WO2019034177A1 - Antibody drug conjugate having two different drugs - Google Patents

Abstract

The invention discloses an antibody drug conjugate having two different drugs. According to the present invention, a drug is linked to a cysteine residue in an antibody in a fixed-point coupling manner, and a second cytotoxic drug having a different action mechanism is linked to the cysteine residue in the antibody in a non fixed-point coupling manner.

Description

Antibody drug conjugate with two different drugs Technical field

The present invention relates to an antibody drug conjugate having two different drugs.

Background technique

As a novel targeted drug, antibody-conjugated drugs (ADCs) generally consist of three parts: antibody or antibody ligands, small molecule drugs, and linkers that couple ligands to drugs. The antibody-conjugated drug utilizes the specific recognition of the antigen by the antibody, transports the drug molecule to the vicinity of the target cell and effectively releases the drug molecule for therapeutic purposes. In August 2011, the US Food and Drug Administration (FDA) approved the launch of the new ADC, Adecteis ^TM , developed by Seattle Genes for the treatment of Hodgkin's lymphoma and relapsed large cell lymphoma (ALCL). The safety and efficacy of such drugs.

The ADC drug antibody mainly functions as a targeted delivery function, and finally the drug effect is the coupled drug molecule. The drug molecules currently used in the field of ADC are classified according to different mechanisms of action: microtubule inhibitors, DNA damaging agents, topoisomerase inhibitors, RNA polymerase inhibitors, protein translation inhibitors, and the like.

In ADC drugs, antibodies carry drug molecules to the vicinity of tumor cells, releasing drug molecules around or within the tumor cells. At the same time, however, tumors have developed different mechanisms against drug molecules during this treatment, including pumping toxin molecules out of the cell with a PgP protein pump to escape killing. At present, ADC drug development mainly focuses on single antibody linking single-acting mechanism drugs. Modern medicine has proved that the combination of different drugs in the chemotherapy process can significantly enhance the efficacy. However, as the number and type of drugs in the ADC increase, the lipid solubility of the molecule increases, the stability in plasma is significantly reduced, and the efficacy is not improved, but also causes side effects.

In the currently known literature, the patent of US Sorrento Therapeutics Co., Ltd. CN106132431 (A) utilizes the amino residue of lysine (also known as K-LOCK technology) and cysteine sulfhydryl residue (C) in the antibody molecule, respectively. -LOCK technology) enables the connection of different drugs to antibodies. Due to the limitations of the coupling method, the C-LOCK technique often results in a DAR of 2-4, and the K-LOCK technique also has obvious limitations: when the lysine-linked drug antibody coupling ratio (DAR) is >2 Since a single antibody molecule contains about 88 lysines, such a large amount of lysine results in poor amino coupling selectivity, and the number of couplings and the coupling position are difficult to determine, although a certain degree of control can be achieved by coupling conditions. However, there are significant challenges in preparing complex antibody conjugates with two different drugs. And the patent CN106132431 does not report the pharmacodynamic data and plasma stability in animals.

In ADC drugs, an innovative coupling method is needed to achieve plasma stabilization, antibody-conjugated drugs coupled to two different drugs, and a more convenient combination of drugs is achieved. The present invention surprisingly meets the above needs.

Summary of the invention

The present invention aims to provide an antibody-drug conjugate having two different drugs. Two different drug molecules are linked to the same antibody by designing the conjugate. Due to the combined use of different mechanisms of action, the conjugate can effectively improve the efficacy.

In particular, the present invention provides an antibody drug conjugate comprising a different drug, or a pharmaceutically acceptable salt thereof, of Formula I

Where Ab is an antibody moiety;

L1, L2 are optional linking units connectable to the drug;

D1 and D2 are drug units;

m and n are integers of 2-8.

Preferably, the drugs D1 and D2 are antitumor drugs having different mechanisms of action.

More preferably, the drugs D1, D2 are preferably selected from the group consisting of tubulin binding agents, DNA alkylating agents, DNA intercalating agents, enzyme inhibitors, immunomodulators, peptides and nucleotides, respectively.

In some preferred embodiments, the m is preferably 2, 4.

In one aspect of the invention, the drug D1 is preferably attached to the antibody in a site-directed coupling manner, and the D2 is coupled to the antibody in a non-targeted manner.

In some embodiments, the preferred site-directed coupling of drug D1 is a half-pointed mutant antibody original amino acid to cysteine or by insertion of a cysteine or a cysteine-containing polypeptide, which is introduced by the above method. The cystine thiol is coupled to the linker-toxin. A preferred non-targeted coupling of D2 is the coupling of the original interchain disulfide bond of the antibody to the linker-toxin.

Preferably, in the present invention, L1 and L2 have the following formula 2:

Wherein C is an optional extendable unit at the end, E is an optional cleavable unit, F is a spacer unit, and subscript e, f is 0 or 1. The wavy line indicates the attachment site to the succinimide and the drug unit.

More preferably, the antibody drug conjugate, the cleavable unit E is absent at e=0 and is present at e=1. When present, the cleavable unit described by E is achieved by a tumor-associated protease or an acidic pH with a drug unit D or a spacer unit F

More preferably, the antibody drug conjugate, the spacer unit F is absent at f=0 and is present at f=1. When F is present, F is selected from the group consisting of p-aminobenzyl alcohol or with ethylenediamine units and derivatives thereof.

More preferably, the drug-ligand conjugate compound of any of the present invention comprises a pharmaceutically acceptable salt thereof, and a pharmaceutical composition of a pharmaceutically acceptable diluent carrier or excipient.

More preferably, the invention comprises administering to the patient the drug-ligand conjugate of any of the preceding claims, wherein the patient has a tumor, an autoimmune disease or an infectious disease, and The antibody-ligand conjugate antibody specifically binds to the target cell of the cancer, autoimmune disease

DRAWINGS

Figure 1 shows the results of the Payload S MS-TOF test.

Figure 2 shows the results of the Payload E MS-TOF test.

Figure 3 shows a graph of the results of the ADC and control ADCs with different drugs.

Detailed ways

After extensive and intensive research, the inventors have surprisingly found that antibody-drug conjugates with two different mechanisms of action have better in vitro and in vivo efficacy than ADC drugs of the traditional single mechanism of action.

Specifically, the antibody drug conjugate represented by the following formula is provided by the present invention

Abbreviations and definitions

Unless otherwise stated, the following terms and phrases as used herein are intended to have the following meanings. When a trade name is used herein, unless otherwise indicated by the context, the trade name includes the product formulation, generic drug, and active pharmaceutical ingredient of the trade name product.

Unless otherwise indicated herein, a "derivative" of a compound as used herein refers to a chemical structure that is similar to a compound but also contains at least one chemical group that is not present in the compound and/or lacks the chemical group present in at least one of the compounds. The substance of the regiment. The compounds to which the derivatives are compared are referred to as "parent" compounds. Generally, a "derivative" can be produced from a parent compound in one or more chemical reaction steps.

As used herein, "antibody" or "antibody unit" is within the scope of its disclosure, including any portion of the antibody structure. This unit can bind, reactively associate, or complex a receptor, antigen, or other receptor unit that the cell population has. The antibody can be any protein or proteinaceous molecule that can bind, complex, or react with a portion of the cell population to be treated or bioengineered. In some embodiments, the linker is covalently attached to the sulfur atom of the antibody. In some aspects, the sulfur atom is a sulfur atom of a cysteine residue that forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom that has been introduced into the cysteine residue of the ligand unit, which forms an interchain disulfide bond of the antibody. In another aspect, the sulfur atom is a sulfur atom that has been introduced into the cysteine residue of the ligand unit (eg, by site-directed mutagenesis or chemical reaction). In other aspects, the linker-bonded sulfur atom is selected from a cysteine residue that forms an interchain disulfide bond of the antibody or a cysteine residue that has been introduced into the ligand unit (eg, by site-directed mutagenesis or chemical reaction). In some embodiments, the EU index in Kabat (Kabat EA et al, (1991)) "Sequences of proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242" Numbering system.

The antibody constituting the antibody drug conjugate of the present invention preferably retains the antigen binding ability in its original wild state. Therefore, the antibody of the present invention can, preferably, specifically bind to an antigen. Antigens involved include, for example, tumor associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell survival regulators, cell proliferation regulators, molecules associated with tissue growth and differentiation (as known or predicted) Functional), lymphokines, cytokines, molecules involved in cell cycle regulation, molecules involved in angiogenesis, and molecules involved in angiogenesis (as known or predicted to be functional). The tumor associated factor may be a cluster differentiation factor (such as a CD protein).

Antibodies for use in antibody drug conjugates described herein include, but are not limited to, antibodies directed against cell surface receptors and tumor associated antigens. Such tumor associated antigens are well known in the art and can be prepared by antibody preparation methods and information well known in the art. To develop effective cell-level targets for cancer diagnosis and treatment, researchers have sought to find transmembrane or other tumor-associated polypeptides. These targets are capable of being specifically expressed on the surface of one or more cancer cells with little or no expression on the surface of one or more non-cancer cells. Typically, such tumor-associated polypeptides are more overexpressed on the surface of cancer cells relative to the surface of non-cancer cells. Confirmation of such tumor-associated factors can greatly enhance the specific targeting characteristics of cancer-based treatment of cancer.

Tumor-associated antigens include, but are not limited to, tumor-associated antigens well known in the art. Nucleic acid and protein sequences corresponding to tumor associated antigens can be found in public databases such as Genbank. Antibody-targeting corresponding tumor-associated antigens include all amino acid sequence variants and isoforms, having at least 70%, 80%, 85%, 90%, or 95% homology to the sequences identified in the references, or The tumor-associated antigen sequences cited in the literature have completely identical biological properties and characteristics.

The term "inhibiting" or "inhibiting" means reducing the detectable amount or completely preventing it.

The term "cancer" refers to a physiological condition or disease characterized by dysregulated cell growth. "Tumors" include cancer cells.

The term "autoimmune disease" is a disease or disorder that results from tissue or protein directed against the individual's own body.

As used herein, "site-directed coupling" is preferably a cysteine introduced by the above method, either by the original amino acid of the site-directed mutant antibody to cysteine or by the insertion of a cysteine or a cysteine-containing polypeptide into the antibody. The thiol group is coupled to the succinimide in the linker. The "non-site-coupled" mode is coupled to the linker-toxin by utilizing the original interchain disulfide bond of the antibody.

The phrase "pharmaceutically acceptable salt" as used herein refers to a pharmaceutically acceptable organic or inorganic salt of a compound (eg, a drug, a drug-linker or a ligand-linker-drug conjugate). The compound may contain at least one amino or carboxyl group and thus may form an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, chlorides, bromides, iodides, nitrates, hydrogen sulfates, phosphates, acids Phosphate, isonicotinic acid, lactate, salicylate, acidic citrate, tartrate, oleate, tannic acid, pantothenate, hydrogen tartrate, ascorbate, salicylate, Formate, orthoformate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium salt, sodium salt, and the like. Additionally, pharmaceutically acceptable salts have more than one dotted atom in the structure. An example in which a plurality of charged atoms are part of a pharmaceutically acceptable salt can have multiple counterexamples. For example, a pharmaceutically acceptable salt has one or more charged atoms and/or one or more counter atoms.

Drugs refer to: a cytotoxic drug used in the treatment of cancer, including but not limited to maytansine or maytansinoid, doxatin 10 (Dolastatin 10) analogs, calicheamicin drugs Pyrrolo[2,1-c][1,4]benzodi-azepines (PBDs) or PBD dimers (PBD dimmers) and derivatives amanita or derivatives thereof Medium active drugs include, but are not limited to, benzopyrrolidone (duocarmycins, CC-1065, etc.) camptothecin compounds including camptothecin, hydroxycamptothecin, SN-38, ezetidine, irinotecan Wait.

On the other hand, the drug is not limited to the above-mentioned categories, but also includes all drugs that can be used for antibody drug conjugates.

According to the mechanism of drug release in cells, as used herein, "linking unit" or "linking unit of antibody drug conjugate" can be divided into two categories: non-cleavable linking unit and cleavable linking unit.

For an antibody drug conjugate containing a non-cleavable linker unit, the drug release mechanism is: after the conjugate is bound to the antigen and endocytosed by the cell, the antibody is hydrolyzed in the lysosome, and the drug is released by the small molecule drug. An active molecule composed of an amino acid residue of an antibody. The resulting change in the molecular structure of the drug does not diminish its cytotoxicity, but since the active molecule is charged (amino acid residues), it cannot penetrate into adjacent cells. Therefore, such active drugs cannot kill tumor cells (bystander effect) adjacent to the non-expressing target antigen (antigen-negative cells) (Ducry et al, 2010, Bioconjugate Chem. 21: 5-13).

A cleavable linker unit, as the name implies, can cleave in the target cell and release the active drug (small molecule drug itself). Breakable linkers can be divided into two main classes: chemically labile linkers and enzyme labile linkers.

Chemically labile linkers can be selectively cleaved due to differences in plasma and cytoplasmic properties. Such properties include pH, glutathione concentration, and the like.

pH sensitive linkers are often referred to as acid cleavage linkers. Such a linker is relatively stable in the neutral environment of blood (pH 7.3-7.5), but will be in the weakly acidic endosomes (pH 5.0-6.5) and lysosomes (pH 4.5-5.0). hydrolysis. Most of the first generation of antibody drug conjugates use such linkers, such as hydrazine, carbonate, acetal, ketal. Antibody drug conjugates based on such linkers typically have a shorter half-life (2-3 days) due to the limited plasma stability of the acid-cleaved linker. This shorter half-life limits the use of pH-sensitive linkers in a new generation of antibody drug conjugates to some extent.

For glutathione-sensitive linkers, it is also called disulfide bond. Drug release is based on a difference between the high concentration (in millimolar range) of intracellular glutathione and the relatively low concentration of glutathione (micromolar range) in the blood. This is especially true for tumor cells, where low oxygen levels result in enhanced reductase activity, resulting in higher glutathione concentrations. Disulfide bonds are thermodynamically stable and therefore have better stability in plasma.

Enzyme-labile linkers, such as peptide linkers, provide better control of drug release. Peptide linkers are capable of being efficiently cleaved by lysosome in vivo proteases such as cathepsin B or plasmin (an increase in the amount of such enzymes in some tumor tissues). This peptide linkage is believed to be very stable in the plasma circulation because the extracellular pH is inappropriate and the serum protease inhibitors cause the protease to be generally inactive. In view of the high plasma stability and good intracellular cleavage selectivity and effectiveness, enzyme-labile linkers are widely used as cleavable linkers for antibody drug conjugates. Typical enzyme-labile linkers include Val-Cit (vc), Phe-Lys, and the like.

Suicide linkers are typically chimeric between the cleavable linker and the active drug, or are themselves part of a cleavable linker. The mechanism of action of the suicide linker is that when the cleavable linker is broken under suitable conditions, the suicide linker can spontaneously rearrange the structure and release the active drug attached thereto. Common suicide linkers include p-aminobenzyl alcohols (PAB) and beta-glucuronides.

This patent may use the following abbreviations and have the specified definitions: Boc, tert-butoxycarbonyl; DCC, cyclohexylcarbodiimide; DCM: dichloromethane; DIPEA: diisopropylcarbodiimide; DMF: N , N-dimethylformamide; DMAP: 4-(N,N-dimethylamino)pyridine; HATU: 2-(7-oxobenzotriazole)-N,N,N',N'- Tetramethylurea hexafluorophosphate; HPLC: high performance liquid chromatography; PEG: polyethylene glycol; TFA: trifluoroacetic acid; THF: tetrahydrofuran; PBS: phosphate buffer solution (pH 7.0-7.5).

The pharmaceutically acceptable excipients include any carrier, diluent, adjuvant or excipient such as preservatives and antioxidants, fillers, disintegrating agents, wetting agents, emulsifying agents, suspending agents, solvents, dispersion media. , coating agents, antibacterial and antifungal agents and absorption delaying agents, and the like. The use of such media and agents for the use of pharmaceutically active substances is well known in the art. In addition to any conventional media or agents that are incompatible with the active ingredients, their use in therapeutic compositions is also contemplated. As a suitable therapeutic combination, the additional active ingredient can also be incorporated into the compositions.

The main advantages of the invention are:

1. The antibody drug conjugate provided by the invention with two different drugs can effectively enhance the drug effect and obtain better therapeutic effect because of different action mechanism of the drug.

2. The method of the present invention provides a combination of site-directed coupling and non-spotting coupling, and the antibody drug conjugate containing two different drugs can be obtained by simple coupling.

The invention is further illustrated by the following examples, which are intended to illustrate the invention and not to limit the scope of the invention. The test methods which do not specify the specific conditions in the following examples are usually carried out according to conventional conditions or according to the conditions recommended by the manufacturer. All percentages, ratios, ratios, or parts are by weight unless otherwise indicated.

All the professions and sciences used herein are used in the same sense as those skilled in the art, unless otherwise defined. In addition, any methods and materials similar or equivalent to those described may be employed in the methods of the invention. The preferred embodiments and materials described herein are for illustrative purposes only.

The general steps employed in the following embodiments of the invention are:

General Procedure A Antibody Site-directed Mutagenesis

The PCR reaction system (50 μL) was prepared in the following proportions, mixed thoroughly and then centrifuged instantaneously. The system contained:

After PCR amplification, 1 μL of DpnI (10 U/μL) was added and incubated at 37 ° C for 3 h; 1 μL of DpnI-treated PCR product was taken for E. coli transformation experiment as follows: ice bath 30 min; heat shock 42 ° C 30 sec; ice bath 2 min Add 150 μL of LB liquid medium, incubate at 37 ° C, 150 rpm for 30 min; centrifuge at 3000 × g RT for 3 min, remove the supernatant, leave 50 μL of the culture supernatant and suspend the cells, and apply to Amp-containing LB solid medium; Cultured at °C for 16h; single colonies were picked and transferred to 5 mL of LB liquid medium containing Amp (pTT5). After overnight culture, the plasmid was extracted and identified by PCR sequencing. General Step B Coupled with Different Toxins to Prepare ADC

The antibody molecules having a monomeric ratio of more than 95% after preliminary purification were exchanged into a phosphate buffer solution containing EDTA using an ultrafiltration centrifuge tube at a concentration of 10 mg/ml. TCEP was added 10 times the number of molecules of the antibody, and reacted at room temperature for 2 h. The solution was changed to a phosphate buffer of pH 6.5 using an ultrafiltration centrifuge tube, and DHAA was added 10 times the number of molecules of the antibody, and reacted at room temperature for 2 hours. Then, a payload 1 of 3 times the number of molecules of the antibody was added, and the reaction was carried out for 4 hours at room temperature. After the reaction, the solution was exchanged into a phosphate buffer containing EDTA using an ultrafiltration centrifuge tube having a molecular weight cut off of 30 KDa, and the unconjugated payload 1 was removed. TCEP was added 10 times the number of molecules of the antibody, and reacted at room temperature for 8 hours. The interchain disulfide bond was opened, and the number of free thiol groups was determined by the Ellman method to determine whether the disulfide bonds were all open. Then, add 2 times the antibody molar number of payload 2, and react at room temperature for 8 hours. After completion of the reaction, the solution was exchanged into PBS using an ultrafiltration centrifuge tube having a molecular weight cut off of 30 KDa, and unconjugated payload 2 was removed.

General procedure C

Pharmacokinetic study

ELISA method for detecting antibodies in serum: anti-antibody 2ug/ml 4°C coating overnight, PBST wash plate 3 times, 1% BSA+PBST blocked at 37°C for 1 hr; serum samples were incubated, PBST was washed 3 times; incubation antibody was incubated at 37°C (Anti-Fc monoclonal antibody or polyclonal antibody (HRP label)) 1 hr, PBST wash plate 3 times, TMB color development, 2M H2SO4 termination, microplate reader reading. General Step D

Hydrophobic Interaction Chromatography (HIC)

Analysis of the ADC was performed using hydrophobic interaction chromatography (HIC). Degassed by 0-100% mobile phase B (MPB) consisting of 1.5 M ammonium sulfate and .025 M sodium phosphate, and MPB consisting of 0.025 M sodium phosphate, 25% isopropanol. The sample loading was approximately 20 μg and the gradient elution was completed in 15 minutes. When the UV 280 nm was used for detection, the stronger the water-transfer sample, the later the peak.

General procedure E

Plasma stability study

Take a certain amount of ADC sample, add it to human plasma from which human IgG has been removed, repeat three tubes of each ADC, incubate in a 37 °C water bath, incubate for 0h, 72h, and then take out the ADC sample, and add ProteinA (MabSelect SuReTM) to each tube. LX Lot: #10221479GE, washed with PBS 100 ul, shaking with a vertical mixer for 2 h, after washing and eluting steps to obtain the ADC after incubation. RP-HPLC detection of ADC samples for specific time of incubation. Plasma stability.

Example 1-6

Example 1 Synthesis of Compound 1

Fmoc-Lys(mmt)-OH (2g, 3.2mmol, 1eq), PABA (788.16mg, 6.4mmol, 2eq), HATU (1.34g, 3.52mmol, 1.1eq), DIEA (1.24g, 9.6) Mmmol, 3 eq) and 20 ml of DMF, stirred and dissolved, nitrogen-protected, 25 ° C reaction; spot plate detection, lysine reaction was complete. After adding 200 ml of water to the reaction mixture, a large amount of solid was precipitated, EA (60 ml×3) was extracted, and the organic phase was washed three times with brine, dried over anhydrous sodium sulfate and evaporated The intermediate in the previous step was dissolved in 20 ml of diethylamine, protected by nitrogen, and reacted at 25 ° C; the plate was tested and the reaction of the starting materials was complete. The solvent was sparged and dissolved in EtOAc (3 mL). Column chromatography purification: Gradient elution with DCM:MeOH = 100:1 to 50:1 as mobile phase to afford 750 mg of product as a white solid.

H ¹ NMR (400 MHz, CDCl ₃ ): 8.01 (s, 1H), 7.63-7.65 (m, 2H), 7.32-7.40 (m, 6H), 7.29-7.31 (m, 8H), 6.87-6.89 (m, 2H), 4.62 (s, 2H), 3.84 (s, 3H), 3.37 (t, 1H), 2.55 (m, 2H), 1.89 (m, 2H), 1.40 (m, 2H), 1.24 (m, 2H) ).

Example 2 Synthesis of Compound 2

Compound 1 (300 mg, 0.574 mmol, 1 eq) N ₃ PEG ₈ COOH (350 mg, 0.631 mmol, 1.1 eq), HOBT (85 mg, 0.631 mmol, 1.1 eq) DIEA (149 mg, 1.15 mmol, 2.0 eq) 4ml dry DMF, nitrogen protection, ice water bath cooling and stirring to dissolve; HATU (240mg, 0.631mmol, 1.1eq), 25 ° C reaction; point plate detection, 005 reaction complete, post-treatment. 50 ml of water was added to the reaction mixture, and the mixture was evaporated. The preparation of the thin layer plate was carried out by using DCM: MeOH = 15:1 as a developing solvent.

Example 3 Synthesis of Compound 3

Compound 2250mg, 0.5mmol, 1.5eq), DMAP (183mg, 1.5mmol, 4.5eq) and re-distilled 10ml DCM were added to the vial, dissolved in nitrogen, and added to phosgene (52mg, 0.175mmol, 0.52eq). After reaction at room temperature, the reaction solution became turbid. After stirring for 30 s, it was clarified. The plate was tested and the 002 reaction was completed. EL-006 (353 mg, 0.333 mmol, 1.0 eq) was added; the plate was detected, the EL-006 reaction was completed, and 3 ml of methanol was added to quench it. , spin dry to a yellow solid 900mg. The preparation of the thin layer plate was carried out by using DCM:MeOH = 10:1 as a developing solvent, and the product was purified to give a pale yellow solid (350 mg).

Example 4 Synthesis of Compound 4

Compound 3 (350 mg, 0.215 mmol, 1.0 eq) and DCM were added to a single-necked flask, and dissolved with nitrogen. After adding 1 M TBAF solution (0.537 ml, 2.5 eq), the reaction mixture turned orange-yellow, and added HAc (1.07 mmol). It turned golden yellow, reacted at 25 ° C; spot plate detection, 007 reaction was complete, and post-treatment. The reaction solution was diluted with 100 ml of DCM, washed three times with brine, dried over anhydrous sodium sulfate

Example 5 Synthesis of Compound 5

Compound 4 (400 mg, 0.273 mmol, 1 eq), SMCC-aminoacetylene (186 mg, 0.682 mmol, 2.5 eq) and 10 ml DMSO were added to the vial, stirred and dissolved, and then protected with nitrogen. CuBr (78 mg, 0.546 mmol, 2 eq) and 2.5 ml were added. Water, stirring reaction at 25 ° C;

The colloid is formed in the reaction solution and attached to the bottom of the bottle; the colloid and the reaction solution are taken, dissolved in DCM, and the plate is washed with water, and the reaction is complete after 008, and the post-treatment is carried out.

The reaction mixture was poured into 100 ml of water, and a large amount of solid was precipitated, which was extracted with DCM (60 ml×3), and the organic phase was washed three times with brine, dried over anhydrous sodium sulfate and evaporated.

Example 6 Synthesis of Compound 6 (Payload S)

Add 550mg EL-009, 1ml anisole and 10ml DCM to the single-mouth bottle, protect with nitrogen, dissolve and cool in ice water bath; add 2ml of dichloroacetic acid, keep the ice water bath, the reaction liquid turns yellow; increase to 25 °C reaction; Detection, EL-009 reaction was complete. Most of the solvent was rotated at 30 ° C, and 20 ml of methyl tertiary ether was added to crystallize. A large amount of yellow solid was precipitated, and the mixture was cooled in an ice water bath for 1 hour, filtered, and dried by an oil pump to obtain 350 mg of a crude yellow powder. HPLC preparation: Gradient elution with acetonitrile-water (0.9% TFA) as mobile phase. After lyophilization, product yellow powder 130 mg (M+H=1480.69).

Example 7-12

Synthesis of Compound 7 of Example 7

10 g of Fmoc-glycyl-glycine (SN-100, 0.028 mol, 1 eq), 300 mL of tetrahydrofuran, 100 mL of toluene were added to a 500 mL three-necked flask, and the mixture was stirred and dissolved, and then 17.5 g of lead tetraacetate (0.0395 mol, 1.4 eq) was added. The mixture was stirred under nitrogen to give a solid, and 2.7 mL of pyridine (0.0338 mol, 1.2 eq) was added to the reflux reaction (external temperature 80 ° C), and the reaction was monitored by TLC. The reaction mixture was concentrated, EtOAc EtOAc m. Purification by silica gel column (PE/EA = 5/1-1/1) gave

H ¹ NMR (400MHz, CDCl ₃ ): 8.11 (s, 1H), 8.08 (s, 1H), 7.87-7.89 (m, 2H), 7.56-7.58 (m, 2H), 7.28-7.33 (m, 4H) , 6.03 (s, 2H), 4.70 (d, 2H), 4.47 (t, 1H), 3.86 (s, 2H), 2.22 (s, 3H).

Synthesis of Compound 8 of Example 8

5 g of SN-101 (13.6 mmol, 1 eq), 150 mL of THF was added to a 250 mL vial, and the mixture was stirred and dissolved, and then 4.5 g of benzyl glycolate (27.2 mmol, 2 eq) and 0.52 g of p-toluenesulfonic acid monohydrate (2.71 mmol, 0.2 eq), room temperature reaction, TLC monitoring. After the reaction was completed, aq. Purification by silica gel column (PE / EA = 5 / 1-1 / 1) gave 2.8 g of white solid. LC-MS: 474.1 [M+H] ⁺

H ¹ NMR (400MHz, CDCl ₃ ): 8.13 (s, 1H), 8.10 (s, 1H), 7.87-7.89 (m, 2H), 7.56-7.58 (m, 4H), 7.28-7.36 (m, 7H) , 5.21 (s, 2H), 4.70 (d, 2H), 4.47 (t, 1H), 4.35 (s, 2H), 3.86 (s, 2H), 3.82 (s, 2H).

Example 9: Synthesis of Compound 9

5 g of Fmoc-glycyl-glycine (SN-100, 14 mmol, 1 eq), 5 g of p-nitrophenol (28 mmol, 2.5 eq), 7.34 g of DCC (28 mmol, 2.5 eq) and 80 mL of THF were added to a 250 mL vial, stirred at room temperature, TLC monitor. After completion of the reaction, the mixture was filtered.

The purer product (14 mmol, 1 eq) obtained in the previous step was added to a 250 mL single-mouth bottle, and then 90 mL of THF, aqueous sodium carbonate (28 mmol, 2.4 g, 2 eq, 30 mL of water), 1.88 g of L-phenylalanine (11 mmol, 0.8) was added. Eq), nitrogen protection, room temperature reaction, TLC monitoring. After the reaction was completed, a 5% aqueous solution of citric acid was added to adjust the pH to about 3, and ethyl acetate was evaporated. Purification by silica gel column (DCM /MeOH = EtOAc / LC-MS: 502.2 [M+H] ⁺

Example 10 Synthesis of Compound 13

1.5 g of SN-102 (0.032 mol, 1 eq), 8 mL of DMF and piperidine (0.8 mL) were added to a 50 mL vial, and reacted at room temperature for about 1 h, and monitored by TLC. At the end of the reaction, DMF and piperidine were removed by concentration under reduced pressure. To the crude was added 15DMF, 1.58 g of SN-106 (0.032 mol, 1 eq), 3.29 g of PyBOP (0.064 mol, 2 eq) and 1 mL of DIEA (0.064 mol, 2 eq). After completion of the reaction, the preparation was purified and lyophilized to give 1 g of a white solid. LC-MS: 758.2 [M+Na] ⁺

Example 11 Synthesis of Compound 16

150 mg of compound 13 (0.2 mmol, 1 eq), 8 mL of methanol and 1 mL of DMF were added to a 25 mL vial, dissolved, and then added with 45 mg of 5% Pd/C, hydrogenated, monitored by TLC, and completed in about 2 h. After filtration, 1.8 mL of diethylamine was added to the filtrate, and the mixture was stirred at room temperature for about 2 hours, and the reaction was completed. Concentrate under reduced pressure to give a crude material. 4 mL of DMF was added to the crude product, and the mixture was dissolved. Then, 62 mg of MC-OSu (0.2 mmol, 1 eq), 66 u L IEA (0.4 mmol, 2e) was added, and the reaction was monitored by HPLC. Prepared and purified, lyophilized to give the product 30 mg. LC-MS: 512.2 (fragment peak)

Example 12 Synthesis of Compound 17

30 mg of compound 16 (0.048 mmol, 1 eq), 25 mg of ezetidine mesylate (0.048 mmol, 1 eq), 50 mg of PyBOP (0.097 mmol, 2 eq), 16 u L IEA (0.097 mmol, 2 eq) and 2 mL DMF were added to a 25 mL vial. React at room temperature. After HPLC, the reaction was completed, and the reaction solution was directly purified by semi-preparation, and lyophilized to obtain 13 mg of the product. LC-MS: 1035.1 [M+H] ⁺

Example 13-17

Example 13 Synthesis of Compound 19 (Fmoc-VC-PABA):

1.5 g of compound 18 (Fmoc-Val-Cit) was dissolved in a mixed solvent of 14 ml of dichloromethane and 7 ml of methanol, 4-aminobenzyl alcohol (445.2 mg, 3.62 mmol) was added, followed by EEDQ (1.5 g, 6 mmol), The reaction mixture was stirred at room temperature overnight, and the solvent was evaporated and evaporated, and then evaporated, and then the mixture was evaporated to the residue, and the mixture was stirred for 30 min, and then the mixture was filtered, and then isopropyl ether was stirred for another 30 min, and filtered to obtain 1.5 g of Fmoc-VC-PABA. . M(+1)=602.6.

Example 14 Synthesis of Compound 20

2 g of Fmoc-VC-PABA was dissolved in 10 ml of DMF, 2 ml of piperidine was added, and the mixture was stirred at room temperature for 30 min. After the reaction was completed by TLC.

Example 15 Synthesis of Compound 21

The VC-PABA obtained in the above step was dissolved in dry DMF, added with McOSu, DIEA, stirred at room temperature for 2 h, and the reaction was monitored by HPLC. After adding isopropyl ether at room temperature, stirring and crystallization for 2 h, then cooling to 0 ° C, stirring for 1 h, filtering, filter cake Wash twice with isopropyl ether and dry under reduced pressure

Example 16: Synthesis of Compound 22

The white solid, Mc-VC-PABA (8 g, 14 mmol) was dissolved in 120 mL dry DMF, and bis(4-nitrophenyl) carbonate (8.5 g, 28 mmol, 2 eq), DIEA (3.66 ml) , 21.0 mmol, 1.5 eq). The reaction mixture was stirred at room temperature for 1 h, then the mixture was evaporated, evaporated, evaporated,,,,,,,,,,,,,,,,,,,,,,, The next step is to react.

Example 17 Synthesis of Compound 23

The above-obtained 500 mg of MC-VC-PAB-PNP was dissolved in 10 ml of dry DMF, and DIEA (0.2 ml) and 300 mg of MMAE were sequentially added to the obtained solution, and the mixture was stirred at room temperature, and the reaction was monitored by HPLC until the MMAE was completely reacted. HPLC The product obtained by purification was prepared, and lyophilized to give a white solid (yield: 300 mg (M+1 = 1302.3)).

Example 18 Preparation of Cet-4S-2M

A single-mutant cetux antibody was prepared by the general method A, and the preparation number Cet-4S-2M of the different toxin ADCs of payload M (DAR=2) and payload S (DAR=4) was completed according to the general method B.

Example 20 Preparation of Cet-8S-4M

A single-mutation cetux antibody was prepared by the general method A, and then the general procedure A was repeated to obtain a double-mutation cetux antibody. The preparation number Cet-8S-4M of the different toxin ADCs of payload M (DAR=4) and payload S (DAR=8) is completed according to the general method B.

Example 21 Preparation of Cet-8E-2M

The single-mutation cetuxima antibody was prepared by the general method A, and the preparation number Cet-8E-2M of the different toxin ADCs of payload M (DAR=2) and payload E (DAR=8) was completed according to the general method B.

Example 22 Preparation of Cet-4M

A single-mutation cetux antibody was prepared by the general method A, and then the general procedure A was repeated to obtain a double-mutation cetux antibody. The payload M (DAR=4) toxin ADC Cet-4M was completed according to General Method B.

Example 23 Preparation of Cet-8E

The Cetuximab antibody having a monomeric ratio of more than 95% after preliminary purification was exchanged into a phosphate buffer solution containing EDTA at a concentration of 10 mg/ml using an ultrafiltration centrifuge tube. TCEP was added 10 times the number of molecules of the antibody, and reacted at room temperature for 8 hours. The interchain disulfide bond was opened, and the number of free thiol groups was determined by the Ellman method to determine whether the disulfide bonds were all open. Then, a payload 10 times the number of molecules of the antibody was added, and the reaction was carried out for 8 hours at room temperature. After the reaction was completed, the solution was exchanged into PBS using an ultrafiltration centrifuge tube having a molecular weight cut-off of 30 KDa, and the unconjugated payload was removed. The non-fixed-point even payload E (DAR=8) number Cet-8E is prepared.

Example 24 Preparation of Cet-8S

The preparation was carried out in accordance with the method of Example 23 to obtain a non-fixed point coupled payload E (DAR = 8) number Cet-8E.

Example 25 Antibody Drug Conjugate DAR Assay

ADC number	Non-fixed point coupling average DAR1	Site-coupled average DAR2
Cet-4S-2M	3.81(S)	1.78(M)
Cet-8S-4M	6.35(S)	2.97(M)
Cet-8E-2M	7.54(E)	1.78(M)
Cet-4M	--	3.67 (M)
Cet-8S	7.56(S)	--
Cet-8E	7.92(E)	--

Example 26 Antibody Drug Conjugate SEC Assay

The SEC assay was performed on the ADC described in the present invention according to the general method. The data is summarized in the following table. From the results, it can be seen that the ADC ratio of the ADC drug with different toxins is slightly lower than that of the single toxin.

Target conjugate	Monomer rate
Cet-4S-2M	84.60%
Cet-8S-4M	83.87%
Cet-8E-2M	92.91%
Cet-4M	99.82%

Example 27 In vitro cell experiment

ADC number	IC 50 (Fadu)(nm)	IC 50 (A431) (nm)
Cet-8S-2M	1.85	4.23
Cet-8S-4M	1.18	0.15
Cet-8E-2M	200~1000	0.08
Cet-4M	200~1000	0.06
Cet-8S	1.02	8.49
Cet-8E	~200	>1000

Example 28 In vivo pharmacodynamic experiment

Human pharyngeal squamous cell carcinoma Fadu was cultured in vitro and inoculated subcutaneously in the back of nude mice according to the number of cells 5×10 ⁶ . After the tumor grew to 70-90 mm ³ , the mice were given a single dose of ADC drug 5 mg/kg (tail vein injection). The vehicle control group was weighed regularly, the tumor volume was measured, and the drug efficacy against the Fadu model was evaluated by investigating the antitumor efficacy of the ADC drug. The results show that ADC drugs with different toxins have better in vivo efficacy than single toxin ADC drugs.

Claims (17)

1. An antibody drug conjugate comprising a different drug, or a pharmaceutically acceptable salt thereof, of formula I

   

   Where Ab is an antibody moiety;

   L1, L2 are optional linking units connectable to the drug;

   D1 and D2 are drug units;

   m and n are each selected from an integer of 2-8.
2. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 1, wherein D1 and D2 are different antitumor drugs.
3. The antibody drug conjugate according to claim 2, comprising an antibody drug conjugate of a different drug or a pharmaceutically acceptable salt thereof, wherein D1 is selected from the group consisting of a tubulin binding agent, a DNA alkylating agent, and DNA. An intercalating agent, an enzyme inhibitor, an immunomodulator, a peptide or a nucleotide.
4. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 2, wherein D2 is selected from the group consisting of a tubulin binding agent, a DNA alkylating agent, a DNA intercalating agent, and an enzyme inhibitor , immunomodulatory agents, peptides or nucleotides.
5. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 1, wherein the drug D1 is attached to the antibody in a site-directed coupling manner, and D2 is coupled in a non-directed manner.
6. An antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 5, wherein the site-directed coupling mode of the drug D1 is a site-directed mutant antibody having an original amino acid as a cysteine or by insertion a cysteine or a cysteine-containing polypeptide, the cysteine thiol group introduced by the above method is coupled with a linker-toxin; the non-site coupling mode of D2 is the original interchain disulfide of the antibody. The bond is coupled to the linker-toxin.
7. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 1, wherein m is preferably 2, 4, 6, or 8; and n is preferably 4, 6, or 8.
8. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 1, wherein L1, L2 are cleavable linkers or non-cleavable linkers.
9. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 1, wherein L1 and L2 have the following formula 2:

   

   Wherein D is succinimide or hydrolyzed ring-opening succinimide, C is an optional extendable unit at the end, E is an optional cleavable unit, F is a spacer unit, subscript e, f is 0 or 1; the left wavy line indicates the attachment site to the antibody thiol group, and the right wavy line indicates the attachment site to the drug unit.
10. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 9, wherein the cleavable unit E is absent at e=0, exists at e=1; At the time, the cleavable unit described in E achieves cleavage with the drug unit D ₁ /D ₂ or the spacer unit F by a tumor-associated protease or an acidic pH.
11. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 9, wherein the spacer unit F is absent at f=0, exists at f=1; when F exists In the case, F is selected from the group consisting of p-aminobenzyl alcohol or an ethylenediamine unit and derivatives thereof.
12. The antibody drug conjugate comprising a different drug, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the antibody light chain comprises a kappa or lambda isoform.
13. The antibody drug conjugate comprising a different drug, or a pharmaceutically acceptable salt thereof, according to claim 1, wherein the antibody heavy chain comprises an IgG1, IgG2, IgG3 or IgG4 isotype.
14. The antibody drug conjugate comprising a different drug or a pharmaceutically acceptable salt thereof according to claim 12, wherein the cysteine is site-introduced into the antibody, wherein the thiol group (-SH) is capable of chemical coupling.
15. A pharmaceutical combination comprising an antibody drug conjugate comprising a different drug according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable diluent carrier or excipient Things.
16. Use of an antibody drug conjugate comprising a different drug, or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 13, for the treatment of a tumor, an autoimmune disease or an infectious disease.
17. The use according to claim 16, characterized in that said antibody comprising an antibody drug conjugate of a different drug or a pharmaceutically acceptable salt thereof specifically binds to said cancer, a target cell of an autoimmune disease.

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