaa Lys ccc Pro aag Lys gac Asp 270 acc Thr etc Leu 816 atg Met ate lie tcc ser 275 egg acc Arg Thr cct Pro gag gtc GIG val 280 aca tgc Thr Cys gtg gtg gtg val val vai 285 gac Asp age Ser 864 cac His gaa Glu 290 gac Asp cct Pro gag Glu fa? aag Lys 295 ttc Phe aac Asn tgg tac gtg Trp Tyr val 300 gac Asp ggc Gly gag Glu 912 305 cat His aat Asn 9cc Ala aag Lys aca Thr 310 aag Lys ccg egg Pro Arg gag gag Glu Glu 315 cag Gin tac Tyr aac Asn age Ser aeg Thr 320 960 tac Tyr cgt Arg gtg gtc val val age Ser 325 etc Leu acc Thr ctg Leu B30 cac His cag Gin gac Asp tgg Trp ctg Leu 335 aat Asn 1008 ggc Gly aag Lys gag Glu tac Tyr 340 aag Lys tgc Cys aag Lys S tcc Ser 345 aac Asn aaa Lys etc Leu cca Pro 350 婼 ccc Pro 1056 ate lie gag Glu aaa Lys 355 acc Thr ate lie tcc Ser aaa Lys 恕 360 aaa Lys ggg Gly cag Gin ccc Pro ega Arg 365 gaa Glu cca Pro cag Gin 1104 fa? tac Tyr 370 acc Thr ctg Leu ccc pro cca Pro tcc Ser 375 egg Arg gat Asp gag Glu ctg Leu acc Thr 380 aag Lys aac Asn cag Gin gti val 1152 age ctg Ser Leu 385 acc Thr tgc Cys ctg Leu 390 aaa Lys ggc Gly ttc tat Phe Tyr ccc Pro 395 age Ser gac Asp ate lie gec gtg Ala val 400 1200 gag Glu tgg Trp gag Glu age Ser aat Asn 405 ccg Pro gag Glu aac Asn 410 aac Asn tac Tyr aag Lys acc Thr aeg Thr 415 cct Pro 1248 ccc Pro fa? ctg Leu gac Asp 420 tcc Ser gac ASp ggc Gly tcc Ser ttc Phe 425 ttc Phe etc Leu tac Tyr age Ser aag Lys 430 etc Leu acc Thr 1296 gac Asp aag Lys 435 age Ser agg Arg tgg Trp cag Gin cag Gin 440 ggg Gly aac Asn 9tc val ttc Phe tea Ser 445 tgc cys tcc Ser 1344 atg Met cat His 450 gag Glu ctg Leu cac His aac Asn 455 cac His tac aeg Tyr Thr cag Gin aag Lys 460 age Ser etc Leu tcc Ser ctg Leu 1392 tet Ser 465 ccg Pro ggt Gly tga 1404 <210> <211> <212> 18 467 PRT <213>智人 <400> 18
Met Gly Trp Ser cys lie lie Leu Phe Leu val Ala Thr Ala Thr Gly 15 10 15 val His Ser Gin val Gin Leu val Gin ser Gly Ala Glu val val Lys 10· 150937-序列表.doc 201117814 20 25 30
Pro Gly Ala Ser val Lys lie Ser cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45
Thr Ala Tyr Tyr Met His Trp val Lys Gin Ser Pro val Gin Ser Leu 50 55 60
Glu Trp lie Gly Leu val Asn Pro Tyr Asn Gly Phe Ser Ser Tyr Asn 65 70 75 80
Gin Lys Phe Gin Gly Lys Ala Ser Leu Thr val Asp Arg Ser Ser Ser 85 90 95
Thr Ala Tyr Met Glu Leu His Ser Leu Thr Ser Glu Asp Ser Ala val 100 105 110
Tyr Tyr Cys Ala Arg Glu Phe Tyr Gly Tyr Arg Tyr Phe Asp val Trp 115 120 125
Gly Gin Gly Thr Ala val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 130 135 140
Ser val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr 145 150 155 160
Ala Ala Leu Gly Cys Leu val Lys Asp Tyr Phe Pro Glu Pro val Thr 165 170 175
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly val His Thr Phe Pro 180 185 190
Ala val Leu Gin Ser Ser Gly Leu Tyr ser Leu ser Ser val val Thr 195 200 205
Val pro Ser Ser ser Leu Gly Thr Gin Thr Tyr lie Cys Asn val Asn 210 215 220
His Lys Pro Ser Asn Thr Lys val Asp Lys Lys val Glu Pro Lys Ser 225 230 235 240
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu 245 250 255
Gly Gly Pro Ser val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu 260 265 270
Met lie Ser Arg Thr Pro Glu val Thr Cys val val val Asp Val ser 275 280 285
His Glu Asp Pro Glu val Lys Phe Asn Trp Tyr Val Asp Gly val Glu 290 295 300 -11 - 150937·序列表.doc 201117814
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr 305 310 315 320
Tyr Arg val val Ser val Leu Thr val Leu His Gin Asp Trp Leu Asn 325 330 335
Gly Lys Glu Tyr Lys Cys Lys val Ser Asn Lys Ala Leu Pro Ala Pro 340 345 350 lie Glu Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin 355 360 365 val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin val 370 375 380
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala val 385 390 395 400
Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro 405 410 415
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 420 425 430 val Asp Lys Ser Arg Trp Gin Gin Gly Asn val Phe Ser Cys Ser val 435 440 445
Met His Glu Ala Leu His Asn His Tyr Thr Gin Lys ser Leu Ser Leu 450 455 460
Ser Pro Gly 465 -12- 150937-序列表.doc
Claims (1)
- 201117814 七、申請專利範圍: 1. 一種式(I)化合物其中: ALK為(Ci-C6)伸烧基; Χι及X2各獨立地為以下基團之一:_ch=ch-、-CO-、 -C0NR-、-NRC0-、_C00-、-0C0-、-0C0NR-、-NRC00- 、-NRC0NR,_、-取-、-S(O)n(n=0、1 或2)或-ο-; R及R,獨立地為Η或(Ci_c6)烷基; I為1至40之整數,較佳為1至2〇之整數,更佳為1至1〇 之整數; 田X2為-CH-CH-時,j為對應於1之整數,當&不 為韻=CH•時,之整t Zb為一簡單鍵、 υ-或-NH-,Rb 為 Η或(CVC6)烧基、 (C3-C7)環烷基、芳基 H、雜芳基或(C3-C7)雜環烷基;或Zb 為單鍵,Rb為Hal。 2 ·如請求項1之化合物 其中 X2 為-CH=CH-或-CONR-,該 co基連接於-χ ALK 〜、基團’ R為Η或(CVC6)烷基。 3.如請求項1或2之化合 物,其中_Xl_ALK-為-S-CH2-。 150937.doc 201117814 4. 如請求項1至3之化合物,其中i為3、4、5、6、7、8、9 或10。 5. 如請求項1至4之化合物,其具有式(II):6 · —種選自以下之化合物:150937.doc 2011178147.如前述請求項中任一項之化合物, 為-COOH、-COCKCVCs)烧基、-COOMe、COOru。 b b % 〇 ^H2CH=CH,、 —coo-N^jM=H或陽離子 Λ 基團 -coo- 其 中GI表示至少一個誘導基團氺〇2、_Hai*_F或上〇 8. 如前述請求項中任一項之化合物,其呈鹼形式或鹽形 式’或該驗或該鹽之溶劑合物或水合物形式。 9. 一種製備結合物之方法,其包含以下步驟: (i)使視情況緩衝之抗體水溶液與如請求項1至8之化合 物之溶液接觸; (ii)隨後視情況使⑴中所形成之結合物與可能存在於 溶液中之未反應試劑及任何聚集物分離。 1 〇·如請求項9之方法,其中反應溫度通常在20。(:至40。(:之 間變化及/或反應時間在1至24小時之間變化。 11.如請求項9至10之方法,其中在步驟⑴或(ii)後,對該含 150937.doc 201117814 有結合物之溶液進行超濾及/或透濾之另外步驟(Hi)。 12·如請求項今至丨丨之方法,其中該抗體為特異性結合於 EphA2受體且包含至少一條重鏈及至少一條輕鏈之抗體 或其抗原決定基結合片段,其中該重鏈包含三個具有 SEQ ID NO. 1、2及3所表示之胺基駿序列的連續互補決 定區’及其中該輕鏈包含三個具有SEQ ID NO: 4、5及6 所表示之胺基酸序列的連續互補決定區。 13_如請求項12之方法,其中該抗體或該抗原決定基結合片 •k為人類化或表面重塑抗體(resurfacecl antib〇dy)或其抗 原決定基結合片段。 14. 如請求項12之方法,其中該重鏈包含由SEq ID N〇: 12組 成之胺基酸序列’及其中該輕鏈包含由Seq id NO: 14組 成之胺基酸序列。 15. 如請求項12之方法,其中該重鏈存在於胺基酸序列SEQ ID NO: 18,及其中該輕鏈存在於胺基酸序列SEq ID NO: 16。 16. —種結合物’其可用如請求項9至1 5之方法獲得。 17. 如请求項16之結合物’用UV分光光度計測量,其平均 DAR超過4,更特別在4與1 〇之間,甚至更特別在4與7之 Ό280, 間’該DAR係依據以下方程式DAR=cD/cA測定,其中 Cd = [(εΑ28。X A252)- (εΑ252 X A280)]/[(sD252 χ εΑ28。)- (εΑ252 — 、1 Ά280 Ca = [Α28。- (CD χ ε^。)]^ ε〇252=26,159 M*lcm ^D28〇=5,180 M cm 150937.doc 201117814 Sa28〇=224,000 丨 εΑ252 = 82,880 M'^cni'1 八252及A28G分別為該結合物在uv分光光度計上在252 nm與2 80 nm所測量之吸光度。 18. 19. 20. 21. 22. 如請求項!7之結合物,其中該證在5與8之間 在5.5與8之間,甚至更特別在5.9與7.5之間。 ,別 -種水溶液,其包含如請求項邮以之結合物。 如:求項1至8之產物’其係用作抗癌劑。 月求項16或1 8之結合物,其係用作抗癌劑。 -種如請求項丨至8之化合物用於製備結合物之用途发 中下式之類美登素(maytansinoid)片段: 其 共價連接於抗體。 150937.doc
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ATE496944T1 (de) | 2003-07-21 | 2011-02-15 | Immunogen Inc | Ca6-antigenspezifisches zytotoxisches konjugat und verfahren zu dessen anwendung |
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SI1928503T1 (sl) * | 2005-08-24 | 2012-11-30 | Immunogen Inc | Postopek za pripravo konjugatov majtansinoid protitelo |
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EP1864682A1 (en) | 2006-06-09 | 2007-12-12 | Sanofi-Aventis | Leptomycin derivatives |
ES2673822T3 (es) | 2006-07-18 | 2018-06-25 | Sanofi | Anticuerpo antagonista contra EphA2 para el tratamiento de cáncer |
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-
2010
- 2010-09-29 AR ARP100103531A patent/AR078471A1/es not_active Application Discontinuation
- 2010-09-29 UY UY0001032913A patent/UY32913A/es not_active Application Discontinuation
- 2010-09-29 TW TW099133090A patent/TW201117814A/zh unknown
- 2010-09-30 MX MX2012003998A patent/MX2012003998A/es unknown
- 2010-09-30 EP EP10768578A patent/EP2483279A1/en not_active Withdrawn
- 2010-09-30 CN CN2010800548853A patent/CN102741260A/zh active Pending
- 2010-09-30 KR KR1020127011167A patent/KR20120091166A/ko not_active Application Discontinuation
- 2010-09-30 WO PCT/IB2010/054417 patent/WO2011039721A1/en active Application Filing
- 2010-09-30 PE PE2012000428A patent/PE20121534A1/es not_active Application Discontinuation
- 2010-09-30 AU AU2010302247A patent/AU2010302247A1/en not_active Abandoned
- 2010-09-30 JP JP2012531540A patent/JP2013506653A/ja active Pending
- 2010-09-30 CA CA2774916A patent/CA2774916A1/en not_active Abandoned
- 2010-09-30 NZ NZ599045A patent/NZ599045A/xx not_active IP Right Cessation
- 2010-09-30 EA EA201270473A patent/EA201270473A1/ru unknown
- 2010-09-30 BR BR112012007305A patent/BR112012007305A2/pt not_active IP Right Cessation
-
2012
- 2012-03-14 TN TNP2012000115A patent/TN2012000115A1/en unknown
- 2012-03-19 IL IL218740A patent/IL218740A0/en unknown
- 2012-03-26 CR CR20120147A patent/CR20120147A/es unknown
- 2012-03-26 EC ECSP12011756 patent/ECSP12011756A/es unknown
- 2012-03-29 US US13/434,363 patent/US20120276124A1/en not_active Abandoned
- 2012-03-30 NI NI201200051A patent/NI201200051A/es unknown
- 2012-03-30 ZA ZA2012/02328A patent/ZA201202328B/en unknown
- 2012-04-02 CL CL2012000820A patent/CL2012000820A1/es unknown
- 2012-04-27 MA MA34818A patent/MA33702B1/fr unknown
Also Published As
Publication number | Publication date |
---|---|
UY32913A (es) | 2011-04-29 |
IL218740A0 (en) | 2012-06-28 |
CL2012000820A1 (es) | 2012-08-31 |
CN102741260A (zh) | 2012-10-17 |
NZ599045A (en) | 2013-09-27 |
EA201270473A1 (ru) | 2013-02-28 |
BR112012007305A2 (pt) | 2016-12-06 |
ECSP12011756A (es) | 2012-07-31 |
PE20121534A1 (es) | 2012-12-03 |
CA2774916A1 (en) | 2011-04-07 |
TN2012000115A1 (en) | 2013-09-19 |
AU2010302247A1 (en) | 2012-04-26 |
MA33702B1 (fr) | 2012-10-01 |
NI201200051A (es) | 2012-08-09 |
WO2011039721A1 (en) | 2011-04-07 |
JP2013506653A (ja) | 2013-02-28 |
EP2483279A1 (en) | 2012-08-08 |
US20120276124A1 (en) | 2012-11-01 |
ZA201202328B (en) | 2014-06-25 |
CR20120147A (es) | 2012-06-01 |
KR20120091166A (ko) | 2012-08-17 |
MX2012003998A (es) | 2012-04-20 |
AR078471A1 (es) | 2011-11-09 |
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