TW200533375A - Freeze dried formulations of antibody conjugates - Google Patents

Abstract

The present invention relates to freeze-dried (=lyophilised) pharmaceutical compositions of antibodies, preferably antibodies which are conjugated to an effector molecule. In particular the present invention relates to stable freeze-dried formulations of antibody-DM1 complexes. Moreover, this invention relates to methods of lyophilising antibody-DM1 complexes.

Description

200533375 IX. Description of the invention: [Technical field to which the invention belongs] The present invention relates to a freeze-dried (= beam-dried) pharmaceutical composition of antibodies. The antibodies are preferably antibodies conjugated to effector molecules. In particular, the present invention relates to stable freeze-dried formulations of the antibody-DM 1 complex. In addition, the present invention relates to a method for lyophilizing an antibody-DM1 complex. [Prior technology] Recombinant antibody molecules have long been known to us in this technology, such as humanized murine antibodies (Shin et al., 1989; Gtissow and Seemann, 1991), bispecific antibodies (Weiner et al., 1993; Goodwin, 1989), single-chain antibodies (scFv, Johnson and Bird, 1991), intact or incomplete immunoglobulins (Coloma et al., 1992; Nesbit et al., 1992; Barbas et al., 1992) or by Antibodies produced by chain shuffling (Winter et al., 1994). Today, fully human antibodies can be obtained, for example, by the phage display method (Aujame et al., 1997; US 5,885,793; US 5,969,108; US 6,3 00,064; US 6,248,516; US 6,291,158) or by means of Transgenic mice (EP 0 438 474; EP 0 463 151; EP 0 546 073) were transcribed with functional human Ig genes. We also know conjugates of antibodies and effector molecules, such as single-chain antibody / toxin fusion proteins (Chaudhary et al., 1990; Friedman et al., 1993); antibodies and radioisotopes or radioactivity such as mI, luIn, 99mTc Compounds (Larson et al., 1991; Thomas et al., 1989; Srivastava, 1988), enzymes such as peroxidase or alkaline phosphatase (Catty and Raykundalia, 1989), fluorescent dyes (Johnson, 1989 97600.doc 200533375) or biotin molecules (Guesdon et al., 1979); toxins linked to cytokines or some other immunomodulatory polypeptides (for example, tumor necrosis factor or interleukin-2) ( Vitetta et al., 1991; Vitetta and Thorpe, 1991; Kreitman et al., 1993; Theuer et al., 1993), cytostatin (Schrappe et al., 1992), prodrugs (Wang et al., 1 992) (Senter et al., 1989), radioactive material.

CD44 is a protein that exhibits different reforming isomers on multiple cells (for example, for further information, see WO02 / 094879A1, WO02 / 094325A2). However, the expression of a junction variant containing the domain v6 (CD44v6) in the extracellular region is limited to a portion of the epithelial tissue. Like other variant exons (CD44v3, CD44v5, CD44v7 / v8, CD44vlO), the CD44v6 line is advantageous in human tumors and normal tissues

Phenotypic tumor-associated antigens (Heider et al., 1995; Heider et al., 1996; Dali et al., 1996; Beham-Schmid et al., 1998; Tempfer et al., 1998; Wagner et al., 1998 ). In the prior art, antibodies specific for specific conjugation variants of CD44 (CD44v, CD44var) are known, and these variants are expressed only on a subset of epithelial cells. For example, CD44v6-specific antibodies (WO02 / 094879A1) can be mentioned, in particular, radioactive conjugates or cytotoxic substances can be mentioned, and specifically, maytansinoids can be mentioned Common filial piety (WOO2 / 094325 A2). In order to administer this antibody conjugate to a patient, it is necessary to develop a stable formulation. A disadvantage of the prior art is that the functional chemical part of the molecule is detached from the antibody in the liquid form of bismuth and is stored in the liquid form at 97600.doc 200533375. It is therefore an object of the present invention to provide novel formulations for antibody conjugates. [Summary of the Invention] This object has been achieved within the scope of the α-Hai patent and the specification and patent application scope. -The present invention relates to a freeze-dried pharmaceutical composition of antibodies. The stability of the product can be increased by adding a cyclodextrin and / or a low pH resistance which is preferably conjugated to the effector molecule; Specifically, the present invention relates to a stable freeze-dried formulation of an antibody complex and a method for preparing the same. [Embodiment] Before describing the preferred embodiment of the present invention, it should be pointed out that the use of the singular ("-⑷ "," the pottery, etc.) in the patent specification and the scope of patent application also includes the plural unless specifically Indicate the opposite or apparently singular in this article. Unless otherwise defined, all terms used have meanings known in the art. Accordingly, all publications and patent specifications mentioned herein are fully incorporated into this specification, and particularly into the content in question. The use of a comma (,) before the decimal place is equivalent to using an English font before the decimal place, and therefore (for example) 0.02 is equivalent to 0.002. The abbreviation% is equivalent to weight percentage. Freeze-dried formulations (= freeze-dried formulations, lyophilized formulations) are advantageous simple, practical and inexpensive preparation forms. 9 Liquid formulations of existing antibody conjugates have a life of only 12 weeks at a storage temperature of 2-8t :. Therefore, the present invention relates to a stable composition of an antibody-effector molecule conjugate 97600.doc 200533375 formulation and a preparation method of a body preparation-effect molecule conjugate. Reduces the formation of free effector molecules during product storage. Two people: startled, this has been achieved by removing water by cold-roll drying. The hunting of the earthy soil is achieved by using a specific formulation (4 w% mannitol, ... / 〇sucrose,: H: 6_5). This increased stability is also achieved by lowering the solution metaspar. -The pharmaceutical composition according to the present invention relates to an antibody conjugate and / or-a mixture of moonlight and / or at least one cyclodextrin, wherein the substance has been cold; dried in the east. Preferably, the medical treatment group 2 according to the present invention relates to a mixture of an antibody co-agent and at least one scaffolding agent, and the drug composition has been dried. Preferably, the mouth mouth material according to the present invention is about the mixing of an antibody co-body with at least one cyclodextrin ', wherein the pharmaceutical composition has been cold; and dried. According to the present invention, the composition preferably contains Tween (Tw is called 2G (corresponding to Poly ^ alcohol ⑽㈣ qing ⑽㈣ soft qing ㈣ ㈣) preferably every sound ::, the vehicle contains 0 · 01 + /_〇.〇1 weight percentage, which means that every i, antibody contains 〇, 〇.〇1 or 0,02 weight percent, particularly preferably each = fine antibody total vehicle contains 0.01 weight Percentage. In particular, it is 20 (() 〇1, 〇2, 〇03, 〇〇 = wide weight percentage) than m0l_0'05 weight percentage. The following uses a more comprehensive description of the Terminology and examples according to the present invention. As described in more detail below, or in combination with reducing the value of the solution 1) ^ 1, in a surprisingly advantageous way, more cyclodextrins or amphiphilic molecules are added to obtain more Stable freeze-dried formulations. 97600.doc 200533375, under / ι has proven to be particularly suitable for stable cold; eastern dry compounds: L lowers the pH to pH 5-6, preferably to 5 · 5, and / or. 2 · Adding a cyclodextrin at a concentration of 0.001 to about 40% by weight (w%), preferably 0.00% such as w%, particularly preferably 01%. , And / or 3. Use mannitol and sucrose as backbone materials, and "Go use a cleaner of Tween 20 (corresponding to polysorbate 20)" preferably every 1 mg / ml antibody co-consumers use 001 + / _ 〇〇1 weight percent, that is, use 0, 001 or 002 weight percent per 1 conjugate, particularly preferably 1 mg / ml antibody total The conjugate uses 0.001 weight percent teflon. Of course, it is also preferably 0.001 to 5% by weight of Tween 2 (0.01, 0.02, 0.03, 0.04, or 0.05 weight). Percentage). Formulations with low pH and / or cyclodextrin can surprisingly prevent detachment of conjugate effector molecules from antibodies. The cyclodextrin (CD) used according to the invention is a ring system consisting of six A cyclic oligosaccharide or cyclic polymer consisting of 1, 7, or 8 仏 1,4-linked glucose units, and is referred to as α, cardio, or > cyclodextrin depending on the number of monomers. Cyclodextrins are known Spermine forms an inclusion complex with different biomolecules (such as fatty acids or amino acids), or it can be encapsulated to the saturation point. The different cyclodextrin available mainly lies in its There are differences in homocyclic sizes (... cyclodextrin, heart cyclodextrin, γ-cyclodextrin). There are also differently substituted cyclodextrins, which have different toxicity and protein-cyclodextrin interaction characteristics. For example Suitable cyclodextrins include substituted heart cyclodextrins (consisting of a glucopyranose unit), hydroxypropylcyclodextrin (Ηρ «〇), 碏 butyl ether- / 3-¾ dextrin ( SBE- / 5-CD), γ-cyclodextrin (composed of 8 glucopyranosyl single 97600.doc -10- 200533375 yuan) and hydroxypropyl_γ-cyclodextrin (HPKD). The cyclodextrin of the present invention is a sulfoalkyl ether_cyclodextrin (SAE-CD) of the formula:

Where n = 4, 5 or 6; the groups Ri, R2, R3, r4, R5, R6, R ?, 8 and in each case each independently represent -0- or -〇- (C2_C6alkylene ) _S〇3 · group, wherein at least one of Ri and R_2 independently represents a _〇_ (C2_C6 alkylene) β03- group, which is preferably -0- (CH2) mS03- group, where m is 4 (For example, '_OCH2CH2CH2S〇3 or _〇cH2CH2CH2CH2s〇 $ and 1 2 S3 s4 S5, S6, s7, 88 and S9 in each case independently represent a pharmaceutically acceptable cation, the material cation contains ⑽ such as) H +, Metal test (such as Ll, Na 'κ +), earth test metal (such as ca ++, Mg ++), ammonium ions and amine cations, such as (ci_c6) carbamoylamine, bridle, roar, radon, tritium, and (C4 heart ). The cations of Cyclamine. In particular, the particularly preferred cyclodextrin is coffee dough small cyclodextrin (D), propyl cyclodextrin (HP CD) 1 propyl # is called cyclodextrin (HP- / 5-CD) or sulfonate Butyl ether + cyclodextrin (E + ⑶). In the very extensive literature that has been published so far concerning the preparation of cyclospermia (especially, / 5-cyclodextrin) 97600.doc -11-200533375 and its use, I will only mention it by way of example. The following publications: Manning, M. et al., P / zarm. (5, pp. 1903-1918, 1989; Yoshida, A. et al., # 7, pp. 217-222, 1988; Brewster, M. Et al., / This J. P / zarm 59, brother 231-243 '1980; Pitha, J. et al., / Η /. «/. PAarm. 2P, pp. 73-82, 1986; Pitha , J. and Pitha J., /. Corpse / zarm ···. 74, pp. 987-990, 1985; Brewster, M. et al., J. PareW. 5cz ·. 7 ^ / ^ /. 43, Pp. 231-240, 1978. In more detail,? Pool &. ?? J.zhi / oc.cz_i. (1986) describes hydroxypropyl-cardiocyclodextrin (Hp «D) And the use of HP- / 3-CD is reported to improve the water solubility of all kinds of active substances and biomacromolecules. For the use of cyclodextrin in the field of medicine, see the following general article: Pitha, j. Et al. C⑽ 〇 0 // by price De / z_vw [BmCk, SD · edit] Volume ，, CRC Publishing House, B〇ca Rat〇n,

Florida ’pp. 125-148, 1983; or Uekama, K. et al., CRC

Critical Reviews in Therapeutic Drug Carrier Sysiems, Volume 3, pages 1_40, 1987; or uekama, K., Tbph h pw then 1987 [Breimer, DD · and p • edit], Elsevier Science Publishers BV (Biomical mvlslon) ), P. 181] 94, 1987. In detail, such as She, ME, et al., Foot, J, pp. 792-795, 1991, described in more detail the use of 2-propyl I cyclodextrin for dissolving and stabilizing different biologically active proteins. It is particularly preferred that the dextrin and yang A < 4 >% dextrin according to the present invention is a cyclodextrin selected from the following table. 97600.doc • 12-200533375 Table 1 Cyclodextrin abbreviated group η ο cyclodextrin a-CD Η 4 0-¾ dextrin β-CO Η 5 7-cyclodextrin γ-CD Η 6 Dextrin CM- / 3-CD CH2C02H or Η 5 carboxyfluorenyl-ethyl- / 3-cyclodextrin CME-fCD CH2C02H, CH2CH3 or Η 5 diethyl- / 3-¾ dextrin DE- / 3-CD CH2CH3 or Η 5 difluorenyl- / 3-cyclodextrin DM- / 3-CD CH3 or Η 5 methyl- / 3-cyclodextrin M- / 3-CD CH3 or Η 5 random fluorenyl_] 8- Cyclodextrin RM- / 3-CD CH3 or Η 5 glucosyl-] 8-cyclodextrin GriS-CD glucosyl or Η 5 maltosyl- / 5-cyclodextrin G2-iS-CD maltosyl or Η 5 via ethyl- / 5-cyclodextrin HE- / 3-CD CH2CH2OH or hydrazone 5 hydroxypropyl cyclic cyclodextrin HP- / 5-CD CH2CHOHCH3 or hydrazone 5 succinylcyclodextrin SBE- / 3-CD (CH2) 4S03Na or Η 5 For example, a particularly preferred hydroxypropyl-γ-cyclopaste having a Mohr substitution content of 0.5 to 0.7 is produced by Messrs Wacker-Chemie GmbH, D-Burghausen under the name `` CAVASOL® W8 HP Pharma ". Surprisingly, formulations using cyclodextrin sulfobutyl ether cyclodextrin (SBE- / 5-CD) can also be used with Captisol (Mes srs CyDex, USA), which has proven to be particularly advantageous. The particularly preferred cyclodextrin is 0.001 to about 40 w% per volume, preferably 0.1-20 wt.%, particularly preferably 0.1- A weight percentage (wt.%) Of 10 wt.% Or 1-5 wt.% Is present in a preferred formulation according to the present invention. Particularly preferably, the cyclodextrin according to the present invention is about 1, 5 or It is present at a content of 15 wt.%. It is particularly preferred that the SBE-jS-CD is present at a content of about 1, 5 or 15 wt.%. The present invention relates to formulations of antibody conjugates. The term π antibody " Equivalent to "antibody molecule". Antibodies can be all immunoglobulins, which contain two heavy chains and 97600.doc -13-200533375 two light chains, fragments of these immunoglobulins (such as Fab, Fab 'or F (ab) 2 fragments), recombinant Sexually produced antibody molecules (such as chimeric, humanized or fully human antibodies). An antibody co-product means a complex of an antibody and an effector molecule. The effector is preferably linked to the antibody via a covalent bond. For example, 'the following refers to antibodies according to the present invention: HER2 antibodies, such as Herceptin (R) (trastuzumab, Genentech, Inc,); VEGF specific antibodies', such as Bevacizumab (Genentech, Inc ·) 'J Rituxan (rituximab), anti-EFGR antibody ABX-EGF; antibody aBX-CBL (Abgenix); antibody ICR-62 (ICR, Sutton, England Xolair MAb, omalizumab)), C242 (cantuzimab),

ImmunoGen), Erbitux (Cetuximab, IMC-C225, ImClone Systems); Monoclonal Antibody 425 (Merck KGaA) ·, Mitumomab (Imclone Systems and Merck KGaA); Antegren (integrin, natalizumab). The antibody conjugate according to the present invention can be prepared from one of the antibodies according to the present invention and an effector molecule, optionally combined with a linking molecule (so-called linker). Examples of effector molecules include: toxins such as the preferred maytansinoids mentioned below or DM1 mentioned by way of example; radioisotopes such as mI, nlIn, 99mTc; such as peroxidase or alkaline phosphatase Enzymes; fluorescent dyes or biotin molecules; cytostatin (adriamycin

(doxorubicin), such as Taxotere (Taxotere⑧) (EP 0 253 738, US 4,814,470), Taxane (1 &) 31 ^) of European paclitaxel ((100613 \ 61), daunorubicin ), Dactinomycin, dactinomycin, 97600.doc -14- 200533375 (plicamycin), mitomycin, bleomycin, idarubicin, cyclotoxin Cyclophosphamide, mechlorethamine, melphalan, chlorambucil, procarbazine, dacarbazine, hexamethylamine (altretamine), cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin, methamamine ( methotrexate, mercaptopurine, thioguanine, fludarabine, lactate, cladribine, pentostatin, fluorouracil ) (5-FU), cytarabine, azacytidine (Azacytidine)); prodrugs; cytokines; immunomodulating polypeptides such as tumor necrosis factor or interleukin-2. Examples of antibody conjugates according to the present invention that may be mentioned include: antibody cohort AS 1406 (antisoma); humanized antibody HMFG1, which is bound to the enzyme RNase; zevalin (replaced by Imbritumomab tiuxetan); bexxar (corixa, tositumomab 1-131); or the maytansinoid or DM1 common vehicle mentioned below . According to the invention, the concentration of the antibody conjugate complex is between 0.01 and 40 mg / ml, preferably between 0.1-20 mg / ml, and especially between 01-10 mg / ml. Another embodiment of the present invention relates to the complexing of an antibody co-prototype with a cyclodextrin and a cationic acid. The amount of cyclodextrin required can be reduced by forming a ternary complex consisting of an antibody conjugate, the cyclodextrin in question, and the hydroxyl 97600.doc -15-200533375 base acid. Suitable dextrin producing agents include, for example, HP | CD, sbe «d, 7_cd, and Hp. Therefore, in addition to the active substance and Rosindube and dextrin, it is intended to The pharmaceutical composition of the invention may contain amenic acid such as malic acid, lactic acid, tartaric acid: succinic acid or citric acid. Ten suitable matrix substances according to the present invention are carbohydrates and combinations thereof (such as mannitol and cane, trehalose, lactose and maltose) or amino acids (such as arginine, glycine) or such as HAS (human serum Albumin or bovine serum albumin itself. The weight ratio of mannitol to sucrose may be from 1 to 100 or 100 to 1, preferably from 60 to 60 to 1, especially from 4 to 1 or 1. To 4 w%. # According to the present invention, the molar ratio of the active substance to the cyclodextrin is between 1: 1 and 1: 330.00. Preferably, the molar ratio of 1: 100 to 1: 150.00 Ear ratio, especially from ^ ⑻ to 1.600. According to the present day and month, the molar ratio of active substance to cyclodextrin in the presence of M-based acid is as described above. For example, a is suitable to replace the ring according to the present invention. Amphiphilic substances for which dextrin is used in the medical music composition according to the present invention are the following: · Choline derivatives (C6 C20) 'Naturally occurring cholecysts (eggs and soybean egg scale fat), such as two Myristyl phospholipids choline (DMPC), dipalmityl phospholipids choline (DPPC), dioleyl phospholipids choline (DOPC); or ethanolamine derivatives (C6 to C2 0); Naturally occurring ethanolamines (eggs and soy ethanolamines), such as dimyristoyl phospholipid 醯 ethanolamine (DMPE), dipalmityl phospholipid 醯 ethanolamine (DPPE), dioleyl phospholipid 醯 ethanolamine ( DopE), where DopE is particularly good. In addition, the corresponding glycerol derivatives of saturated and unsaturated fatty acids 97600 doc 200533375 (C6-C20) or linoleic acid (C6-C20) can be used as further phospholipids The pharmaceutical composition according to the invention also optionally contains conventional excipients and backing agents, such as isotonic glucose, mannitol or sodium chloride or sodium acetate or sodium acetate trihydrate, which is associated with acetic acid or (for example) A citric acid / phosphate buffer consisting of citric acid and disodium hydrogen phosphate or disodium dihydrogen dihydrate is used as a buffering agent. The solvent is usually water for injection. The concentration of such excipients and carriers is familiar. Those skilled in the art are aware and can be found, for example, in Remington, s Pharmaceutical Sciences (1990), 18th edition, Mack Publ., Easton.

The following agents can be used to lower the pH (made acidic, depending on the initial pH) · HC1, H3P04 and other phosphate derivatives, HNO3, H2s〇4, CHsCOOH or all known for this purpose A pharmaceutically acceptable acid, or the value of 卩 11 can be adjusted by selecting a suitable buffer system selected from a buffer system based on the following: monobasic acid · acetic acid, benzoic acid, glucose Acids, glycerol, lactate; dibasic acids: aconitic acid, adipic acid, ascorbic acid, carbolic acid, glutamic acid, fruit-like acid, succinic acid, tartaric acid; polyacids: citric acid, phosphoric acid; or alkali: ammonia , Diethanolamine, glycine, triethanolamine, tromethamine and their salts. If it is necessary to raise the positive value because it is too low, a known suitable substance or a solution thereof, such as various solvents, KOH, ammonia solution, etc. may be used. Like other ingredients, such as additional stabilizers, the pharmaceutical composition may also contain salts or saline solutions, and may especially contain pharmaceutically acceptable salts, such as inorganic salts such as hydrochloride, sulfate, phosphate, diphosphate , Hydrobromic acid and / or stone salt. The liquid suspension may also contain organic salts, such as, for example, 97600.doc • 17- 200533375 salts, maleates, fumarate, tartrate, succinate, ethyl axate , Citrate, acetate, lactate, mesylate, benzoate, ascorbate, p-toluidine sulfonate, palmoate, salicylate, stearate , Estolate, gluceptate, or labionate. Examples of the matrix substance optionally contained in the pharmaceutical composition according to the present invention are amino acids, polymers, sugars or sugar alcohols. Therefore, a certain composition according to the present invention may contain different amino acids such as arginine and glycine. Other suitable amino acids are known to those skilled in the art. Examples of the polymer optionally contained in the pharmaceutical composition according to the present invention include: $ acetylene slightly. Ding_; Derived cellulose, such as methyl, ethyl, or propyl ethyl cellulose; polymerized sugars , Such as polysucrose (£ 1 state or dextran; Dian powder, such as ethyl ethyl propyl starch); dextrin, such as cyclodextrin (2. Polyethylene glycol; Polyethylene glycol; Polyglucosamine; Collagen; Hyaluronic acid; Polypropylene; Polyvinyl alcohol and / or pectinic acid. Examples include soap sugars, disaccharides, oligosaccharides or polysaccharides., Is 釈 P ^ times, and, and examples of early sugar are preferably one, ..., sugars and their derivatives and optical isomers, fructose, Almond, galactose, glucose, D_mannose, sorbose, d (; D (+) _ mannose, D (+)-galactose m, Tianmendian sugar analogues. () + Engage, D (· ) · Fructose, exo) _ Examples of sorbose and its double enzymes are lactose, sucrose, and the like. 533375 Examples of suitable polysaccharides include, in particular, raffinose, melezitose, dextrin, powder, and the like. According to the medical practice of the present invention * and the human beings-the sugar alcohols in the mouth may optionally contain Examples include the addition of mannitol, mannitol, xylitol, maltitol, galactitol, arabitol, ribitol, lactitol, sorbitol (glucosyl alcohol), piperanyl sorbitol, cellulosic alcohol (In〇sit〇1), inositol (myoinositol) and the like. / Like other ingredients, the pharmaceutical composition may also contain cleanliness, which is preferably selected from a concentration of 0.01-5 weight percent, more than The Tween is preferably between 0.000 weight and 100 weight ratios, and particularly preferably between 0.001 and 0.05 weight percentage (00, 0.02, 0.03, 0.04, or 0.05 weight percentage). Tween 60, Tween 80 (polysorbate 20, 60, 80, poloxamer (Pluronic). In a preferred pharmaceutical composition, the concentration of the detergent 0 01 + / _ 〇〇1 weight percentage per 1 mg / ml antibody conjugate (meaning, 0, 001 or 002 weight percent), particularly preferably 0.001 weight percent per 1 mg / rnl antibody conjugate. It has also been proven that the weight percent used) is particularly preferred. Medicine according to the invention The composition is useful in the context of the present invention for parenteral systemic (eg, intravenous, intravascular, intramuscular, intraarterial, intraperitoneal, or intralesional) muscarinics. A more topical application of a pharmaceutical composition according to the present invention may be Achieved via subcutaneous, intradermal, intracardiac, intralobular, intramedullary or intrapulmonary pathways or by direct access to or access to the organ to be treated (connective tissue, bone pathways, muscle, nerve or epithelial tissue). 97600.doc -19- 200533375 The pharmaceutical preparations according to the present invention can be used in a variety of clinical or non-clinical applications, where toxic compounds will be directed to CD4 cells. In another embodiment, the present invention relates to a method for treating cancer by administering a therapeutically effective amount of a pharmaceutical preparation according to the present invention to a patient in need of treatment. &Quot; Hei cancer is preferably a culture plate epithelial cancer ( For example, "Head and Neck Squamous Cell Carcinoma (scc)"), esophagus scc, lung-like, skin-seeking, breast cancer, lung adenocarcinoma, uterine cancer c, adenoma, colon adenocarcinoma, gastric adenocarcinoma). In the cancer treatment of cancer, the pharmaceutical preparations according to the present invention are, for example, administered according to the following treatment regimens, such as intravenous bolus administration, once a week for the mother, for 1 to 6 weeks or 5 consecutive times Day infusion. The bolus dose can be administered in 50 to 100 ml of physiological saline to which 5 to 10 ml of human serum albumin is added. Continuous infusion can be administered in 25 to 50 ml of physiological saline in which 25 to 50 ml of human serum albumin is added every 24 barks. The dosage of the antibody conjugate is generally 10 mg to 400 mg / m2 body surface area per application. On the one hand, the dose must be high enough to be effective, but it must also be lower than the so-called "dose limit toxicity" (DLT). The maximum tolerable dose below DLT is the "maximum tolerated dose" (MTD). Those skilled in the art know how to determine MTD (Lambert et al., 1998). For example, for weekly dosing, the MTD can be between 100 and 200 mg / m2. Alternatively, the time interval between administrations may be longer, for example 2 to 4 weeks, preferably 3 weeks. In this case, the desired MTD is between 200 and 300 mg / m2. Alternatively, it may be administered in divided doses of 5 times per day, followed by intervals of several weeks. Next, the expected MTD is less than 100 mg / m2. For example, the medicine according to the invention 97600.doc -20-200533375 can be administered as a single intravenous (IV) infusion at a rate of 3 mg / min every 21 days. Given up to 7 treatment cycles. If clinical conditions require, the range used can vary outside a certain range. For example, if MTD has been found to be higher than a given value ', a single dose may also be higher than 400 mg / m2 or a weekly dose may be higher than 200 mg / m2. Examples of parenteral compositions of the antibody co-products according to the present invention contain 1 mg / kg body weight to 40 mg / kg body weight, preferably 3-15 mg / kg in mother tongue / biological shellfish. Weight. The following doses are also better, expressed as [mg / m2]: 10 [mg / m2] to 200 [mg / m2]; more preferably 20s100 [mg / m2]; most preferably 10, 20, 40 Or 50 mg / m2. In a preferred embodiment, the formulation is administered by continuous infusion of 0.5 to 24 hours or possibly several days to maintain a stable plasma level. The volume used is between 50 and 500 ml, preferably between 100 and 250 ml, meaning that the concentration of the active substance used for administration is between 80 mg / 500 ml ==. 16 mg / ml (0.015% mi500 mg / 100ml = 15mg / ml (15%). The concentration of the active substance is preferably 0.5 mg / mi = 0.050 / 0 ( g / v) to 35 mg / ml = 0.35% (g / v). Therefore, in a preferred embodiment, the present invention relates to a formulation or a pharmaceutical composition according to the present invention for preparing a medicine for treating cancer. (The definition is given above). The key point of the present invention consists of a formulation or pharmaceutical composition of an antibody conjugate composed of antibodies coupled to maytansinoids. The antibody_DM1 conjugate is especially Best. An example of an antibody-DM1 conjugate according to the present invention is an antibody conjugate MLN2704 composed of a single antibody vehicle T-MAV, which specifically binds to a prostate-specific membrane protein 97600.doc -21-200533375 , This protein line maytansinum DM1, C242 / CanAg-specific humanized monoclonal antibody-DM1 conjugate cantuzumab_DMl (Liu et al., 1996; Lambert et al., 1998), Humanized monoclonal antibody-DM1 conjugate destined for CD56 antigen f'lmN901-DMln (Chari et al., 2000), humanized monoclonal antibody-DM1 conjugate specifically for CD33 antigen My9-6- DM1 (Aventis), humanized monoclonal antibody-DM1 conjugate (MorphoSys / ImmunoGen) specific for IGF-1 receptor "anti-IGF1 hu MAbs", humanized monoclonal antibody with target molecule EGFR-DM1 Antibiotic EGFRvIII-DMl (Abgenix) conjugate. Particularly preferred is a pharmaceutical composition of an antibody conjugate of the formula: A (LB) n where A is an antibody molecule; L is a linker substance; B is toxic to cells And η is a decimal number, η = 1 to 10. Also particularly preferred is a pharmaceutical composition of the above formula, wherein the linker substance contains a chemical bond that can be decomposed in a cell. A pharmaceutical of the above formula is also particularly preferred A composition in which the chemical bond is a disulfide bond. Advantageously, the antibody-DM1 conjugate can be formulated in one of the above manners by the above-mentioned excipients, etc. Antireceptor A has about CD44, preferably a variant CD44 Binding specificity. "Specific for ⑽4" means that the antibody is specific in the CD44 region 97600.doc -22- 200533375 bound to the epitopes. Binds to an amino acid sequence variant of the CD44 gene encoding human body bad J Xi Bu Xianzi antibody according to the present invention the preferred system. The preferred antibody molecule specifically binds to the sequence SEQ ID NO: 1 containing the attached sequence listing or a dual gene variant of the sequence or a peptide consisting thereof. Particularly preferably, the antibody molecule according to the invention specifically binds to the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3. Such antibodies can be produced by methods known in the prior art (WO 95/33771 > WO 97/21104), for example, by the immune action of a laboratory animal having a chemically synthesized peptide having the above-mentioned sequence to produce such antibodies.

Preferred antibodies are fusion tumor cells deposited (deposited on June 7, 1994, accession DSM ACC2174 5 DSM-Deutsche Sammlung fur Mikroorganismen and Zellkulturen GmbH 5 Mascheroder Weg lb, D-38 124 Braunschweig, Germany) [ Original text] Murine monoclonal antibody VFF-1 8. Also preferred are humanized recombinant antibodies, in which the so-called VFF-18 complementarity determining region (CDR) has been applied to the corresponding regions of the light and heavy chains of the human immunoglobulin gene. The definition of the complementary decision area is similar to that of Kabat et al. (1991) and Chothia and Lesk (1987). Most preferably, the antibody A is an antibody containing the light chain of the amino acid sequence SEQ ID NO: 4 and the heavy chain of the amino acid sequence SEQ ID NO: 6. This resistance system is called BIWA4 and is a humanized variant of VFF-18. It is also particularly preferred that the antibody A is an antibody containing the light chain of the amino acid sequence SEQ ID NO: 8 and the heavy chain of the amino acid sequence SEQ ID NO: 6. This resistance system is called BIWA8 and is a humanized variant of VFF-18. The antibody according to the present invention may be similar to WO02 / 094879 A1 and stomach 〇02 / 094325 Λ2, respectively, by the nucleotide sequence SEQ 4 SEQ ID NO: 5 and SEQ ID 97600.doc -23-200533375 NO: 7 (BIWA4) and SEQ ID NO: 9 and SEQ ID NO: 7 (BIWA8) were generated. WO02 / 094325 A2 also shows in detail the binding to a toxin according to the present invention, and its entirety is incorporated into this application by reference. It is "toxic" if it inhibits or completely obstructs cell function and / or causes cell destruction. Toxic substances can act as ligands with regard to cytostatic or cytotoxicity and cause cell cycle arrest or cell death. These substances can interfere with the cell cycle to varying degrees by interacting with nucleic acid synthesis, inactivating nucleic acids, or by binding to microtubule proteins. The toxic compound B is preferably maytansine, meaning a maytansinoid (CAS 3 5 8465 3 8). B is preferably a C-3 ester of maytansinol. Antibodies maytansinoids have been described (Chari et al., 1992; Liu et al., 1996; U.S. Patent No. 5,208,020). Such maytansinoids can be used in the present invention. The toxic compound B is preferably N2'-desethylfluorenyl-N2 '-(3-fluorenyl-1-oxopropyl) -maytansine (CAS number: 139504-50-0), also referred to as DM1. Preferably, the maytansinoid is a maytansinol derivative that is coupled to the antibody via a disulfide bridge at the C-3 position of maytansinol. Particularly preferably, the antibody / maytansin conjugate according to the present invention is prepared from maytansinoid of the formula: 97600.doc 24-200533375 formula (II) QH, 〇

(CH2) mSR1 R! Represents fluorene or SR4 'wherein R4 represents a chain alkyl group, a ring group, a single or silk group, a linear alkyl group, a branched square group, or a heterocyclic ring; R2 represents C1 or fluorene; R3 represents Η or CH3; and m represents 1, 2 or 3.

Ri is preferably Η, CH3 or SCH3, and R ^ c and R ^ CH4m = 2. In the literature, the compounds in which UK is r3 = CH3 and m = 2 are called DM1. In a preferred embodiment, the compound according to the invention has sin 97600.doc -25- 200533375

CH0 〇 ㈧ CH. (Formula III) wherein A is an antibody molecule specifically for CD44, preferably specifically for variant exon v6, and preferably for the amino acid sequence SEQ ID NO: 3; (Lf) is Linker molecules as appropriate; P is a decimal number, where p = 1 to 10. Preferably, p = 3 to 4, and particularly preferably about 3.5. Methods for preparing such maytansinoids are known in the art (detailed US 5,208,020, Example 1; WO02 / 094325A2). In a preferred embodiment, compounds of formula (I) are less toxic than compounds B, BX or BL "-X released after intracellular breakdown. Antibodies / maytansin conjugates linked by a disulfide bond It is preferred that it releases maytansinoid molecules. Such cell-bound co-cells are prepared by methods described in the prior art (US 5,208,020; WO02 / 094325A2). In another aspect, the conjugate It is composed of CD44v6 specific antibody molecule and maytansinoid. "CD44v6 specific" means that the antibody has an amine group encoded by exon v6 with a variant of 97600.doc -26- 200533375 CD44 (preferably human CD44). The [sic] epitope in the acid sequence peptide has a specific binding affinity. A preferred antibody molecule of the present invention specifically binds to a peptide or polypeptide comprising or exactly having the amino acid sequence SEQ ID NO: 1 or a dual gene variant of that sequence. The antibody molecule preferably specifically binds to an antigen-determining site in the sequence. The antibody molecule according to the present invention is particularly preferably specifically bound to the epitope of the amino acid sequence SEQ ID NO: 2 or SEQ ID NO: 3. The antibody molecule according to the present invention is preferably a monoclonal antibody VFF-18 (DSM ACC2174) or a recombinant antibody having a complementary binding region (CDR) of VFF-18 in the conjugate. Particularly preferably, the antibody molecule according to the present invention contains the light chain of the amino acid sequence SEQIDNO: 4 and the heavy chain of the amino acid sequence SEQIDNO: 6. It is also particularly preferred that the antibody molecule according to the present invention contains a light chain of the amino acid sequence SEQ ID NO: 8 and a heavy chain of the amino acid sequence SEQ ID NO: 6. The maytansinoid is preferably coupled to the antibody via a disulfide bond and has the following formula:

The formula (IV) 97600.doc 200533375 wherein the connection to the antibody via a sulfur atom in formula ιν is affected by another sulfur atom in the antibody molecule. As described in WO02 / 094325A2, in order to obtain such a sulfur atom for binding in an antibody molecule, the antibody molecule may be modified by a suitable linker. The maytansinoid is preferably bound to the antibody molecule by an S-CH2CH2-CO-, -S-CH2CH2CH2CH2-CO-, or -S-CH (CH3) CH2CH2-CO- group. The sulfur atom in such a linker group forms a disulfide bond with maytansine, however, the carbonyl functional group may be bonded to an amine function which may be present in the amino acid side chain. Several maytansinoid groups can be bound to antibody molecules in this manner. Preferably 3 to 4 binding maytansinoid groups are present in each antibody molecule. The most preferred is a conjugate of a CD44V6 specific antibody molecule and maytansinin, wherein the antibody molecule contains the light chain of the amino acid sequence SEQ ID NO: 4 and the heavy chain of the amino acid sequence SEQ ID NO: 6, and wherein Maytan has the following formula:

Formula (IV) is coupled to the antibody via a disulfide bond. 97600.doc -28- 200533375 Preferably, the linker group is _S CH2CH2CH2CH2_c0_ or S-CH (CH3) CH2CH2-CO- and the number of maytansinoid groups per antibody molecule is 3 to 4 . The preferred purpose of the present invention is to reduce the chemical (preferably hydrolytic) decomposition of maytansinoids or antibody-maytansinoids or matine 1 complexes, and thereby avoid free maytansinoids or DM 1. Therefore, various physical and chemical conditions must be made feasible to ensure high storage stability of the antibody-DM1 complex. This can be achieved by providing lyophilized formulations, using new I-member excipients, and developing a tumble-dried antibody _ maytansinol or _DM1 complex. In the field of 7 people, a lyophilized formulation or a pharmaceutical composition according to the present invention can be advantageously prepared by a method using a steam-permeable packet (for example, Medipee can also refer to examples). The preferred method of producing stable antibody-maytansin or heptaM1 complexes results in reduced formation of free maytansinol or Dmi and its derivatives during storage of the product. This can be achieved by freeze-drying to remove water and use This is achieved with selected excipients. Also, should it be lowered? 11 value. Alternative solution? A decrease in the value of 11 or a combination thereof results in a surprisingly advantageous way by adding one or more of the above cyclodextrins or amphiphilic molecules as detailed above to obtain more stable freeze-dried formulations. The preferred cyclodextrin is in particular 7-cyclodextrin (yCD), transpropyl decacyclodextrin (HP-γ-CD), hydroxypropyl · cyclodextrin (Hp «D) or sulfobutane Ether ether cyclodextrin (SBE- / 3-CD). The use of cyclodextrin-capitasol (αΐη1δ01) (δΒΕ «Ο: sulfobutyl ether_excess_ 97600.doc -29- 200533375 cyclodextrin) (〇, 〇〇1 to 40.69 W%) has been proven Especially advantageous. The blending of border temples in the formulation is also important for the addition steps before and during lyophilization. Surprisingly, compared to the reference formulation (liquid form,. ^), The corresponding combination of the above methods has the potential to prolong the effective life of the lyophilized formulation (stored under 2_8.) By 6 to 8 times (see experimental section) . The following steps have proven to be particularly suitable for stabilizing compounds in the cold-rolled dry state: 1. Reduce _ to pH 5-6, preferably to 55, particularly preferably with the aid of a sodium succinate buffer (such as succinic acid hexahydrate) Disodium), and / or 2. added cyclodextrin, the concentration of which is from 0.1 to 40% by weight, preferably from 0.1 to 20% by weight, particularly preferably from 0 to 0% by weight, and / or 3. Mannitol and sucrose are used as backbone materials, and / or, the main = use a cleaning agent preferably Tween 20 (corresponding to polysorbate ester), preferably 1 mg / ml antibody total The conjugate uses 〇001 + / _ 〇〇1 weight 100 μ, that is, the mother 1 mg / ml antibody, a total weight of 〇, 001, or 002 weight 100 knife ratio, particularly preferably per 1 mg / ml antibody The conjugate uses a Tween of 0.001 weight percent and particularly preferably a weight percentage of 0.001 to 0.05 weight percent (e.g., OM, 0.02, 0.003, 0.004, or 0.05 weight percent). According to a preferred embodiment, the concentration of the antibody conjugate complex is between 0.01 and 40 mg / mi, preferably between 〇1_20 mg / ml and 405_i0g ml, especially between between. This concentration is particularly preferably 2 3, 4, 5, 6, 7, 8, 9, or 1011 ^ / 1111 (each case corresponds to g / 1). ~ The preferred implementation of such formulations is the same as the other formulations mentioned above 97600.doc -30- 200533375. Examples also preferably contain other ingredients cleaners, which are preferably selected from the concentration of 0.001 to 5 weight percent Time, preferably in an amount of 0 〇1_〇1 (〇〇〇〇002, 〇 03, 〇 04, H 〇 〇 6 '0, 07, 08, 〇 09, 〇__ amount Tween 20, Tween 60, Tween 60, and T.L. 60, especially weight percent (0.01, 0.02, 0.03, 0.04, or 0.05 weight percent). Tween 80 (polysorbate 0680, poloxamer (Pluromc)). The amount of the cleaning agent is also preferably-antibody co- The conjugate is 0.01 +/- 0.01 weight percent, which means that the total amount of the antibody per antibody is 0, 0.001, or 0.02 weight percent, and it is particularly preferably 0.01 weight percent per 1 mg / ml antibody conjugate. Formulations with low pH and / or cyclodextrin can surprisingly prevent the decomposition of the conjugate effector molecule from the antibody. A Compared to the reference compound (liquid form), the method described above The effective life of the lyophilized formulation (stored under 2_8) should be extended by 6 to 8 times in an advantageous way (7 months to 7 months) (refer to the experimental part). According to another embodiment, the present invention relates to a The method of freeze-drying the antibody-D Μ1 complex compound in the formulation is based on the combination of the following two stabilization methods mentioned above; κ present · lower the pH value and make Cape Trisox exists in the formulation. According to the present invention, there are methods that can prevent the decomposition of DM1 from the antibodies in the freeze-dried formulation. Features of the method according to the present invention Highly stable heart for the antibody-DM1 complex | Wang's effective dance life at 2-8 C is 18 to 2 ^ months. Freeze-dried preparations are made with water (WFI, injection field ^^ water for injection) or saline solution After reconstitution, a liquid formulation for non-fine eye m ^, bismuth, preferably intravenous administration, 97600.doc • 31- 200533375 was obtained. Surprisingly, it has been found that the decrease in pH and And / or / and the presence of kaizen ladder will stabilize the cold-dried formulation of the antibody-DM 1 complex According to a preferred embodiment, the concentration of the antibody-DM1 complex is between 0.001 and 40 mg / m, preferably between 0.1 and 10 mg / ml. This concentration is particularly preferably i, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / ml. The following steps have proven to be particularly suitable for compounds that stabilize the freeze-dried state: 1. Set the pH value Reduce to pH 5-6 ', preferably to 5,5, and / or 2. Add capecide (0.01 to about 40w%), preferably at a concentration of 01-20w%, especially It is preferably from 0 to 10 w%, and / or 3. using mannitol and sucrose as a skeleton substance, and the weight ratio of mannitol and sucrose is preferably 4: 1 w / w. As with the other formulations mentioned above, the preferred embodiments of such formulations preferably also contain other ingredient cleaners, which are preferably selected from concentrations ranging from 0.001 to 5 weight percent, preferably from 0.0} _0 "(0. (Π, 0.002, 0.03, 0.04, 0.005, 0.06, 0.07, 0.08, 0.09, 0.1)), particularly preferably 0.01-0.05 weight percent Tween 20, Tween 60, Tween 80 (0.01, 0.02, 0.03, 0.04, or 0.05 weight percent) (polysorbate 20, 60, 80, poloxacin (Poronic)). The amount of the cleaning agent is also preferably 0.001 +/- 0 〇1 weight percent per 1 mg / ml antibody conjugate, which means that the antibody conjugate per 1 mg / ml. , 〇 丨 or 〇002 weight percent, particularly preferably 0.01 weight percent per 1 mg / ml antibody conjugate. 97600.doc 200533375 A particularly preferred formulation is a formulation, which:- It is formulated by lowering the pH to pH 5-6 (preferably 5.5),-a succinate buffer containing preferably disodium succinate hexahydrate, which has a pH of 10.0. 〇mmol / L; or Phosphate buffer containing 1-1.9 mM NaH2P〇4 X H20, 4-5 mM KH2P04, 3.5-4.5 mM Na2HP04, and / or-containing 4: 1 w / w mannitol and sucrose , And / or-contain Tween 20 in an amount of 0.001, 0.002, 0.03, 0.004 or 0.005 weight percent or 0.005 per 1 mg / ml antibody conjugate. 〇ι + / _ 〇 · 〇ι weight percentage of Tween 20, which means that every 1 mg / ml antibody totals 〇, 〇〇〇ι or 〇 02 weight percent, especially preferably every 1 mg / ml antibody co-products 丨 weight percent 'and / or- together with one of the above-mentioned antibody conjugates, an antibody co-product that is specifically coupled to a CD44v6 specific antibody to maytansinoid DM1 as described above is particularly preferred. Contains one of phosphate buffer, table salt, Tween 20, 4: iw / w of mannitol, sucrose, and the above antibody, especially preferably maytansin coupled to the above-mentioned Antibodies for CD44v6 specific antibodies to DM 1 | Euregen. Particularly preferred formulations contain a carbamate buffer containing 1 _ 1.9 mM NaH2P〇4 x H20-4-5 mM KH2P04-3.5-4.5 mM Na2HP04, also Contains 4: 1 w / w of mannitol and vegetable _, 3__loo mM NaC, 〇.〇 ·· 0.05 wt.% Tween 20 (0.01 +/- 0.01 per 1 mg / ml antibody conjugate Tween 20 in weight percent means 0, 0.01, or 0.02 weight percent per 1 mg / ml antibody coche, and particularly preferably 1 97600.doc -33-200533375 mg / ml antibody conjugate. (Π weight percent) and antibody conjugates, preferably containing the antibody-DM1 conjugate, particularly preferably an antibody conjugate containing a CD44v6 specific antibody coupled to maytansinoid DM1 as described above, at a concentration of卩 115-6 is 1-5 11 ^ / 1111. Especially preferred? 115.5. Another preferred formulation contains the following: a phosphate buffer containing about 45 mM NaH2P04 x H20, about 4.19 mM KH2P04, and about 3 91 mM Na2HP04; 4: 1 w / w mannose Alcohol and sucrose; and about 60 mM NaCl; and about 0.02 w% Tween 20 or 0.01 w% Tween 20 per mg / ml antibody conjugate; and antibody conjugate, preferably Is an antibody-DMi conjugate, particularly preferably an antibody co-conjugate coupled to the cD44v6 specific antibody of maytansinoid Dmi as described above, whose concentration is 1-2.5 mg / ml, preferably 1.8 mg at pH 6.5 / ml. Another preferred formulation contains 10% "succinate buffer (such as sodium succinate buffer), 2-5 mg / ml antibody conjugate, and 4: 1 w / w mannitol However, the antibody conjugate is preferably antibody-DM] [conjugate, particularly preferably a cd44v6 specific anti-version antibody coupled to maytansinoid DM1 as described above. This is particularly preferred Other optional ingredients of the pharmaceutical preparation are: NaC1, which is NaCl at a concentration between 30-100 mM, preferably 60 mM NaCl 'and / or Tween 20, which is at a concentration of 0.001-0.05 vomit between w% The temperature is 20, and the amount of Tween 20 is preferably 0.001, 0.002, 0.003, 0.004, or 0.05 weight percent or 0.001 per 1 mg / ml antibody conjugate. -〇1 weight percent Tween 20 'meaning that the antibody conjugate is 0, 001, or 002 weight percent per 1 mg / ml of antibody conjugate' is particularly preferably 0.001 per 1 mg / ml antibody conjugate. Percent by weight. The pH value is adjusted to 5-6, especially preferably 5.5. 97600.doc -34- 200533375 The following examples are used to illustrate the present invention in more detail, without limiting the scope of the invention in any way. Illustrative EXAMPLES The following examples are used to illustrate the purpose and method according to the present invention. Preparation of liquid formulations (as control formulations) and freeze-dried formulations. Equipment and methods: Chemicals: The excipients used are in accordance with the medical license Excipient requirements. Table 2 shows the excipients used. WFI (water for injection) was purchased from Boehringer Ingelheim, Biberach, DE 〇 Table 2 Material supplier molecular weight (g / mol) KH2P〇4 BTP BI 136.09 NaH2P04 x 2H2OBTP BI 156.0 Na2HP04 x 2H2OBTP BI 177.99 NaCl BTP BI 58.44 Manna Sugar alcohol BTP BI 182.17 Sucrose BTP BI 342.3 Orthophosphate 85% Fluka HP- / 3-CD BI (DE) 1521.0 HP-γ-CD Wacker (DE) 1297.0 T-CD Wacker (DE) 1576.0 Cape Trisso (SBE -/ 3-CD) CyDex (USA) 2163.0 HP: hydroxy-propyl SBE: sulfobutyl ether CD: cyclodextrin All cyclodextrin used was purchased (see Table 1).

The antibody-DM1 complex used is a co-roller of the CD44v6 specific antibody molecule and maytansinoid DM 1, wherein the antibody molecule contains the light chain and amino group of the amino acid sequence of SEQ ID 97600.doc -35-200533375 NO: 4 The heavy chain of the acid sequence SEQ ID NO: 6, and wherein the maytansine has the formula:

? h3 〇 〇 3 Η C

S- Formula (IV) and is coupled to the antibody via a disulfide bond. A. Preparation of antibody-DM1 complex studies Preparation of antibody-DM1 preparation: To prepare a 1.8 mg / ml antibody-DM1 solution, first (by standard methods) buffer the starting solution to the solution to be tested recently, then Add or add corresponding excipients. The following examples describe the precise composition of these formulations. Analytical measurement methods:-Measure pH, permeability, appearance, color and transparency according to the universal PharmEu and USP methods. -HP-SEC (High Performance Exclusion Chromatography). HP-SEC was used to determine the monomer content in the solution. T2 was moderately dissociated from the SEC column and the monomer peak was calculated by the integration method. 97600.doc -36- 200533375

-RP-HPLC (reverse phase high performance liquid chromatography): RP-HPLC was used to determine the content of free DM1. The analytical method is based on a combination of HPLC and a '' shielded ff reverse phase column, which makes it possible to identify small chemical molecules that can be absorbed by UV in the presence of protein molecules. This combined method combines the advantages of size exclusion chromatography with reversed-phase chromatography. Macromolecules cannot penetrate the porous column matrix and as a result, macromolecules cannot bind to the hydrophobic interior of the matrix. On the other hand, small molecules can enter the column matrix and bind to the hydrophobic interior. An antibody-DM1 complex (' large ') solution was applied to the column in the presence of free DM1 ("small"). Only free DM1 can bind and elute the antibody-DM1 complex. DM1 and the corresponding ansamitocine derivative were dissolved from the column by means of an acetonitrile gradient. Major Encapsulations for Stress Stability Studies

Vial: 20/25 ml standard vial (20R), colorless, GA1 Inj. (Messrs Schott? DE) Terminator: Gusto WS 579, W 1 888, grey, Teflon (Messrs West, USA) flange Cap: Kombika / Alu-KU (Messrs West, DE) Freeze-dried formulation: Vial: 20/25 ml standard vial (20R), colorless, GA1 Inj. (Messrs Schott, DE) Terminator: Gusto WS 1797 PH 4023 / 50, gray, coated (Messrs West, USA) 97600.doc -37- 200533375 Flange cover Kombika / Alu-KU (Messrs West, DE) Conducted a stress stability study to sterilize the different formulations to be tested (22 μm, Mα.

Mmipore) was sterile filtered and transferred to a vial. The volume content of the liquid formulation was 20 ml, and the freeze-dried formulation was 0 ml. The 10 ml lyophilized formulation in the vial was then lyophilized. Use a terminator and a flanged cap to seal the vial and store it upside down. The storage temperature for the stress stability study was 40 ° C. 〇. The samples were taken after 0, 4 and 8 weeks. Example 1 · Effect of pH and beam drying on the stability of Zhao-DM1 complex Protein concentration (antibody-DM 1 complex) is 1.8 mg / ml for all liquid formulations and for lyophilized formulations It is 4.65 mg / ml. Therefore, the protein $ in each vial is the same. 1.8 mg / ml < 20 1111 = 36 mg / vial (liquid formulation), 4.65 mg / ml x 10 ml = 46.5 mg / vial (lyophilized formulation ). The control formulation (Table 3) consisted of the following excipients. Table 3: Substance component protein concentration: 1.8mg / mL KH2P〇4 4.19 mM NaH2P〇4 x H2〇1.45 mM Na2HP〇4 3.91 mM NaCl 139.6 mM Tween 20 0.02 w% pH____ 6.5 The pH was adjusted to pH 6 · 0 and 5 · 5 and the reaction was started with the control formulation (Table 3) and 1 N phosphoric acid.

Transfer 20 ml of each formulation to a 20 & vial and store these vials upside down at 40 ° C 97600.doc 200533375. The frozen formulation consists of (Table 4). Table 4:-Component 3 White matter ~~~ '-Concentration: 4.65 mg / mL KH2P04 4.19 mM NaH2P04 X H20 1.45 mM ^ a2HP〇4 --- 3.91 mM Mannitol 4w%' — 1 w% Tween 20 — 0.02 w% ~ ρϊϊ-6.5 Figures 1 and 5 show the results of the stress study. Table 5: Time pH 5, 5 pH 6, 0 pH 6, 5 weeks HP-SEC 1 body [%] ACC pH HP-SEC monomer [%] ACC pH HP-SEC monomer [%] ACC pH ^ 0 94 1,3 93 1.2 5.9 92-L6 _ 6.3 4 84 2,1 5.3 84 2.0 5.9 82 2.6 6.4 8 87 1,4 Γ5.4 85 1 2.8 5.8 81 5.2 6.4 Figure 1 shows the difference in free DM 1 content. It shows the difference between the free dmI value between 0 and 4 weeks and the difference between 0 value and $ week. The evaluation of the DM1 content is described by adding up the measured areas of different DM1 molecules for each sampling time. This results in the sum of the area of DM1 derivatives in individual samples. The graph shows that for each formulation, the areas between 0 and 4 weeks (bright) increased (the increase in free maytansin in the corresponding formulation) and those between 0 and 8 weeks The dark (dark) area has also increased. Surprisingly, it has been found that pH has a major effect on the stability of the antibody_DM1 complex. Figure 2 clearly shows the effect of pH. in? ] 9 [5.5 Free in the formulation 97600.doc -39- 200533375 DM1 content is 2 to 3 times lower than that in the pH 6.5 formulation. More surprising results were found when studying the DM] data for tumble dry formulations. It can clearly confirm that the cold green dry method significantly stabilizes the stability of the antibody-coffin. This method was developed as cold; East drying method and mixing ㈣ to produce stable products. The monomer content in the pH 5.5 formulation is 2-6% higher than the monomer content in the pH 6.5 formulation. Low ρΗ values also have a beneficial effect on the Acc (Appearance, Color, Transparency) parameters (see Table 4). " After 8 weeks (stored under thief), the monomer content is reduced by 12% ⑽ 6.5 formulation). Correspondingly, Donggan formulations did not show low monomer content. In summary, it can be said that the pH of the stomach from 5.5 to 5.5 has a positive effect on the stability of the antibody-then complex. Compared with the formulation at pH 6 5, its effective life can be extended by 2-3 times when stored under 2_ generation. However, hunting achieved significant stabilization effects by removing water by freeze-drying. The stress stability data of the freeze-dried formulations (Table 4) provided above clearly show the effect of freeze-drying on the DM1 value and the stability of the unchanged monomer content. The results of the research on combined liquid formulations can be expected to have other beneficial effects' meaning that the pH value of the lyophilized formulation is reduced to pH 5.5. Example 2: The effect of cyclodextrin on the stability of the antibody-DM1 complex In one test, the effect of cyclodextrin on the stability of the antibody complex was evaluated in a stress study. Four different cyclodextrins were tested: 97600.doc -40- 200533375 • hydroxy-propyl- / 5 • Cyclodextrin (HP_ / 5_CD) • Hydroxy-propyl-γ-cyclodextrin (HP-γ-CD) • γ-cyclodextrin (γ-CD) • Sulfobutyl cyclodextrin (SBE- / 3 -CD, Cape Tissole) The basic formulation is composed of the following excipients (Table 6). Table 6 ··

Substance White matter ~ ~ ΚΗ2Ρ〇4 rNaH2P04 x H20 ^ a2HPQ4 ~ NaCl keeps the pH constant at pH 6.5 in all CD formulations. The cyclodextrin content varies between 1 and 15 wt.% (Table 7). Table 7: ------------------------------- pH = 6. ^ I A1 K ^ Ay; D w% I ΪΡ -γ-C MB),: D w% 7-CD (from C) w% sa BE- / 3-CD t to D) w% A5 A15 B1 B5 B15 Cl C5 • k D1 D5 D15 L 1— 5 15 1 5 15 1 5 k 1 5 15 Not feasible due to solubility limitations. According to the basic formulations in Table 6, the 11 (31) liquid formulations shown in Table 7 were all prepared by adding a corresponding amount of cyclodextrin. 20 ml of each formulation was placed in a vial (vial, n = 2), the vials were sealed and stored upside down at 40 ° C. Table 6 shows the names of the various formulations. Formulation D 丨 means: 1 wt% of cyclodextrin D (SBE-i8-CD, Cape Tissole). Figures 2 and 8-11 show the results of the stress study (representing the results of Comparative Example 1). 97600-doc -41-200533375 Table 8 Time Α1 Α5 Α15 Weeks HP-SEC monomer [% 1 ACC HP-SEC monomer ACC HP-SEC monomer f0 / 〇1 ACC 0 93 1.0 '93 1.1 — 93 0.9 4 85 5.0 9.0 85 6.7 8 ~ 83 '4.9 85 4.3 86 6.3 Table): Time Β1 Β5 B15 week HP-SEC monomer "% 1 ACC HP-SEC monomer [% 1 ACC HP-SEC monomer" yol ACC 0 93 1.0 93 1.0 " 11 ----------- 94 0.9 4 86 5.4 87 7.0 88 6.2 8 82 4.3 83 6.2 82 8.5 Time C1 ~ C5-Zhou YPP-SEC monomer [· 〇 / 〇! 93 ACC 11.1 One HP-SEC unit [%] ACC 72 93 — " One 2S4 4 88 114 89 195 8 矣 1 1 84 50 " 86 '~ 171 Time ~ D5 ~ ~ D15 Week 0 Λ wr Monomer 93 ~ ACC ΗΡ-SEC Monomer ACC 1.5 ACC 1.0 92 ^ 1 Λ 4 Q 87 〇1 Γ4.2 85 3.6 ~ 85 ^ 兮 • Xi 9 R 〇〇1 5.0 178 '~ 5.2 Ύ Δ.Ο 4.9 Figure 2 shows that the content of free DM1 is the lowest in formulation D (Cape Texo). For example, a comparison of π week formulations D1 and B 1 (the same content of cyclodextrin) shows that the content of free DM1 is less than that of the formulation_ Half. Compared to formulations without cyclodextrin, some The formulation even showed an instability effect on the antibody-DM1 complex. Beta week D and the cyclodextrin-free formulation (control in Figure u shows that, for the 8-week value, the The DM1 content has an increased (double) value. The monomer content in different cyclodextrin formulations can be formulated with the control of ρΗ 6 · 5

(Table 3) for comparison. Cyclodextrin formulations c㈣d) were excluded due to their extremely high AN values. In summary, it can be described as a formulation containing cyclodextrin (especially Cape Texo) 97600.doc -42- 200533375

Contributes to stabilizing the effective life of the antibody-DM1 Li 舳 m L product, which can be extended compared to the control formulation when using Captizole in the formulation; once again, the excipient is expected to stabilize It was Leng Li "state antibody. ^^. B. Lyophilization of the antibody-DM1 complex The process by which the antibody gamma complex can be tumble-dried will now be described. The product consisting of antibody side conjugates is a toxic product that involves specific risks in processing. In the test, a tumble dry process and its required steps should be developed to eliminate it; the dry process caused by product contamination Danger to guards. This is done by heat-sealing " HAIurtj # 瓦 来 达 < " in a tightly sealed high-pressure bag throughout the freeze-drying process. All steps in the #high pressure bag around the product are performed in an insulator. The process has been modified to make the finished product bundle dry cakes-only slightly different from the product cakes produced without high pressure bags. The test was performed in a TINY lyophilizer. The main packaging components used are: vial: 20/25 ml standard vial (20R), colorless, GA1 Inj · (Messrs Schott, DE) terminator: Gusto 1797 PH 4023/50, gray, coated (Messrs West, USA) Flange cap: Kombika / Alu-KU (Messrs West, DE) Test individual lyophilized formulations with and without actives. 1. Lyophilize with mannitol buffer. Two metal plates were loaded with 64 vials each. A 10 mi buffer was placed in each vial (Table 12). 97600.doc -43-200533375 Table 12: Substance composition KH2P〇4 4.19 mM NaH2P04 x H20 1.28 mM Na2HP04 3.12 mM mannitol 4w% NaCl 139.6 mM ~ PH! 6.5 Leave a plate in the chamber and heat seal it in High pressure bag made of Messrs Sengewald (Medipeel). Use normal film seals to make seams on high pressure bags. Three side-by-side seams are made. The following additional parameters apply. Table 13 summarizes the lyophilization procedures used. The procedure was further refined during the study (see below). It includes an incubation step (-12 ° C / E ·· 3 h, 45 min) and lasts for a total of about 56 h. Table 13: Step name Processing time [hh: mm] Temperature [° c] Vacuum degree [%] Vacuum degree alarm [%] 1 Load 00:01 +5--2 Freeze 01:30 -50--3 Freeze 02: 00 -50 surface-4 insulation 00:45 -12--5 insulation 03:00 -12--6 frozen 01:00 -50--7 frozen 00:10 -50-8 cold; east 00:10 -50- -9 frozen 00:10 50--10 frozen 00:20 -50--11 dry 01:30 -25 44 (0.3 mbar) 99,9 12 dry 02:00 soil 0 44 (0.3 bar) 48 (0.4 Mbar) 13 Dry 10:00 ± 0 44 (0.3 mbar) 48 (0.4 mbar) 14 Dry 10:00 ± 0 44 (0.3 mbar) 48 (0.4 mbar) 15 Dry 10:00 ± 0 44 ( 0.3 mbar) 48 (0.4 mbar) 16 dry 03:00 +30 44 (0.3 mbar) 48 (0.4 mbar) 17 dry 04:00 +30 44 (0.3 mbar) 48 (0.4 mbar) 18 dry 04:00 +30 44 (0.3 mbar) 48 (0.4 mbar) 19 Dry 02:00 +30 44 (0.3 mbar) 48 (0.4 mbar) 20 Remove 00:20 +5 98 99.9 Total time: 55 : 56 97600.doc -44- 200533375 The high pressure bag appears to be under a certain overpressure (slight expansion) during processing. Therefore, the lyophilization procedure was optimized in subsequent tests. Observe the following phenomena:-The product in the high-pressure bag seems to dry significantly more slowly than the product without a high-pressure bag-The product in the high-pressure bag rises at the edge of the glass-The product in the high-pressure bag forms a concave surface and seems to become lumpy. Very different freeze-drying occurred in two tests (with and without high-pressure bags). Therefore, all the lyophilized materials in the high-pressure bag have a large hole structure, which can become lumpy with the lightest contact. In contrast, samples that were not encapsulated in high-pressure bags formed the best cakes. However, it is also prone to splitting upon contact. This is due to the extremely low solids content of the sample. Reconstitution of some samples (Table 14), on average, resulted in longer sample dissolution times. Admittedly, although the most active phase remains subjectively unchanged until all residues are completely dissolved, all residues dissolve over a significantly longer period of time than the product in the high-pressure bag. Table 14: Reconstruction time (random sample) Sample without south pressure bag Sample with south pressure bag is most active after [sec] and completely after [sec] Most active phase after [sec] Dissolve the active phase to dissolve 10 60 05 45 10 50 12 85 15 62 15 115 10 84 10 90 011.25 063.5 010.5 083J5 In order to determine the residual moisture based on KaH-Fischei *, select some vials for residual moisture analysis. Based on these results, the products were all in a similarly dry state and contained an average residual moisture of 0.23% (Table 15). 97600.doc -45-200533375 Table 15: I Residual moisture in random samples without high pressure bags [%] Random samples with high pressure bags 1 1 1 1 γΓ ^ Τ ~ 2 " 020 ~~ 0.31 Λ ^ 3 " 〇 27 " ~ 3 — (\ Λ Π 4 5 ~ 023 1 ~~ U.1 7 ~ 026 — ~ 6 ~ i average value of high pressure bag ~~ total average value--one ====== 0.22 ΎΪ9 ~ '~~ 024 1 ~ " 023 " ~ 6 The average value of two pressure bags ..... J ~ 026 ~ * 022 ~' The following conclusions can be drawn from this experiment: The pressure in the high pressure bag indicates that the sublimation water cannot be easily removed. It escapes from the high-pressure bag. The reason is that the plastic film is impermeable to water vapor. The increase in pressure in the high-pressure bag is also the reason for the formation of a constricted structure. Figure 3 shows that when the main drying process has been completed, the drying seems to be incomplete. Therefore 'Drying time doubled from 30 hours to 60 hours. At the same time, in order to offset any pressure booster in the high-pressure bag, try to reduce the system pressure from 44% to 37.5%. 2 · Mannitol / sucrose buffer Lyophilization In another test, several parameters were changed / tested simultaneously. These parameters were: type of high pressure bag, effect of metal plate, some lyophilization parameters Changes and changes in formulations. These changes come from considering the results of the first test. Three metal plates are loaded with 64 vials. Filling volume: 10 ml Filling level: 16 mm high-pressure bag 1: MedipeeKMessrs sengewald), acc DIN 58953, 30x6.5x58 High Pressure Bag 2: Ultraclean Cleansteam BAGS, newf〇rm (B_332〇Hoegaarden) 97600.doc -46- 200533375 Solution pH: 6.78 (measured with pH meter) Skeleton substance: 4% mannose Alcohol and 1% sucrose Table 16: Substance composition KH2P〇4 6.4 mM NaH2P04 x H20 1.97 mM Na2HP04 4.8 mM mannitol 4w% sucrose 1 w% PH 6.7 Table 17 summarizes the lyophilization procedures used. Table 17: Step name Processing time [hh: mm] Temperature [° C] Vacuum degree [%] Vacuum degree alarm [%] 1 Load 00:01 +5--2 Freeze 01:30 -50--3 Freeze 02: 00 -50--4 Insulation 00:45 -12 Wrap-5 Insulation 03:00 -12--6 Freezing 01:00 -50--7 Freezing 00:10 -50-8 Freezing 00:10 -50--9 Frozen 00:10 -50--10 Frozen 00:20 -50 _-11 Dry 01:30 -25 44.0 (0.3 mbar) 99.9 12 Dry 02:00 ± 0 37.5 (0.15 mbar) 48 (0.4 mbar) 13 Dry 20:00 ± 0 37.5 (0.15 mbar) 48 (0.4 mbar) 14 Dry 20:00 ± 0 37.5 (0.15 mbar) 48 (0.4 mbar) 15 Dry 20:00 ± 0 37.5 (0.15 mbar) ) 48 (0.4 mbar) 16 dry 03:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 17 dry 04:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 18 dry 04:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 19 Dry 02:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 20 Remove 00:20 +5 98 99.9 Total time: 85:56 according to Two considerations are used to optimize the Cambodian drying process. The drying time is doubled and the airspace is halved from 0.3 mbar to 0.15 mbar. There is no external signal of any overpressure. 97600.doc -47- 200533375 Use this process, especially # 士 _ 5 |. ^ There are two people. In the test (no high pressure bag, high pressure bag 1 and two ^, convincing Compare the results. The tumble dry in the high-pressure bag played a role in the improvement and the dry process. As far as the product is concerned, the two high-pressure bags do not seem to have any difference. The plate under the vial (two test tubes) such as Guanghai has been proven In the improved procedure, «adverse effects. Therefore, Gan-Ling /, is located under the vial in the high-pressure bag during the whole process. The change of the process parameters in the drying process (Figure 4) shows a significantly better Ergo 1 & 'I think that all three tests in the 4 drying program are completely dry; 在 the product temperature rises above the nominal area under each I · month condition) 3. Lyophilization of the antibody-DM1 complex As mentioned above, as a result of Donggan's military test, the process was performed in a high-pressure bag (⑽ipeei). Figure 6 shows the configuration placed in the lyophilizer. A total of 70 vials were lyophilized in this manner. The finished formulation has a protein concentration of 4.65 mg / ml.夂 ^ ,, 丄, put 10 ml solution in each vial. The formulations used (Table 18) are described below. Table 18:

The second experiment; Donggan procedure (Table 19) produced good results and was therefore used unchanged. 97600.doc -48- 200533375 Table 19: Step name processing time [hh: mm] temperature [° c] vacuum degree [%] vacuum degree alarm [%] 1 load 00:01 +5--2 cold; east 01: 30 -50--3 frozen 02:00 -50--4 insulated 00:45 -12--5 insulated 03:00 -12--6 cold 01:00 -50--7 frozen 00:10 -50- -8 frozen 00:10 -50--9 frozen 00:10 -50--10 frozen 00:20 -50--11 dry 01:30 -25 44.0 (0.3 mbar) 99.9 12 dry 02:00 ± 0 37.5 (0.15 mbar) 48 (0.4 mbar) 13 Dry 20:00 ± 0 37.5 (0.15 mbar) 48 (0.4 mbar) 14 Dry 20:00 ± 0 37.5 (0.15 mbar) 48 (0.4 mbar) 15 Dry 20:00 Soil 0 37.5 (0.15 mbar) 48 (0.4 mbar) 16 Dry 03:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 17 Dry 04:00 +30 37.5 (0.15 mbar ) 48 (0.4 mbar) 18 Dry 04:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 19 Dry 02:00 +30 37.5 (0.15 mbar) 48 (0.4 mbar) 20 Remove 00: 20 +5 98 99.9 Total time 85:56 Figure 6 shows the progress of the beam drying process. Analytical study of the freeze-dried antibody-DM1 complex according to Test 3 (stress study over 8 weeks at 40 ° C). The stability data are described in area A. In summary, it can be said that lyophilization of the antibody-DM1 complex using a high-pressure bag gave comparable results for the product obtained by lyophilization without a high-pressure bag. Example 3 The lyophilization process of the antibody-DM1 complex of two different formulations will now be described. 1 · Succinate formulations: 97600.doc -49-200533375 Table 2 0 gives the formulations of the hyaluronate formulations. Table 20:

Component concentration [mmol / 1] Concentration [g / 1] Nominal weight [mg / vial], V = 10.0ml Disodium succinate hexahydrate (C4H4Na204 X 6 Η20) 10.00 2.700 27.00 Mannitol 219.58 40.0 400.0 Sucrose 29.21 10.0 100.0 Tween 20 0.4 (0.04 w%) 4.00 Antibody-DM1 4.0 40.0 WFI Add 10.0 ml WFI Target volume: V = 10.5 ml (0.5 ml overfilled). Reconstituted with 10.5 mlWFI. Physical properties of this formulation (Table 20): pH = 5.5 Permeability = 296 mOsmol Density = 1.0183 g / ml. The main package is composed of the following: Vial: 50/60 m, colorless, GA1 Inj. Terminator: Gusto 1797 PH 4023/50, gray coating, ready-to-sterilize easy-open lid: Kombika / Alu / ku, white, Messrs West Company. 2. Phosphate formulations: Table 21 gives the formulations of phosphate formulations. 97600.doc 50- 200533375 Table 21: Component concentration [mmol / 1] Concentration [g / 1] Nominal weight [mg / vial], V = 10.0 ml KH2P〇4 4.19 0.570 5.70 NaH2P04 x 2 H20 1.45 0.226 2.26 Na2HP04 x 2H20 3.91 0.696 6.96 Mannitol 219.58 40.0 400.0 Cane St 29.21 10.0 100.0 Tween 20 0.4 (0.04 w%) 4.00 Antibody-DM1 4.0 40.0 WFI Add 10.0 ml WFI Target volume: V = 10.5 ml (0.5 ml overfilled). Reconstituted with 10.5 ml WFI. The physical properties of this formulation (Table 21) are: ρΗ = 6.5; permeability = 287 mOsmol density = 1.0184 g / ml. The main package is composed of the following: Vial: 50/60 m, colorless, GA1 Inj. Terminator: Gusto 1797 PH 4023/50, gray coating, ready-to-sterilize easy-open lid: Kombika / Alu / ku, white, Messrs West Company. Both formulation examples were lyophilized by the following lyophilizer and lyophilization procedure. 97600.doc -51-200533375 Table 22: Lyophilizer

Freeze dryer STOKES 132 sq. Ft, BVL program is shown in Table 23. Processing time of freeze-drying is about 52h. Loading without freeze-drying plate vacuum adjustment without N2 injection vacuum measurement. Pirani probe annealing temperature -16 ° C. Ventilation pressure 600 mbar (below) Ventilation medium n2 Storage temperature 2 ° C to 8 ° C Table 23 summarizes the precise lyophilization parameters. Table 23 ·· Step Processing Time Temperature Pressure [hh: mm] [° C] [mbar] 1 Load 00:01 +5 2 Freeze 01:00 +5 3 Freeze 03:00 -48 4 Freeze 02:00 -48 5 Frozen 00:45 -16 6 Frozen 03:00 -16 7 Frozen 01:45 -48 8 Frozen 00:30 -48 9 Preparation 00:30 -48… 10 Primary drying 1 00:30 -40 0.15 11 Primary drying 2 02:00 0 0.15 12 Primary drying 3 24:00 0 0.15 13 Secondary drying 1 03:00 +30 0.15 14 Secondary drying 2 10:00 +30 0.15 15 Storage 1 00:05 +5 0.15 Processing time 52: 05 The adjustment of the process allows the washing and drying process to be performed without metal plates and without Me dipeel bags. The lyophilized product obtained by means of the above-mentioned lyophilization process produces a surprisingly stable product that meets the medical requirements. The choice of the formulation is also the main reason for the high quality of the product, such as using the correct amount of detergent in the antibody 97600.doc -52- 200533375 -DM1 complex. literature

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Ye et al., Gene synthesis and expression in E. coli for pump, a human matrix metalloproteinase o Biochem Biophys Res Commun 186 (1): 143-9 (1992). [Schematic description] Figure 1: Figure 1 shows the difference from the content of free DM 1. It shows the difference between the value of free DM 1 between 0 and 4 weeks and the value between 0 and 8 weeks. Figure 2: Figure 2 shows the lowest free DM1 content in formulation D (Cape Texo). For example, a comparison of formulations D1 and B1 (same cyclodextrin content) shows that the free DM 1 content is halved compared to formulation B1. Compared to formulations without cyclodextrin, some formulations even show destabilizing effects on the antibody-DM 1 complex. The comparison of Formulation D with the formulation without cyclodextrin (Figure 1) shows that for the value of 97600.doc -65- 200533375 for 8 weeks, the DM1 content in the latter formulation has an increase (double) value. Figure 3 ·· Freeze-drying process. Figure 3 shows that when the amount of trunk welding is too high, the desiccant does not show completeness. ⑴ λα λ, C each. Changes in program parameters during drying-good results. No obvious inflammation head Figure 5 · Figure 5 shows 1 Figure 6 · Soil, non-tumble dryer placement configuration, Donggan process. Continue 97600.doc -66- 200533375 Sequence Listing

< 110 > Boehringer Ingelheim Pharma GmbH & Co. KG < 120 > Lyophilized formulation of antibody conjugate < 130 > Case 1 / 1602-2 TK / Kn

< 140 > 093140566 < 141 > 2004-12-24 < 160 > 9 < 170 > Patentln version 3.1

< 210 > 1 < 211 > 42 < 212 > PRT

< 213 > human CD44 exon v6 < 400> 1

Gin Ala Thr Pro Ser Ser Thr Thr Glu Glu Thr Ala Thr Gin Lys Glu 15 10 15

Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr Pro Arg 20 25 30

Glu Asp Ser His Ser Thr Thr Gly Thr Ala 35 40 97600.doc 200533375 < 210 > 2 < 211 > 14 < 212 > PRT < 2i3 > Homo sapiens < 400〉 2

Gin Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg Gin Thr 1 5 10 < 210 > 3 < 211 > 11 < 212 > PRT < 213 > Homo sapiens < 400 > 3

Trp Phe Gly Asn Arg Trp His Glu Gly Tyr Arg 1 5 10

< 210 > 4 < 211 > 213 < 212 > PRT < 213 > Humanized antibody BIWA4 light chain < 400> 4

Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 10 15 97600.doc • 2- 200533375

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser lie Asn Tyr lie 20 25 30

Tyr Trp Leu Gin Gin Lys Pro Gly Gin Ala Pro Arg lie Leu lie Tyr 35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro Glu 65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Leu Gin Trp Ser Ser Asn Pro Leu Thr 85 90 95

Phe Gly Gly Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala Pro 100 105 110

Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140

Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160

Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190

Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 97600.doc 200533375

Asn Arg Gly Glu Cys 210

< 210 > 5 < 211 > 702 < 212 > DNA < 213〉 Humanized antibody BIWA4 light chain < 400 > 5 atggaagccc cagctca £ ct tctctttcctc ctgctgctct ggctcact cctcc agt cctaccggt cctaccggt ggtaccagca gaagccagga 180 caggctccta gactcttgat ttatctcaca tccaacctgg cttctggagt ccctgcgcgc 240 ttcagtggca gtgggtctgg aaccgacttc actctcacaa tcagcagcct ggagcctgaa 300 gattttgccg tttattactg cctgcagtgg agtagtaacc cgctcacatt cggtggtggg 360 accaaggtgg agattaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ga 702

< 210 > 6 < 211 > 444 < 212 > PRT 97600.doc -4- 200533375 < 213 > Humanized antibody BIWA4 or BIM8 heavy chain < 400> 6

Glu Val Gin Leu Val Glu Ser Gly Gly Gly Gly Leu Val Lys Pro Gly Gly 15 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30

Asp Met Ser Trp Val Arg Gin Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45

Ser Thr lie Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Leu Asp Ser lie 50 55 60

Lys Gly Arg Phe Thr lie Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80

Leu Gin Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Gin Gly Leu Asp Tyr Trp Gly Arg Gly Thr Leu Val Thr Val 100 105 110

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser 115 120 125

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys 130 135 140

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu 145 150 155 160 97600.doc 200533375

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu 165 170 175

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr 180 185 190

Gin Thr Tyr lie Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val 195 200 205

Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 210 215 220

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 225 230 235 240

Pro Pro Lys Pro Lys Asp Thr Leu Met lie Ser Arg Thr Pro Glu Val 245 250 255

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 260 265 270

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 275 280 285

Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 290 295 300

Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 305 310 315 320

Ser Asn Lys Ala Leu Pro Ala Pro lie Glu Lys Thr lie Ser Lys Ala 325 330 335

Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Arg 340 345 350 97600.doc 200533375

Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly 355 360 365

Phe Tyr Pro Ser Asp lie Ala Val Glu Trp Glu Ser Asn Gly Gin Pro 370 375 380

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 385 390 395 400

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gin Gin 405 410 415

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 420 425 430

Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440

≪ 210> 7 < 211 > 1392 < 212 > DNA < 213 > humanized antibody BIWA4 or BIWA8 heavy chain < 400 > 7 atggagtttg ggctgagctg gctttttctt gtggctattt taaaaggtgt ccagtgtgaa 60 gtgcagctgg tggagtctgg gggaggctta gtgaagcctg gagggtccct aagactctcc 120 tgtgcagcct ctggattcac tttcagtagc tatgacatgt cttgggttcg ccaggctccg 180 gggaaggggc tggagtgggt ctcaaccatt agtagtggtg gtagttacac ctactatcta 240 gacagtataa agggccgatt caccatctcc agagacaatg ccaagaactc cctgtacctg 300 caaatgaaca gtctgagggc tgaggacacg gccgtgtatt actgtgcaag acaggggttg 360 97600.doc 200533375 gactactggg gtcgaggaac cttagtcacc gtctcctcag ctagcaccaa gggcccatcg 420 gtcttccccc tggcaccctc ctccaagagc acctctgggg gcacagcggc cctgggctgc 480 ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 540 agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctactc cctcagcagc 600 gtggtgaccg tgccctccag cagcttgggc acccagacct acatctgcaa cgtgaatcac 660 aagcccagca cct agctt aggt gggac cgtcagtctt cctcttcccc 780 ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacatg cgtggtggtg 840 gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt acgtggacgg cgtggaggtg 900 cataatgcca agacaaagcc gcgggaggag cagtacaaca gcacgtaccg tgtggtcagc 960 gtcctcaccg tcctgcacca ggactggctg aatggcaagg agtacaagtg caaggtctcc 1020 aacaaagccc tcccagcccc catcgagaaa accatctcca aagccaaagg gcagccccga 1080 gaaccacagg tgtacaccct gcccccatcc cgggatgagc tgaccaagaa ccaggtcagc 1140 ctgacctgcc tggtcaaagg cttctatccc agcgacatcg ccgtggagtg ggagagcaat 1200 gggcagccgg agaacaacta caagaccacg cctcccgtgc tggactccga cggctccttc 1260 ttcctctaca gcaagctcac cgtggacaag agcaggtggc aggacagggga cgtcttctca 1320 tgctccgac ac tg

< 210 > 8 < 211 > 213 < 212 > PRT < 213> Humanized antibody BIWA8 light chain 97600.doc 200533375 < 400 > 8

Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 15 10 15

Glu Arg Ala Thr Leu Ser Cys Ser Ala Ser Ser Ser lie Asn Tyr lie 20 25 30

Tyr Trp Leu Gin Gin Lys Pro Gly Gin Ala Pro Arg lie Leu lie Tyr 35 40 45

Leu Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60

Gly Ser Gly Thr Asp Phe Thr Leu Thr lie Ser Ser Leu Glu Pro Glu 65 70 75 80

Asp Phe Ala Val Tyr Tyr Cys Leu Gin Trp Ser Ser Asn Pro Leu Thr 85 90 95

Phe Gly Gly Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala Pro 100 105 110

Ser Val Phe lie Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly Thr 115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140

Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin Glu 145 150 155 160

Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 97600.doc 200533375

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190

Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205

Asn Arg Gly Glu Cys 210

≪ 210> 9 < 211 > 702 < 212 > DNA < 213 > humanized antibody BIWA8 light chain < 400> 9 atggaagccc cagctcagct tctcttcctc ctgctgctct ggctcccaga taccaccgga 60 gaaattgttc tcacccagtc tccagcaacc ctgtctctgt ctccagggga gagggccacc 120 ctgtcctgca gtgccagctc aagtataaat tacatatact ggctccagca gaagccagga 180 caggctccta gaatcttgat ttatctcaca tccaacctgg cttctggagt ccctgcgcgc 240 ttcagtggca gtgggtctgg aaccgacttc actctcacaa tcagcagcct ggagcctgaa 300 gattttgccg tttattactg cctgcagtgg agtagtaacc cgctcacatt cggtggtggg 360 accaaggtgg agattaaacg tacggtggct gcaccatctg tcttcatctt cccgccatct 420 gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc 480 agagaggcca aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag 540 agtgtcacag agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg 600 agcaaagcag actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg 660 agctcgcccg tcacaaagag cttcaacagg ggagagtgtt ga 702 97600.doc -10-

Claims (1)

200533375 10. Scope of patent application: 1. A freeze-dried pharmaceutical composition containing a conjugate of an antibody and maytansmoid, which is characterized in that it is adjusted to 5-6. 2. A composition as claimed, characterized in that it contains a buffer, preferably a succinate, phosphate or acetate buffer. 3. The composition according to any one of the items 1 or 2, characterized in that it contains bone shellfish, and the substance is preferably mannitol and / or sucrose. 4. The composition of any one of the items in claim ⑷, characterized in that it contains, and a cleaning agent, and the cleaning agent is preferably a polysorbate § purports to add coffee, any of June 0 items 1 or 2 The composition of the item 'characterized in that the total amount of the product is 1 mg / m 2 mg / mi before freeze-drying. u 6. If any one of items 1 or 2 is cold, the solution is characterized by containing 4w% mannitol and 1 in the solution. For example, if the concentration of any one of the items 1 or 2 is frozen at A, ^,, and 54, it is characterized by the cleaning agent 8. Before ~ lyophilization, it is from 0.001 to 0.1 w%. This kind of preparation is as follows: The method is to make the solution containing the antibody: US 4 = compound 'its characteristic solution is to cool; to dry the water with a pH value of 5-6 9. ::: cold =: pharmaceutical composition The four-inch sign that it contains antibodies and maytansinol is that it contains cyclodextrin. 1 〇 · If you look for item 9 and it ’s human fighting, the special feature is that the cyclodextrin is butyl scale 97600.doc 200533375 cyclodextrin, super-propyl-β_cyclodextrin 0 hydroxyl The propyl-γ-cyclodextrin or bamboo cyclodextrin composition according to any one of item 9 or 10, characterized in that the "paste companion is in an amount of 0.01 · 40% by weight and the percentage by weight before the chilling is dried presence. Preferably, · 1, I 'is a method for preparing a composition as claimed in any one of the following items, characterized in that an aqueous solution containing a conjugate of an antibody and maytansin and a cyclodextrin is freeze-dried. It is characterized by the light key of the ID NO: 8. 13. The composition of any one of the claims, 2, 9, or 10. The antibody contains an amino acid sequence of SEQ ID NO: 4 ^ SEq and has an amine. Heavy chain of the amino acid sequence SEQ ID NO: 6. 14 · A use that will be as requested in items 1, 2. The pharmaceutical composition of 9 or 10 for treating cancer 15. A packaging unit containing the pharmaceutical composition and water of any one of claims 1, 2, 9, or 10 in a separate container. 97600.doc