Multi-drug-loading-site, high drug-loading capacity ligand-drug conjugate

Abstract

The present invention provides a multi-drug-loading site and high drug-loading capacity ligand-drug conjugate. The ligand-drug conjugate has a structure of general formula (I). The ligand-drug conjugate has the characteristics of high loading capacity, high drug efficacy, low toxicity, and low risks. The ligand-drug conjugate can be used particularly to connect to a low toxicity chemical molecule, thereby extending a therapeutic window. Furthermore, the present invention provides an antibody-drug conjugate molecule. The antibody-drug conjugate molecule has the characteristics of multiple drug-loading ability and high drug-loading capacity, such that the antibody-drug conjugate can carry a large amount of a low toxicity chemical molecule and achieve a therapeutic effect without depending on antibody targeting or high toxicity chemicals.
TM-{R.sup.2-PEG1-[R.sup.1-PEG2-(R.sup.3-A′-D).sub.n].sub.m}.sub.l  (I

Claims

1. A ligand drug conjugate, having a structure represented by general formula I:
TM-{R.sup.2-PEG1-[R.sup.1-PEG2-(R.sup.3-A′-D).sub.n].sub.m}.sub.l   (I) wherein: TM is a recombinant anti-HER2 humanized monoclonal antibodies; PEG1 is a multi-branched polyethylene glycol residue having a structure represented by general formula (V-1): ##STR00041## wherein: k1 is an integer from 1 to 240, j1 is an integer from 3 to 8, R.sub.1 is a core molecule of a multi-branched polyethylene glycol, and R.sub.1 is pentaerythritol; PEG2 is a multi-branched polyethylene glycol residue having a structure represented by general formula (V-2): ##STR00042## wherein: k2 is an integer from 1 to 240, j2 is an integer from 3 to 8, R.sub.2 is a core molecule of multi-branched polyethylene glycol, and R.sub.2 is pentaerythritol; 1 is an integer from 11 to 100; m is an integer from 1, 3, 5 or 7; n is an integer from 1, 3, 5 or 7; A′ is a valine residue; R.sup.1 is a linking unit linking PEG1 and PEG2; R.sup.2 is a ligand unit linking the ligand unit and PEG1; R.sup.3 is a linking unit linking PEG2 and spacer A′ or a drug; and D is irinotecan, or a pharmaceutically acceptable salt thereof; wherein R.sup.1 has a structure of -A.sub.1-B-A.sub.2-, wherein A.sub.1 is selected from the group consisting of: —(CH.sub.2).sub.iNHCO—; A.sub.2 is selected from the group consisting of: —(CH.sub.2).sub.i—; B is selected from a structure of: ##STR00043## R.sup.2 is selected from the group consisting of: —(CH.sub.2).sub.iNHCO— and —(CH.sub.2).sub.iCONH; R.sup.3 is selected from the group consisting of: —(CH.sub.2).sub.iCO—; i is an integer from 0 to 10.

2. The ligand drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the PEG1 and/or PEG2 have a molecular weight of from 1 to 50 kDa.

3. The ligand drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the ligand drug conjugate has a structure represented by formula VIII: ##STR00044## Z is ##STR00045## wherein e1 to e4 and f1 to f4 are independently selected from an integer of 1-240; -A′-D has a structure of: ##STR00046##

4. The ligand drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, wherein the pharmaceutically acceptable salt is one or more selected from the group consisting of sodium salt, potassium salt, cesium salt, calcium salt, magnesium salt, triethylamine salt, pyridine salt, methylpyridine salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, N,N-dibenzylethylenediamine salt, hydrochloride, hydrobromide, sulfate, nitrate, phosphate, formate, acetate, trifluoroacetate, pantothenate, succinate, citrate, tartrate, fumarate, maleate, gluconate, glucuronate, saccharate, benzoate, lactate, methanesulfonate, ethanesulfonate, besylate, p-toluenesulfonate, argininate, aspartate, glutamate, pantothenate, and ascorbate.

5. A pharmaceutical composition comprising a ligand drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, and a pharmaceutically acceptable carrier or excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a graph of rat plasma drug-time profiles (total antibody concentration, g/mL Vs time, hr), intravenous injection dose of Ab and antibody conjugate 30 mg/kg.

(2) Ab: naked anti-antibody (recombinant anti-HER2 humanized monoclonal antibody); APEGA-8: antibody drug conjugate (single arm+single arm, prepared in Example 6); APEGA-9: antibody drug conjugate (four arm+single arm, prepared in Example 7; APEGA-10: antibody drug conjugate (four arms+four arms, prepared in Example 8).

(3) FIG. 2 is a graph of rat plasma drug-time (Irinotecan concentration, ng/mL Vs time, hr). The intravenous injection doses of PEG-Irinotecan and APEGA-9 were both 4.2 mg/kg in term of Irinotecan.

(4) APEGA-9: antibody drug conjugate (four arms+single arm, prepared in Example 7); PEG-Irinotecan (PEG5K-PEG3.5K-Irinotecan): PEG drug conjugate (four arms+singlearm, prepared in Example 5).

(5) FIG. 3 is a graph showing the mean tumor volume in the colorectal cancer model (HCT-116) corresponding to the days after tumor transplantation, the dose of Ab and the antibody conjugate 30 mg/kg, and the PEG-Drug administered 30 mg in term of Irinotecan.

(6) Ab: naked anti-antibody; APEGA-8: antibody drug conjugate (single arm+single arm, prepared in Example 6); APEGA-9: antibody drug conjugate (four arms+single arm, prepared in Example 7); APEGA-10: antibody drug conjugate (four arms+four arms, prepared in Example 8); PEG-Irinotecan (PEG5K-PEG3.5K-Irinotecan): PEG drug conjugate (four arms+single arm, prepared in Example 5).

DETAILED DESCRIPTION OF THE INVENTION

(7) Unless otherwise stated, the terms hereinafter have the following meanings.

(8) The term “antibody” as used herein is used in its broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (eg, bispecific antibodies) and antibody fragments, as long as they show desired biological activity (Miller et al. (2003) Jour. of Immunology, 170: 4854-4861). The antibody can be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that recognize and bind specific antigens (Janeway, C. et al. (2001) ImmunoBiology, 5th Ed., Garland Publishing, New York). Target antigens generally have a large number of binding sites, also referred to as epitopes, which are recognized by the CDRs of a variety of antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen can correspond to more than one antibody.

(9) Antibodies include full length immunoglobulin molecules or immunologically active portions of full length immunoglobulin molecules, i.e, molecules containing antigens or portions thereof that specifically bind to a target of interest. Such targets includes, but not limited to cancer cells or cells that produce autoimmune antibodies associated with immune diseases. In particular, the antibodies of the invention are reactive against cancer cells, malignant cells, infectious organisms or antigens associated with autoimmune diseases or epitopes thereof. The immunoglobulins disclosed herein can have any type of immunoglobulin molecule (for example, IgG, IgE, IgM, IgD, and IgA), classes (for example, IgG1, IgG2, IgG3, IgG4, IgA, IgA2) or subclasses. The immunoglobulins can be derived from any species. However, in one aspect, the immunoglobulin is derived from a human, a mouse or a rabbit.

(10) The term “antibody fragment” herein encompasses a portion of a full length antibody, typically its antigen binding or variable region. Examples of antibody fragments include: Fab, Fab′, F(ab′)2 and Fv fragments; diabodies; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4): 315-323); fragments prepared from Fab expression libraries; anti-idiotype (anti-Id) antibodies; CDRs (complementarity determining regions); and any of the above epitope-binding fragments immunologically specifically binding to cancer cell antigens, viral antigens or microbial antigen; a mono-chain antibody molecule; and a multispecific antibody formed from the antibody fragment.

(11) The term “monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the antibodies contained in the population are identical except for naturally occurring possible mutations that may be present in minor amounts. Monoclonal antibodies are highly specific antibodies that target a single antigenic site. Moreover, in contrast to polyclonal antibody preparations which typically include different antibodies that target different antigenic determinants (epitopes), each monoclonal antibody only targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized in a manner that is not contaminated by other antibodies. The modifier “monoclonal” denotes the property of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as producing the antibody by any particular method. For example, a monoclonal antibody used in the present invention can be prepared by the hybridoma method first described by Kohler et al. (1975) Nature 256:495 or can be prepared by recombinant DNA methods (for example: U.S. Pat. Nos. 4,816,567; 5,807,715). For example, monoclonal antibodies can be separated from phage antibody libraries by the technique described by Clackson et al. (1991) Nature, 352: 626-628; Marks et al. (1991) J. Mol. Biol., 222: 581-597.

(12) A “monoclonal antibody” herein specifically includes a “chimeric” antibody wherein a portion of the heavy and/or light chain is identical or homologous to a corresponding sequence of antibody derived from a particular species or belonging to a particular antibody type or subtype, and the remainder of the strand is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody type or subtype. The fragments of the chimeric antibodies are also included herein so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al. (1984) Proc. Natl. Acad. Sci. USA, 81: 6851-6855). Chimeric antibodies of interest herein include “primatized” antibodies comprising antigen-binding sequences of variable region derived from non-human primates and human constant region sequences.

(13) The term “intact antibody” herein comprises antibodies of the VL and VH domains as well as the light chain constant domain (CL) and the heavy chain constant domains CH1, CH2 and CH3. The constant domain can be a native sequence constant domain (for example, a human native sequence constant domain) or an amino acid sequence variant thereof. An intact antibody may have one or more “effector functions”, meaning those biological activities due to the Fc constant region of the antibody (the native sequence Fc region or the amino acid sequence variant Fc region). Examples of antibody effector functions include Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell mediated cytotoxicity (ADCC); endocytosis; and cell surface receptors, such as B cell receptors and down-regulation of body and BCR.

(14) Depending on the amino acid sequence of its heavy chain constant domain, intact antibodies can be assigned to five major classes of intact immunoglobulin antibodies: IgA, IgD, IgE, IgG, and IgM, and several of them can be further divided into “subclasses” (subtypes) such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to different antibody classes are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Type Ig includes hinge-modified or hinge-free (Roux et al. (1998) J. Immunol. 161: 4083-4090; Lund et al. (2000) Eur. J. Biochem. 267: 7246-7256; US 2005/0048572; US 2004/0229310).

(15) The term “parent antibody” as used herein is an antibody in which one or more amino acid residues in the amino acid sequence is replaced with one or more cysteine residues. The parent antibody can include a native or wild type sequence. The parent antibody may comprise a native or wild type sequence. The parent antibody may have pre-existing amino acid sequence modifications (such as additions, deletions, and/or substitutions) relative to other native, wild-type or modified forms of the antibody. The parent antibody can be directed against a target antigen of interest, such as a biologically important polypeptide. Antibodies directed against non-polypeptide antigens, such as tumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178, are also of interest. Exemplary parent antibodies include selective antibodies that have affinity for cell surface and transmembrane receptors and tumor associated antigens (TAA).

(16) The term “antigen bound to an antibody” as used herein includes, but is not limited to, HER-2/neu, carbonic anhydrase IX, B7, CCCL19, CCCL21, CSAp, BrE3, CD1, CD1a, CD2, CD3, CD4, CD5, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD44, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CEACAM5, CEACAM-6, alpha-fetoprotein (AFP), VEGF, ED-B fibronectin, EGP-1, EGP-2, EGF receptor (ErbB1), ErbB2, ErbB3, factor H, FHL-1, Flt-3, folate receptor, Ga733, GROB, HMGB-1, hypoxia-inducible factor (HIF), HM1 0.24, insulin-like growth factor (ILGF), IFN-γ, IFN-α, IFN-J3, IL-2R, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-2, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, IGF-1R, Ia, HM1.24, ganglioside, HCG, HLA-DR, CD66a-d, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, macrophage migration inhibitory factor (M IF), MUC1, MUC2, MUC3, MUC4, MUC5, placental growth factor (PIGF), PSA, PSMA, PSMA dimer, PAM4 antigen, NCA-95, NCA-90, A3, A33, Ep-CAM, KS-1. Le(y), mesothelin, S100, tenascin, TAC, Tn antigen, Thomas-Friedenreich antigen, tumor necrosis antigen, tumor angiogenic antigen, TNF-α, TRAIL receptor (R1 and R2), VEGFR, RANTES, T101, cancer stem cell antigen, complement factor C3, C3a, C3b, C5a, C5 and oncogene products, etc.

EXAMPLE

(17) The various embodiments of the invention are illustrated by the following examples, but are not intended to limit the invention.

(18) The irinotecan used in the Examples was purchased from Shanghai Longxiang Biomedical Development Co, Ltd, 4-dimethylaminopyridine (DMAP) and 1-hydroxybenzotriazole (HOBT) from Shanghai Covalent Chemical Technology Co, Ltd. The polyethylene glycol derivative was purchased from Beijing Keykai Technology Co, Ltd, and other reagents were purchased from Sinopharm Group.

[PEG-1] Synthesis Example

Example 1: Synthesis of Four-Arm Polyethylene Glycol Maleimide-Succinimidyl Acetate (V-3)

(19) ##STR00024##

(20) Procedure:

(21) Four-arm polyethylene glycol maleimide-succinimidyl acetate (V-3) was prepared by following the procedure of Examples 1-9 of Patent Application CN201610398765.4.

(22) NMR (DMSO) δ: 2.32

(23) ##STR00025##
2.82

(24) ##STR00026##
3.15 (q, 6H, CH.sub.2CH.sub.2NH), 4.60 (s, 2H, CH.sub.2COO), 6.99

(25) ##STR00027##

[PEG-2] Synthesis Example

Example 2: Preparation of SH-PEG-(CONHI).SUB.3.(5K)(T2-4)

(26)
HS-PEG-(CONHI).sub.3   T2-4

(27) SH-PEG-(CONHI).sub.3(5K)(T2-4) was prepared by following the procedure of Examples 36-39 of the patent application CN201610398765.4.

(28) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 8.09 (d, 3H), 7.92 (d, 3H), 7.67 (m, 3H), 7.43 (m, 3H), 7.12 (s, 3H), 5.37 (s, 6H), 5.03 (s, 6H), 4.35-4.22 (m, 9H), 4.19 (s, 6H), 4.07 (s, 6H), 3.13 (s, 9H), 2.86-2.67 (m, 9H)), 2.35 (m, 4H), 2.28 (d, 3H), 2.09-1.98 (m, 6H), 1.76-1.53 (m, 30H), 1.31 (s, 9H), 0.91 (m, 9H).

Example 3: Preparation of SH-PEG-CONHI (3.5K) (T5-6)

(29)
HS-PEG-CONHI   T5-6

(30) SH-PEG-CONHI (3.5K) (T5-6) was prepared by following the procedure of Examples 46-51 of the patent application CN201610398765.4.

(31) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 8.09 (d, 1H), 7.92 (d, 1H), 7.67 (m, 1H), 7.43 (m, 1H), 7.12 (s, 1H), 5.37 (s, 2H), 5.03 (s, 2H), 4.35-4.22 (m, 3H), 4.19 (s, 2H), 4.07 (s, 6H), 3.81-3.49 (m, 150H), 3.13 (s, 3H)), 2.86-2.67 (m, 3H), 2.35 (m, 4H), 2.28 (d, 1H), 2.09-1.98 (m, 2H), 1.76-1.53 (m, 10H), 1.31 (s, 9H), 0.91 (m, 9H).

Example 4: Preparation of SH-4ARMPEG5K-(CONH-YSV)3

(32) (1) Preparation of HCl.Tyr-Ser-Val-OMe

(33) 8.4 g (17.5 mmol) of Boc-Tyr-Ser-Val-OMe (prepared according to CN100519576C) was added portionwise to the cooled 4M HCl/ethyl acetate. After the addition, the mixture was stirred at the same temperature for 2 hours, and the TLC was monitored until the disappearance of the starting material; then filtrated and the precipitate was washed with ethyl acetate (50 mL*3) and dry diethyl ether (50 mL*3), and dried. 5.7 g product was obtained, which was used directly for the next reaction.

(34) (2) Preparation of Py-S-S-PEG-(CONH-Tyr-Ser-Val-OMe).sub.3(5K)

(35) 2.5 g (0.5 mmol) of Py-SS-PEG-(COOH).sub.3 and 2.3 g (4.5 mmol) of tyroservatide methyl ester hydrochloride (HCl.Tyr-Ser-Val-OMe), 932 mg (7 mmol) of HOBt were placed into the reaction flask, dissolved in dichloromethane, then added with 1.8 mL (10.5 mmol) of diisopropylethylamine, homogenized by stirring, added with 1.34 g (7 mmol) of EDCI, after that, stirred at room temperature overnight, and concentrated to dryness. The residue was crystallized with isopropanol, filtered and dried to give a white solid.

(36) .sup.1H NMR (300 MHz, DMSO-d.sub.6) δ: 8.57 (s, 1H), 8.23 (s, 3H), 8.17 (s, 3H), 8.12 (s, 3H), 7.72 (m, 1H), 7.47 (d, 1H), 7.26 (d, 1H), 7.12 (d, 6H), 6.98 (d, 6H), 5.03 (m, 3H), 4.88 (m, 3H), 4.53 (m, 3H), 4.19 (d, 6H), 3.87 (s, 9H), 3.18 (m, 9H), 1.12 (d, 18H).

(37) (3) Preparation of Py-S-S-PEG-(CONH-Tyr-Ser-Val-OH).sub.3(5K)

(38) 2.1 g (0.4 mmol) of Py-SS-PEG-(CONH-Tyr-Ser-Val-OMe).sub.3 was dissolved in 20 mL of methanol, cooled to 0° C., and slowly added with 2 mL of 2M NaOH aqueous solution, and stirred at the same temperature after the completion of the dropwise addition. After 6 hours, TLC was monitored until the disappearance of the starting material. The reaction solution was adjusted to neutral with KHSO.sub.4 aqueous solution. The mixture was concentrated under reduced pressure to remove methanol, then adjusted to pH 1-2 with KHSO.sub.4 aqueous solution, extracted with dichloromethane. The organic layers were combined, washed to become neutral, dried, and filtered. The filtrate was evaporated to dryness, and the residue was crystallised by using isopropyl alcohol, filtered, and dried to give 1.8 g product as a white solid.

(39) .sup.1H NMR (300 MHz, DMSO-d6) δ: 8.57 (s, 1H), 8.23 (s, 3H), 8.17 (s, 3H), 8.12 (s, 3H), 7.72 (m, 1H), 7.47 (d, 1H), 7.26 (d, 1H), 7.12 (d, 6H), 6.98 (d, 6H), 5.03 (m, 3H), 4.88 (m, 3H), 4.53 (m, 3H), 4.19 (d, 6H), 3.18 (m, 9H), 1.12 (d, 18H).

(40) (4) Preparation of SH-PEG-(CONH-Tyr-Ser-Val-OH).sub.3(5K)

(41) 1.5 g (0.3 mmol) of Py-SS-PEG-(CONH-Tyr-Ser-Val-OH).sub.3 as the raw material was taken, dissolved in dichloromethane, added with DTT and triethylamine, stirred at room temperature overnight. The reaction solution is concentrated to dry, the residue was crystallised with isopropanol, filtered, and dried to give 1.2 g product as a white solid.

(42) .sup.1H NMR (300 MHz, DMSO-d6) δ: 8.23 (s, 3H), 8.17 (s, 3H), 8.12 (s, 3H), 7.12 (d, 6H), 6.98 (d, 6H), 5.03 (m, 3H), 4.88 (m, 3H), 4.53 (m, 3H), 4.19 (d, 6H), 3.18 (m, 9H), 1.12 (d, 18H).

COUPLING—EXAMPLES

Example 5: Preparation of [PEG-1A]-[PEG-2A]-Drug

(43) [PEG-1A]-[PEG-2A]-Drug was prepared from(MAL)3-4ARMPEG5K-NHS (V-3, prepared in Example 1) and SH-PEG3.5K-CONHI (T5-6, Example 3)), which has a structural formula of:

(44) ##STR00028##

(45) wherein,

(46) Y is

(47) ##STR00029##

(48) Iri is

(49) ##STR00030##

(50) Step 1, feed at a molar ratio of V-3:T5−6=1:3.6 (a mass ratio of about 1:2.8), the total volume of 14 mL;

(51) The V-3 solution (125 mg/mL) was freshly prepared using 1 mM diluted hydrochloric acid, and 3.44 mL (430 mg) was weighed and rapidly added to 10.64 mL of equilibration buffer (PEG1 concentration: 30.7 mg/mL). A T5-6 solution (172 mg/mL) was freshly prepared using 1 mM diluted hydrochloric acid, and 7 mL (1204 mg) was weighed and rapidly added to the above V-3 solution to be used as PEG5K-PEG3.5K-Irinotecan reaction group. (T5-6 final concentration 43 mg/mL, V-3 final concentration 15.4 mg/mL).

(52) Step 2, remove free PEG-Irinotecan by ultrafiltration

(53) (1) Membrane ultrafiltration, molecular weight cutoff 50 KD;

(54) (2) The rotation speed is 100 rpm, and the concentration multiple of each time is recorded;

(55) (3) The filtrate was quantified by UV carried out until the free PEG-Irinotecan did not interfere with the quantification, and the solution was replaced with 50 mM PB, pH 6.0, 97 mM NaCl solution, and the Irinotecan concentration was adjusted to 3.0 mg/mL. A further filtration and sterilization was carried out to give a conjugate.

Example 6: Preparation of a Ligand Drug Conjugate (APEGA-8)

(56) (TM-[PEG-1A]-[PEG-2A]-Drug) was prepared from MAL-PEG6-NHS (purchased from Pomeranian), SH-PEG3.5K-CONHI (T5-6, prepared in Example 3) and recombinant anti-HER2 humanized monoclonal antibody (TM), which has a structural formula of:

(57) ##STR00031##

(58) wherein,

(59) Iri is

(60) ##STR00032##

(61) TM is a recombinant anti-HER2 humanized monoclonal antibody.

(62) Step 1, the preparation of one-step coupling product (PEG1-antibody coupling reactant)

(63) Feed at a molar ratio of TM:MAL-PEG6-NHS=1:120 (a mass ratio of about 1:0.48). 86 mg/mL MAL-PEG6-NHS solution was prepared in DMSO, and 11.2 μL was quickly added to the antibody coupling buffer system (50 mM sodium phosphate pH 6.0, 50 mM sodium chloride, 1 mM EDTA) (80.6 μL 24.8 mg/mL antibody. In 308 μL of coupling buffer). The mixture was gently shaken at room temperature, react for 2 hours to obtain a one-step coupling product; the reaction was stopped at −20° C. to terminate the reaction.

(64) Step 2, solution replacement

(65) The one-step coupled product was replaced by ultrafiltration to 50 mM sodium phosphate, pH 6.0.

(66) Step 3, two-step coupling

(67) Feed at a molar ratio of the PEG1-antibody coupling reactant:T5-6=1:1.

(68) 42 mg/mL of T5-6 solution was prepared by using 1 mM HCl, and 40 μL was quickly added to the PEG1-antibody coupling reaction solution (80 μL), and the mixture was gently shaken at room temperature, react for 2 hours; and stored at −20° C. to terminate the reaction. The free T5-6 molecule was removed by ultrafiltration and the solution was replaced with 50 mM PB, pH 6.0, 97 mM NaCl solution. A further filtration and sterilization was carried out to give a conjugate.

Example 7: Preparation of a Ligand Drug Conjugate (APEGA-9)

(69) (TM-[PEG-1A]-[PEG-2A]-Drug) was prepared from (MAL) 3-4ARMPEG5K-NHS (V-3, prepared in Example 1), SH-PEG3.5K-CONHI (T5-6, prepared in Example 3), recombinant anti-HER2 Humanized Monoclonal Antibody (TM), which has a structural formula of:

(70) ##STR00033##

(71) wherein,

(72) Y is

(73) ##STR00034##

(74) Iri is

(75) ##STR00035##

(76) TM is a recombinant anti-HER2 humanized monoclonal antibody.

(77) Step 1, the preparation of one-step coupling product (PEG1-antibody coupling reactant)

(78) Feed at a molar ratio of antibody:V-3=1:60 (a mass ratio of about 1:2). 358 mg/mL V-3 solution was prepared in DMSO, and 11.2 μL was quickly added to the antibody coupling buffer system (50 mM sodium phosphate pH 8.0, 50 mM sodium chloride, 1 mM EDTA) (80.6 μL 24.8 mg/mL antibody in 308 μL Coupling buffer), gently shake at room temperature, react for 2 hours to obtain a one-step coupling product; store at −20° C. to terminate the reaction.

(79) Step 2, solution replacement

(80) The one-step coupled product was replaced by ultrafiltration to 50 mM sodium phosphate, pH 6.0.

(81) Step 3, two-step coupling

(82) Feed at a molar ratio of PEG1-antibody coupling reactant:T5-6=1:1. 14 mg/mL of T5-6 solution was prepared by using 1 mM HCl, and 40 μL was quickly added to the PEG1-antibody coupling reaction solution (80 μL), and the mixture was gently shaken at room temperature, react for 2 hours; and stored at −20° C. to terminate the reaction. The free T5-6 molecule was removed by ultrafiltration and the solution was replaced with 50 mM PB, pH 6.0, 97 mM NaCl solution. A further filtration and sterilization was carried out to give a conjugate.

Example 8: Preparation of Antibody Drug Conjugate (APEGA-10)

(83) (TM-[PEG-1A]-[PEG-2A]-Drug), was prepared from (MAL) 3-4ARMPEG5K-NHS (V-3, prepared in Example 1), SH-4ARMPEG5K-(CONHI)3 (T2-4, prepared in Example 2), and recombinant anti-HER2 humanized monoclonal antibody (TM). The preparation method steps 1 and 2 refer to Example 47. It has a structural formula of:

(84) ##STR00036##

(85) wherein

(86) Z is

(87) ##STR00037##

(88) Iri is

(89) ##STR00038##

(90) TM is a recombinant anti-HER2 humanized monoclonal antibody.

(91) Step 3, two-step coupling

(92) Feed at a molar ratio of PEG1-antibody coupling reactant:T2−4=1:1. 20 mg/mL of T2-4 solution was prepared by using 1 mM HCl, and 40 μL was quickly added to the PEG1-antibody coupling reaction solution (80 μL), and the mixture was gently shaken at room temperature for 2 hours; and stored at −20° C. to terminate the reaction. The free T2-4 molecule was removed by ultrafiltration and the solution was replaced with 50 mM PB, pH 6.0, 97 mM NaCl solution. A further filtration and sterilization was carried out to give a conjugate.

Example 9: Determination of Antibody Drug Conjugate Antibody Content in Examples 6, 7, and 8

(93) Method: UV/Vis Method

(94) In formula (1), the sum of the absorbances of the drug and the antibody at 280 nm constitutes the total absorbance (A.sub.280):
A.sub.280=(ε.sub.drug.sup.280C.sub.drug+ε.sub.mAb.sup.280C.sub.mAb)l  (1)

(95) wherein ε.sub.drug.sup.280 is the extinction coefficient of the drug at 280 nm; C.sub.drug is the drug concentration (mg/mL); E bis the extinction coefficient of the antibody at 280 nm; C.sub.mAb is the concentration of the antibody.

(96) Equation (2) is the parallel equation for the total absorbance of the drug at the maximum absorption λ(D):
A.sub.λ(D)=(ε.sub.drug.sup.λ(D)C.sub.drug+ε.sub.mAb.sup.λ(D)C.sub.mAb)l  (2)

(97) wherein ε.sub.drug.sup.λ(D) is the extinction coefficient of the drug at λ(D)nm; C.sub.drug is the drug concentration (mg/mL); ε.sub.mAb.sup.λ(D) is the extinction coefficient of the antibody at λ(D)nm; C.sub.mAb is the concentration of the antibody (mg/mL).

(98) The concentration of antibody and drug can be calculated separately by two equations (1) and (2).
C.sub.mAb=(A.sub.280ε.sub.drug.sup.λ(D)−A.sub.λ(D)ε.sub.drug.sup.280)/[(ε.sub.mAb.sup.280ε.sub.drug.sup.λ(D)−ε.sub.mAb.sup.λ(D)ε.sub.drug.sup.280)l]   (3)
C.sub.drug=(A.sub.280ε.sub.mAb.sup.λ(D)−A.sub.λ(D)ε.sub.mAb.sup.280)/[(ε.sub.drug.sup.280ε.sub.mAb.sup.λ(D)−ε.sub.drug.sup.λ(D)ε.sub.mAb.sup.280)l]   (4)

(99) The average drug antibody coupling ratio (DAR) calculated by dividing

(100) $\frac{C_{\mathrm{drug}}}{{\mathrm{Mr}}_{\mathrm{drug}}}\mathrm{by}\frac{C_{\mathrm{mAb}}}{{\mathrm{Mr}}_{\mathrm{mAb}}}$
is expressed as the number of moles of drug divided by the number of moles of antibody:

(101) $\begin{array}{cc}\mathrm{DAR}=\frac{C_{\mathrm{drug}}\times {\mathrm{Mr}}_{\mathrm{mAb}}}{C_{\mathrm{mAb}}\times {\mathrm{Mr}}_{\mathrm{drug}}}& \left(5\right)\end{array}$

(102) The result is shown in Table 1:

(103) TABLE-US-00001 TABLE 1 The summary of the quantification of two-step coupling products by UV-Vis C.sub.drug C.sub.mAb Drug (mg/mL) (mg/mL) DAR λ(254) λ(280) APEGA-8 (1ARM + 0.119 0.193 22.2 0.722 0.313 1ARM) APEGA-9 (4ARM + 0.184 0.186 35.6 1.226 0.505 1ARM) APEGA-10 (4ARM + 0.251 0.056 95.3 1.556 0.37 4ARM)

Example 10: Preparation of Antibody Drug Conjugate (APEGA-11)

(104) (TM-[PEG-1A]-[PEG-2A]-Drug) was prepared from (MAL)3-4ARMPEG5K-NHS (V-3, prepared in Example 1), SH-4ARMPEG5K-(CONH-YSV)3 (prepared in Example 4), Recombinant Anti-HER2 Humanized Monoclonal Antibody (TM), which has a structural formula of:

(105) ##STR00039##

(106) wherein

(107) T is

(108) ##STR00040##

(109) P is tyroservatide, YSV,

(110) TM is a recombinant anti-HER2 humanized monoclonal antibody.

(111) Step 1, preparation of one-step coupling product (PEG1-antibody coupling reactant)

(112) Feed at a molar ratio of antibody:V-3=1:600 (a mass ratio of about 1:2). 358 mg/mL V-3 solution was prepared with 1 mM HCl, and 55.8 μL was quickly added to the antibody coupling buffer system (50 mM sodium phosphate pH 8.0, 50 mM sodium chloride, 1 mM EDTA) (40.3 μL 24.8 mg/mL antibody in 104 μL of coupling buffer), gently shaken at room temperature, react for 2 hours to obtain a one-step coupling product; storage at −20° C. to terminate the reaction.

(113) Step 2, solution replacement

(114) The one-step coupled product was replaced by ultrafiltration to 50 mM sodium phosphate, pH 6.0.

(115) Step 3, two-step coupling

(116) Feed at a molar ratio of the PEG1-antibody coupling reactant:SH-4ARMPEG5K-(CONH-YSV)=1:1, a solution of 20 mg/mL SH-4ARMPEG5K-(CONH-YSV)3 was prepared with 1 mM HCl, and 40 μL was quickly added to the PEG1-antibody coupling reaction solution (80 μL), and gently shaken at room temperature, react for 2 hours; stored at −20° C. to terminate the reaction. The free SH-4ARMPEG5K-(CONH-YSV) 3 molecule was removed by ultrafiltration and the solution was replaced with 50 mM PB, pH 6.0, 97 mM NaCl solution. A further filtration and sterilization was carried out to give a conjugate.

Example 11: Determination of Drug Loading for the Antibody Drug Conjugate in Example 10

(117) Principle:

(118) $\mathrm{coupling}\mathrm{rate}=\frac{C_{\mathrm{drug}}\times {\mathrm{Mr}}_{\mathrm{mAb}}}{C_{\mathrm{mAb}}\times {\mathrm{Mr}}_{\mathrm{drug}}}$

(119) wherein: C(drug) is the concentration of YSV coupled to the Ab in the reaction solution, and is obtained by HPLC subtractive feeding method;

(120) C(Ab) is the concentration of Ab in the reaction solution;

(121) Mr (Ab) is the molecular weight of Ab, 150 KD;

(122) Mr (Drug) is the molecular weight of YSV.

(123) The coupling rate was 97.6 as a result of the determination.

Pharmacokinetics, Pharmacodynamics Experiment

Example 12: Antibody Pharmacokinetic Assay of Antibody Drug Conjugates Prepared in Examples 6, 7, and 8

(124) Experimental method: SD rats were anesthetized by intraperitoneal injection of 40 mg/kg of 1% pentobarbital sodium, and the skin was prepared by the neck and the front of the neck, and disinfected by iodophor. Cut the skin at the right of the neck and expose the jugular vein. After the venous catheter is inserted into the blood vessel, it is ligated and the skin at the opening is sutured. After the end of the operation, about 0.2 mL of the heparin sodium solution and 0.1 mL of the blocking solution were injected into the catheter, and thereafter replaced every day for one week. After one week, the surgical wounds of the rats were all healed, the catheter was fixed, and the blood was taken repeatedly to be used for the pharmacokinetic study of this project. Recombinant anti-HER2 humanized monoclonal antibody (hereinafter abbreviated as Ab) and APEGA-8, 9, 10 (prepared in Examples 6-8, respectively) were administered via the tail vein, respectively, and the blood was taken from the animals at a predetermined time after administration, to perform an analysis.

(125) 100 μL/well of 0.05 μg/mL human HER2 protein was coated in microwells with 0.05 M carbonate buffer and incubated overnight at 4° C. Wash the plate with 400 μL PBST, add 300 μL of blocking solution, block at 37° C. for 1 h, and wash the plate with 400 μL of PBST. 100 μL/well of the standard and the sample to be tested were added to the above-mentioned coated reaction wells, and incubated at 37° C. for 1 hour. Then wash. Add an enzyme-labeled antibody: 100 μL/well of goat anti-human IgG enzyme-labeled antibody was added to each reaction well. Incubate for 1 hour at 37° C. and wash. Add substrate liquid to develop: Add 50 μL/well of the temporarily prepared TMB substrate solution to each reaction well at 37° C. for 15-30 minutes. Reaction termination: 50 μL/well of 2 M sulfuric acid was added to each reaction well. The absorbance was measured at 450 nm on an ELISA detector. The linear regression of the standard absorbance value corresponding to the absorbance value (subtracting the blank) was performed, and the regression equation is obtained; the absorbance value of the product to be inspected (subtracting the blank) is incorporated into the standard curve equation to obtain the antibody concentration in the sample to be tested.

(126) The Experimental results were shown in FIG. 1.

(127) The results showed that the elimination rate of APEGA-8, APEGA-9 and APEGA-10 in rat plasma increased to a certain extent compared with the control Ab, but the decline trend was not significant.

Example 13: Irinotecan Pharmacokinetic Test of Antibody Drug Conjugate Prepared in Example 7

(128) Experimental method: SD rats were anesthetized by intraperitoneal injection of 40 mg/kg of 1% pentobarbital sodium, and the skin was prepared by the neck and the front of the neck, and disinfected by iodophor. Cut the skin at the right of the neck and expose the jugular vein. After the venous catheter is inserted into the blood vessel, it is ligated and the skin at the opening is sutured. After the end of the operation, about 0.2 mL of the heparin sodium solution and 0.1 mL of the blocking solution were injected into the catheter, and thereafter replaced every day for one week. After one week, the surgical wounds of the rats were all healed, the catheter was fixed, and the blood was taken repeatedly to be used for the pharmacokinetic study of this project. Ab, APEGA-9 (prepared in Example 47) and PEG5K-PEG3.5K-Iri (hereinafter abbreviated as PEG-Irinotecan, prepared in Example 45) were administered via the tail vein, respectively, and the blood was taken from the animals at a predetermined time after administration, to perform an analysis.

(129) The assay used a validated HPLC method to detect the content of irinotecan.

(130) Test results were shown in FIG. 2.

(131) The results showed that, compared with PEG-Irinotecan (4.2 mg/kg), the equivalent dose of APEGA-9 after the antibody was conjugated slowed the elimination of irinotecan in plasma over time. The terminal elimination half-life of APEGA-9 was significantly longer than that of PEG-Irinotecan. It is suggested that APEGA-9 elimination rate is significantly slower than PEG-Irinotecan, and the amount of irinotecan exposed to plasma is significantly higher than PEG-Irinotecan.

Example 14: Efficacy Test of Antibody Drug Conjugate Prepared in Examples 6, 7, and 8

(132) Experimental method: Evaluation of the efficacy of the sample was performed using a colorectal cancer model in which HER2 was not expressed. The cell line at logarithmic growth phase was inoculated subcutaneously into the right side of the immunodeficient mice, and the cell inoculation amount was 5×10.sup.6 cells/mouse. After the transplanted tumor was formed, allow it grow in the mice for 2 passage before use. The tumor tissue with strong growth period was cut into tumor pieces with a diameter of about 2 mm, and inoculated subcutaneously into the right torso of nude mice under sterile conditions. The formed tumor tissue is measured by a vernier caliper, and the long and short diameters are represented by a and b, respectively, and the tumor volume (TV) is calculated as: TV=½×a×b.sup.2. Animals were randomized after tumor growth to 100-150 mm.sup.3. Both models were divided into vehicle group, control group (recombinant anti-HER2 humanized monoclonal antibody Ab, 30 mg/kg) and test group I (administered with APEGA-8, 9, 10, respectively (prepared in Example 6-8), the dose was 30 mg/kg in terms of Ab, and the test group II (PEG-Irinotecan (prepared in Example 5), the dose was 30 mg/kg). Both the test article and the control drug were administered once a week vial the tail vein for a total of three administrations. The diameter of the transplanted tumor was measured twice a week during the entire experiment, and the body weight of the mice was weighed. After the end of the administration, continue to observe the animals for a week, and then they were sacrificed.

(133) Experimental results: The test results were shown in FIG. 3 and Table 2.

(134) TABLE-US-00002 TABLE 2 Comparison of tumor inhibition rates in the third week of administration of test articles in colorectal cancer model (HCT-116) Drug loading (based on Tumor inhibition Test article irinotecan, mg/kg) rate (%) Vehicle / 0 Ab 30 mg/kg QW×3 / −11.7 APEGA-8 (1ARM + 1ARM) 2.6 40.1 30 mg/kg QW × 3 APEGA-9 (4ARM + 1ARM) 4.2 49.5 30 mg/kg QW × 3 APEGA-10 (4ARM + 4ARM) 11.2 81.7 30 mg/kg QW × 3 PEG-Irinotecan 30 mg/kg 30 36.7 QW × 3

(135) The results showed that the antitumor activities of the tested APEG-8, APEGA-9 and APEGA-10 increased with the drug loading, and APEGA-10 had stronger anticancer activity; the antibody control did not show antitumor activity. In the case that the dose of irinotecan was lower than that of group II, the efficacy of group I (APEG-8, APEGA-9 and APEGA-10, at a dose of 2.6, 4.2, 11.2 mg/kg, respectively) was still better than group II (PEG-Irinotecan, at a dose of 30 mg/kg). During the whole experiment, no obvious abnormal responding of the animals was observed, and the tolerance to the drugs was good.