Pharmaceutical composition of antibody drug conjugate

Abstract

The invention discloses a pharmaceutical composition of an antibody drug conjugate as well as a preparation method and application of the pharmaceutical composition. Specifically, the pharmaceutical composition comprises the antibody drug conjugate as shown in the formula I, the pharmaceutical composition is an injection, the preparation method of the pharmaceutical composition and the application of the pharmaceutical composition in preparation of drugs for treating proliferative diseases. # imgabs0 #

Description

Pharmaceutical composition of antibody-drug conjugate Technical Field

[0001] The present invention relates to a pharmaceutical composition of an antibody-drug conjugate and a method for preparing the composition. Background Art

[0002] Antibody-drug conjugates (ADCs) consist of three different components: antibodies, linkers, and drugs. ADC technology couples antibodies and drug molecules together through linkers, utilizing the antibody's specific targeted transport of drug molecules to target tissues to exert its effects, thereby reducing the drug's systemic toxic side effects, expanding the drug's therapeutic window, and enhancing the therapeutic potential of antibodies.

[0003] EGFR is the expression product of the proto-oncogene c-erbB1 and a member of the epidermal growth factor receptor (HER) family. This family includes HER1 (erbB1, EGFR), HER2 (erbB2, NEU), HER3 (erbB3), and HER4 (erbB4). EGFR is a transmembrane protein that plays a vital role in cell growth, development, and differentiation. Overexpression of EGFR and binding of its specific ligands, such as EGF and TGFα, can lead to abnormal activation of EGFR. Activated EGFR can further activate and regulate multiple signal transduction pathways, thereby inhibiting tumor cell apoptosis, promoting tumor cell proliferation and migration, and promoting tumor angiogenesis by mediating VEGF expression. Consequently, it plays a crucial role in tumor development, progression, malignancy, and prognosis. Therefore, blocking EGFR-mediated signal transduction pathways can achieve therapeutic effects on tumors.

[0004] HER3 (also known as ERBB3, human epidermal growth factor receptor 3) is a transmembrane receptor and a member of the epidermal growth factor receptor family (EGFR). Its gene is located on the long arm of human chromosome 12 (12q13), and the protein it encodes has a molecular weight of 160 or 180 kDa. HER3 is known to be expressed in various cancers, such as breast cancer, lung cancer, and colorectal cancer, and to form heterodimers with tyrosine kinase receptors such as HER2 and EGFR. HER3 is then phosphorylated, thereby inducing cancer cell growth or apoptosis inhibition signals.

[0005] CN202211461614.0 discloses an antibody-drug conjugate targeting EGFR:

[0006] Wherein Ab is an antibody or antigen-binding fragment thereof targeting HER2, HER3 or EGFR, and R is selected from C 1-6Alkyl, n is selected from an integer or a decimal of 1 to 8. In vitro studies have shown that the ADC has a good inhibitory effect on various tumor cells.

[0007] In the research of antibody and antibody-drug conjugate (ADC) formulations, the formation of aggregates and the production of decomposition products cause pharmaceutically undesirable side effects, which bring increased immunogenicity or venous disease-related risks to patients receiving drug treatment. For these reasons, when formulating related formulations, it is necessary to inhibit the formation of aggregates and the production of decomposition products. In particular, when studying the pharmaceutical formulations of antibody-drug conjugates, more technical difficulties are faced. Not only the specific properties of the antibody part need to be considered, but also the specific properties of the drug-linker part need to be considered. For example, during the storage of antibody-drug conjugates, small molecule toxins may be shed, and this shedding affects the effectiveness and toxicity of the related drugs.

[0008] Our research direction is to develop stable pharmaceutical compositions and solve the aggregation and degradation problems of ADC molecules in the pharmaceutical compositions of antibody-drug conjugates.

[0009] Summary of the Invention

[0010] The present invention provides a pharmaceutical composition comprising an antibody-drug conjugate represented by Formula I:

[0011] Wherein Ab is an antibody or antigen-binding fragment thereof targeting HER2, HER3 or EGFR, and R is selected from C 1-6 Alkyl, n is selected from an integer of 1-8 or a decimal of 1-8.

[0012] Furthermore, n is selected from an integer of 4-8 or a decimal of 4-8.

[0013] Further, when n is an integer selected from 1-8, it can be 1, 2, 3, 4, 5, 6, 7, 8; when n is an integer selected from 4-8, it can be 4, 5, 6, 7, 8.

[0014] Furthermore, when n is a decimal, it refers to the average number of linker-drug molecules conjugated per antibody unit.

[0015] In a preferred embodiment of the present invention, R is selected from C 1-3 alkyl.

[0016] In a preferred embodiment of the present invention, R is selected from methyl, ethyl, propyl, and isopropyl.

[0017] In a preferred embodiment of the present invention, R is selected from methyl.

[0018] Furthermore, the antibody drug conjugate shown in Formula I has the structure shown in Formula Ia or Formula Ib:

[0019] Wherein, in Formula Ia and Formula Ib, Ab, R and n are as defined in Formula I.

[0020] Furthermore, the antibody-drug conjugates of Formula I have the structures shown in Formulas I-1, Ia-1, and Ib-1:

[0021] Wherein, in Formula I-1, Formula Ia-1 and Formula Ib-1, Ab and n are defined as in Formula I.

[0022] In a preferred embodiment of the present invention, in the above-mentioned antibody-drug conjugate, when the Ab is an antibody or antigen-binding fragment thereof targeting HER2, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain variable region sequence comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3), and the light chain variable region comprises a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 1, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 2, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 3, and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 4, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 5, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 6.

[0023] Further preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting HER2, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 7, and the amino acid sequence of LV is as shown in SEQ ID NO: 8.

[0024] More preferably, when the Ab is an antibody targeting HER2 or an antigen-binding fragment thereof, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 9, and the amino acid sequence of LC is as shown in SEQ ID NO: 10.

[0025] In a preferred embodiment of the present invention, when the Ab is an antibody targeting HER3 or an antigen-binding fragment thereof, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain variable region sequence comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3), and the light chain variable region comprises a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 11, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 12, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 13, and / or the amino acid sequence of LCDR1 is shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 16.

[0026] Further preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting HER3, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 17, and the amino acid sequence of LV is as shown in SEQ ID NO: 18.

[0027] More preferably, when the Ab is an antibody targeting HER3 or an antigen-binding fragment thereof, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 19, and the amino acid sequence of LC is as shown in SEQ ID NO: 20.

[0028] In a preferred embodiment of the present invention, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain variable region sequence comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3), and the light chain variable region comprises a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 21, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 22, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 23, and / or the amino acid sequence of LCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 26.

[0029] In a preferred embodiment of the present invention, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 31 or SEQ ID NO: 35 or SEQ ID NO: 38, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 22, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 23 or SEQ ID NO: 32, and / or the amino acid sequence of LCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 26.

[0030] In a preferred embodiment of the present invention, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO: 31 or SEQ ID NO: 35 or SEQ ID NO: 38, the amino acid sequence of HCDR2 is shown in SEQ ID NO: 22, the amino acid sequence of HCDR3 is shown in SEQ ID NO: 32, and the amino acid sequence of LCDR1 is shown in SEQ ID NO: 24, the amino acid sequence of LCDR2 is shown in SEQ ID NO: 25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO: 26.

[0031] Further preferably, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 27, and the amino acid sequence of LV is as shown in SEQ ID NO: 28.

[0032] Further preferably, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it includes a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 33 or SEQ ID NO: 36 or SEQ ID NO: 39, and the amino acid sequence of LV is as shown in SEQ ID NO: 28.

[0033] More preferably, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 29, and the amino acid sequence of LC is as shown in SEQ ID NO: 30.

[0034] More preferably, when the Ab is an antibody targeting EGFR or an antigen-binding fragment thereof, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 34 or SEQ ID NO: 37 or SEQ ID NO: 40, and the amino acid sequence of LC is as shown in SEQ ID NO: 30.

[0035] When Ab is an antibody targeting HER2, HER3 or EGFR, the antibody is preferably a monoclonal antibody, and the monoclonal antibody is preferably selected from human antibodies, humanized antibodies, and chimeric antibodies.

[0036] When Ab is an antigen-binding fragment targeting HER2, HER3 or EGFR, the antigen-binding fragment is preferably selected from Fab', (Fab')2, Fab, Fv, scFv, and dAb.

[0037] In the present invention, the numbering convention for antibody amino acid sequences adopts the Kabat numbering convention.

[0038] Furthermore, the antibody-drug conjugate of Formula I has the following structure:

[0039] Furthermore, the antibody-drug conjugate of Formula I has the following structure:

[0040] In the above-mentioned antibody-drug conjugates:

[0041] DP001 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 9 and two light chains with the amino acid sequence shown in SEQ ID NO: 10;

[0042] Patritumab is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 19 and two light chains with the amino acid sequence shown in SEQ ID NO: 20;

[0043] SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 29 and two light chains with the amino acid sequence shown in SEQ ID NO: 30;

[0044] SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 34 and two light chains with the amino acid sequence shown in SEQ ID NO: 30;

[0045] SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 37 and two light chains with the amino acid sequence shown in SEQ ID NO: 30;

[0046] SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30;

[0047] n is an integer or a decimal selected from 1-8.

[0048] Furthermore, n is selected from an integer of 4-8 or a decimal of 4-8.

[0049] Furthermore, n is 8.

[0050] Further, when n is an integer selected from 1-8, it can be 1, 2, 3, 4, 5, 6, 7, 8; when n is an integer selected from 4-8, it can be 4, 5, 6, 7, 8.

[0051] Furthermore, the pharmaceutical composition is an injection, preferably an injection or a lyophilized injection, more preferably a lyophilized injection.

[0052] The lyophilized injection is a solid injection (ie, lyophilized powder) that can be used by dissolving in water (preferably water for injection) at the time of use, which is obtained by lyophilizing an aqueous solution containing a predetermined amount of a drug component.

[0053] In the present invention, water for injection refers to water that complies with the provisions of the Water for Injection section of the Chinese Pharmacopoeia (2020).

[0054] Furthermore, the pharmaceutical composition comprises the antibody-drug conjugate shown in Formula I and a buffer.

[0055] Furthermore, the pharmaceutical composition further comprises a stabilizer.

[0056] Furthermore, the pharmaceutical composition further comprises a surfactant.

[0057] Furthermore, the pharmaceutical composition may further comprise water.

[0058] In some embodiments, the pharmaceutical composition comprises the antibody-drug conjugate of Formula I and a buffer, a stabilizer, and a surfactant.

[0059] In some embodiments, the pharmaceutical composition further comprises water.

[0060] In some embodiments, in the pharmaceutical composition, the antibody-drug conjugate represented by Formula I is selected from ADC-1, ADC-2, ADC-3, ADC-4, and ADC-5.

[0061] In some embodiments, the buffer includes, but is not limited to, acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, tromethamine (Tris), morpholineethanesulfonic acid (MES), and other organic acid buffers.

[0062] In some embodiments, the histidine salt buffer is a buffer containing histidine ions. Examples of histidine salt buffers include histidine-histidine hydrochloride, histidine-acetate, histidine-phosphate, and histidine-sulfate buffers. Histidine-histidine hydrochloride buffer is a mixture of histidine and histidine hydrochloride, and histidine-acetate buffer is a mixture of histidine and acetic acid. Similarly, a succinate buffer can be succinic acid-sodium succinate, and a citrate buffer can be citric acid-sodium citrate.

[0063] In some embodiments, histidine-histidine hydrochloride buffer is preferred.

[0064] In some embodiments, the stabilizer includes but is not limited to sugars (such as sucrose, trehalose), polyols (such as mannitol, sorbitol), and amino acids (L-serine, sodium glutamate, alanine, glycine, sarcosine, etc.).

[0065] In some embodiments, the surfactant includes, but is not limited to, polysorbates (eg, polysorbate 20, polysorbate 80).

[0066] In the following embodiments describing pH, mass percentage (in mass %) and content (in mass / volume such as mg / ml), in the absence of contrary teachings, the pH value of the pharmaceutical composition is the pH value of the aqueous solution of the pharmaceutical composition (the water therein is water for injection); in the absence of contrary teachings, when describing the mass percentage, the total weight of the pharmaceutical composition is 100 wt % as the benchmark and the pharmaceutical composition does not include water for injection; in the absence of contrary teachings, when describing the content, the pharmaceutical composition includes water for injection and is based on the volume of the pharmaceutical composition;

[0067] In some embodiments, the pH of the pharmaceutical composition is 4.0-7.5 (e.g., about 4.0, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, about 7.0), preferably 4.5-7.0, further preferably 4.5-5.5, further preferably 5.0-5.5, and most preferably about 5.0.

[0068] In the above embodiments and similar embodiments below, the word "about" preceding a numerical value includes the value itself and each value within the error range understood by a person skilled in the art.

[0069] In some embodiments, the mass percentage of the antibody-drug conjugate of Formula I (such as ADC-1, ADC-2, ADC-3, ADC-4, and ADC-5) in the pharmaceutical composition is 1-30% (such as about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%), preferably 5-25%, 8-20%, 8-15%, 8-10%, and 15-25%.

[0070] In some embodiments, in the pharmaceutical composition, the content of the antibody-drug conjugate of Formula I (such as ADC-1, ADC-2, ADC-3, ADC-4, ADC-5) is 1-30 mg / ml (such as about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 10.5 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13mg / ml, about 14mg / ml, about 15mg / ml, about 16mg / ml, about 17mg / ml, about 18mg / ml, about 19mg / ml, about 20mg / ml, about 21mg / ml, about 22mg / ml, about 23mg / ml, about 24mg / ml, about 25mg / ml, about 26mg / ml, about 27mg / ml, about 28mg / ml, about 29mg / ml, about 30mg / ml), preferably 5-25mg / ml, further preferably 5-20mg / ml, 5-15mg / ml, 15-25mg / ml.

[0071] In some embodiments, in the pharmaceutical composition, the mass percentage of the buffer (such as histidine-histidine hydrochloride) is 1-10% (such as about 1%, about 2%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, about 10%), preferably 1-9.9%, 2-6%, 2-5%, 4-6%.

[0072] In some embodiments, in the pharmaceutical composition, the content of the buffer (such as histidine-histidine hydrochloride) is 1-10 mg / ml (such as about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml), preferably 2-6 mg / ml, 2-4 mg / ml, 3-6 mg / ml, 4-6 mg / ml.

[0073] In some embodiments, in the pharmaceutical composition, the buffer is histidine-histidine hydrochloride, wherein the mass percentage of the histidine is 0.1-1% (such as about 0.1%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%), preferably 0.1-0.5%, 0.2-0.4%.

[0074] In some embodiments, in the pharmaceutical composition, the buffer is histidine-histidine hydrochloride, wherein the content of histidine is 0.1-1 mg / ml (e.g., about 0.1 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, about 0.3 mg / ml, about 0.31 mg / ml, about 0.32 mg / ml, about 0.33 mg / ml, about 0.34 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml), preferably 0.1-0.5 mg / ml, 0.1-0.3 mg / ml, 0.2-0.4 mg / ml.

[0075] In some embodiments, in the pharmaceutical composition, the buffer is histidine-histidine hydrochloride, wherein the mass percentage of the histidine hydrochloride is 1-10% (such as about 1%, about 2%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.5%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, about 10%), preferably 1-9.9%, 2-6%, 2-4%, 3-6%, 3.5-5.5%, 4-5%.

[0076] In some embodiments, in the pharmaceutical composition, the buffer is histidine-histidine hydrochloride, wherein the content of the histidine hydrochloride is 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 2.6 mg / ml, about 2.7 mg / ml, about 2.8 mg / ml, about 2.9 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml), preferably 2-6 mg / ml, 2-4 mg / ml, 3-6 mg / ml, about 4-5.5 mg / ml, about 4.5-5 mg / ml.

[0077] In some embodiments, in the pharmaceutical composition, the mass percentage of the stabilizer (such as trehalose, sucrose) is 50-90% (such as about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%), preferably 60-90%, 70-90%, 80-90%.

[0078] In some embodiments, in the pharmaceutical composition, the content of the stabilizer (such as trehalose, sucrose) is 50-110 mg / ml (such as about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml, about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, about 94 mg / ml, about 95 mg / ml, about 100 mg / ml, about 105 mg / ml, about 110 mg / ml), preferably 70-100 mg / ml, 80-100 mg / ml, 75-95 mg / ml.

[0079] In some embodiments, in the pharmaceutical composition, the mass percentage of the surfactant (such as polysorbate 80) is 0.1-1% (such as about 0.1%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, About 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%), preferably 0.2-0.4%, further preferably 0.15-0.4%, 0.15-0.35%, 0.25-0.35%.

[0080] In some embodiments, in the pharmaceutical composition, the content of the surfactant (such as polysorbate 80) is 0.1-1 mg / ml (such as about 0.1 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml), preferably 0.1-0.4 mg / ml, 0.15-0.25 mg / ml, 0.2-0.4 mg / ml, 0.25-0.35 mg / ml.

[0081] In some embodiments, the pharmaceutical composition comprises, by weight percentage, 1-30% (e.g., about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%) of an antibody-drug conjugate (e.g., ADC- 1, ADC-2, ADC-3, ADC-4, ADC-5), and 0.1-1% (e.g., about 0.1%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%) of histidine, 1-10% (e.g., 1-9.9%, or about 1%, about 2%, about 2.5%, about 3%) of histidine, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.5%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, about 10%) of histidine hydrochloride, 50-90% (e.g., about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%) trehalose, 0.1-1% (e.g., about 0.1%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%) of polysorbate 80.

[0082] In some embodiments, the pharmaceutical composition comprises, by weight percentage, 1-30% (e.g., about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%) of the antibody-drug conjugate (e.g., ADC-1, ADC-2, ADC-3, ADC-4, ADC-5) of Formula I, and 0.1-1% (e.g., about 0.1%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%) of histidine, 1-10% (e.g. 1-9.9%, or about 1%, about 2%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.5%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, about 10%) of histidine hydrochloride, 50-90% (e.g., about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%) of sucrose, 0.1-1% (e.g., about 0.1%, about 0.15%, about 0.16%, about 0.17%, about 0.18%) of sucrose, .18%, about 0.19%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%) of polysorbate 80.

[0083] In some embodiments, the pharmaceutical composition comprises 1-30 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 10.5 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml). ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml) of an antibody-drug conjugate of Formula I (such as ADC-1, ADC-2, ADC-3, ADC-4, ADC-5), and 0.1-1 mg / ml (such as about 0.1 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml) g / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, about 0.3 mg / ml, about 0.31 mg / ml, about 0.32 mg / ml, about 0.33 mg / ml, about 0.34 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml) of histidine, 1 -10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 2.6 mg / ml, about 2.7 mg / ml, about 2.8 mg / ml, about 2.9 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5mg / ml, about 7mg / ml, about 8mg / ml, about 9mg / ml, about 10mg / ml) of histidine hydrochloride, 50-110mg / ml (e.g., about 50mg / ml, about 60mg / ml, about 70mg / ml, about 75mg / ml, about 80mg / ml, about 81mg / ml, about 82mg / ml, about 83mg / ml, about 84mg / ml, about 85mg / ml, about 86mg / ml, about 87mg / ml, about 88mg / ml, about 89mg / ml, about 90mg / ml, about 91mg / ml, about 92mg / ml, about 93mg / ml, about 94mg / ml, about 95mg / ml, about 100mg / ml), g / ml, about 105 mg / ml, about 110 mg / ml) of trehalose, 0.1-1 mg / ml (such as about 0.1 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml) of polysorbate 80.

[0084] In some embodiments, the pharmaceutical composition comprises 1-30 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 10.5 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml). g / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml) of the antibody drug conjugate of Formula I (such as ADC-1, ADC-2, ADC-3, ADC-4, ADC-5), and 0.1-1 mg / ml (such as about 0.1 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, about 0.3 mg / ml, about 0.31 mg / ml, about 0.32 mg / ml, about 0.33 mg / ml, about 0.34 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml g / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml) of histidine, 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 2.6 mg / ml, about 2.7 mg / ml, about 2.8 mg / ml, about 2.9 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5mg / ml, about 7mg / ml, about 8mg / ml, about 9mg / ml, about 10mg / ml) of histidine hydrochloride, 50-110mg / ml (e.g., about 50mg / ml, about 60mg / ml, about 70mg / ml, about 75mg / ml, about 80mg / ml, about 81mg / ml, about 82mg / ml, about 83mg / ml, about 84mg / ml, about 85mg / ml, about 86mg / ml, about 87mg / ml, about 88mg / ml, about 89mg / ml, about 90mg / ml, about 91mg / ml, about 92mg / ml, about 93mg / ml, about 94mg / ml, about 95mg / ml, about 100mg / ml), g / ml, about 105 mg / ml, about 110 mg / ml) of sucrose, 0.1-1 mg / ml (such as about 0.1 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml) of polysorbate 80.

[0085] In some embodiments, the pharmaceutical composition, after dissolution, comprises about 10 mg / ml of the antibody drug conjugate of Formula I, about 0.33 mg / ml histidine, about 4.79 mg / ml histidine hydrochloride, about 0.3 mg / ml polysorbate 80, about 90 mg / ml trehalose, and has a pH of about 5.0.

[0086] In some embodiments, the pharmaceutical composition comprises about 10 mg / ml of an antibody drug conjugate of Formula I, about 0.33 mg / ml histidine, about 4.79 mg / ml histidine hydrochloride, about 0.3 mg / ml polysorbate 80, and about 90 mg / ml trehalose, and has a pH of about 5.0.

[0087] In some embodiments, the pharmaceutical composition is a lyophilized injection. When the lyophilized injection is diluted with water for injection, the diluted pharmaceutical composition comprises about 10 mg / ml of the antibody-drug conjugate of Formula I, about 0.33 mg / ml histidine, about 4.79 mg / ml histidine hydrochloride, about 0.3 mg / ml polysorbate 80, and about 90 mg / ml trehalose. The pH of the diluted pharmaceutical composition is about 5.0 based on the total volume of the diluted pharmaceutical composition. Preferably, the antibody-drug conjugate of Formula I is selected from the group consisting of SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably SWY2110-JSSW-001.

[0088] In some embodiments, the pharmaceutical composition is an injection, comprising about 10 mg / ml of the antibody-drug conjugate of Formula I, about 0.33 mg / ml histidine, about 4.79 mg / ml histidine hydrochloride, about 0.3 mg / ml polysorbate 80, and about 90 mg / ml trehalose, and the pH of the injection is about 5.0. Preferably, the antibody-drug conjugate of Formula I is selected from the group consisting of SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably SWY2110-JSSW-001.

[0089] In other words, in one embodiment of the present invention, the pharmaceutical composition comprises about 10 mg / ml of the antibody drug conjugate of formula I, about 0.33 mg / ml histidine, about 4.79 mg / ml histidine hydrochloride, about 0.3 mg / ml polysorbate 80, about 90 mg / ml trehalose, and the balance water, and the pH of the pharmaceutical composition is about 5.0. Preferably, the antibody drug conjugate of formula I is selected from: SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably Preferably, it is SWY2110-JSSW-001; or the pharmaceutical composition comprises or consists of: about 10 parts by weight of an antibody-drug conjugate of formula I, about 0.33 parts by weight of histidine, about 4.79 parts by weight of histidine hydrochloride, about 0.3 parts by weight of polysorbate 80, and about 90 parts by weight of trehalose. Preferably, the antibody-drug conjugate of formula I is selected from the group consisting of SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, and preferably SWY2110-JSSW-001.

[0090] In some embodiments, the pharmaceutical composition, after dissolution, comprises about 20 mg / ml of the antibody drug conjugate of Formula I, about 0.21 mg / ml histidine, about 2.86 mg / ml histidine hydrochloride, about 0.2 mg / ml polysorbate 80, about 80 mg / ml sucrose, and has a pH of about 5.0.

[0091] In some embodiments, the pharmaceutical composition comprises about 20 mg / ml of an antibody drug conjugate of Formula I, about 0.21 mg / ml histidine, about 2.86 mg / ml histidine hydrochloride, about 0.2 mg / ml polysorbate 80, and about 80 mg / ml sucrose, and has a pH of 5.0.

[0092] In some embodiments, the pharmaceutical composition is a lyophilized injection. When the lyophilized injection is diluted with water for injection, the diluted pharmaceutical composition contains about 20 mg / ml of the antibody-drug conjugate of Formula I, about 0.21 mg / ml histidine, about 2.86 mg / ml histidine hydrochloride, about 0.2 mg / ml polysorbate 80, and about 80 mg / ml sucrose. Based on the total volume of the diluted pharmaceutical composition, the pH of the diluted pharmaceutical composition is about 5.0. Preferably, the antibody-drug conjugate of Formula I is patritumab-JSSW-001.

[0093] In some embodiments, the pharmaceutical composition is an injection, comprising about 20 mg / ml of the antibody-drug conjugate of Formula I, about 0.21 mg / ml histidine, about 2.86 mg / ml histidine hydrochloride, about 0.2 mg / ml polysorbate 80, and about 80 mg / ml sucrose, and the pH of the injection is about 5.0. Preferably, the antibody-drug conjugate of Formula I is patritumab-JSSW-001.

[0094] In other words, in one embodiment of the present invention, the pharmaceutical composition comprises about 20 mg / ml of an antibody-drug conjugate of Formula I, about 0.21 mg / ml histidine, about 2.86 mg / ml histidine hydrochloride, about 0.2 mg / ml polysorbate 80, about 80 mg / ml sucrose, and the balance water, and the pH of the pharmaceutical composition is about 5.0. Preferably, the antibody-drug conjugate of Formula I is patritumab-JSSW-001; or the pharmaceutical composition comprises or consists of: about 20 parts by weight of an antibody-drug conjugate of Formula I, about 0.21 parts by weight of histidine, about 2.86 parts by weight of histidine hydrochloride, about 0.2 parts by weight of polysorbate 80, and about 80 parts by weight of sucrose. Preferably, the antibody-drug conjugate of Formula I is patritumab-JSSW-001.

[0095] In some embodiments, the antibody drug conjugate of Formula I is selected from one of ADC-1, ADC-2, ADC-3, ADC-4, and ADC-5.

[0096] In another aspect, the present invention also relates to a method for preparing the pharmaceutical composition, comprising the steps of:

[0097] S1) preparing the antibody-drug conjugate of Formula I;

[0098] S2) preparing ultrafiltration replacement buffer;

[0099] S3) exchanging the antibody-drug conjugate prepared in S1 with the buffer prepared in S2).

[0100] Optionally, S4) freeze-drying is also included.

[0101] Furthermore, the step S2) includes weighing a buffer and a stabilizer according to the prescribed amount, diluting them with water for injection to a target preparation volume, and stirring and mixing them uniformly to obtain a replacement buffer.

[0102] Furthermore, the step S3) includes using an ultrafiltration membrane to ultrafilter the antibody-drug conjugate prepared in S1 into the buffer prepared in S2) according to a certain volume, diluting the antibody-drug conjugate to the target concentration, and adding a surfactant according to the volume fraction to obtain a stock solution.

[0103] Furthermore, in step S3, the preparation method optionally includes the step of sterile filtering the obtained buffer solution.

[0104] The present invention provides the purposes of the pharmaceutical composition of the present invention in the preparation of the medicine for treating proliferative disease.Described proliferative disease is preferably the disease relevant to HER2, HER3 or EGFR abnormal expression, including cancer, described cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia) bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma or recurrent anaplastic large cell lymphoma).

[0105] The present invention provides a method for treating or preventing a proliferative disease, the method comprising administering a therapeutically effective dose of a pharmaceutical composition of the present invention to a patient in need thereof; the proliferative disease is preferably a disease associated with abnormal expression of HER2, HER3 or EGFR, including cancer. The cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma).

[0106] The present invention provides use of the pharmaceutical composition of the present invention in the preparation of a medicament for treating a proliferative disease that develops drug resistance. The proliferative disease that develops drug resistance is a disease associated with abnormal expression of HER2, HER3, or EGFR, including cancer. The drug-resistant cancer is preferably a drug-resistant cancer caused by a HER2, HER3 or EGFR gene mutation, and the cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia) bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (such as Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma), and is further preferably colon cancer, rectal cancer, lung cancer or pancreatic cancer. The drug resistance is preferably resistance to receptor tyrosine kinase inhibitors, further preferably resistance to first-generation, second-generation or third-generation EGFR inhibitors, further preferably resistance to gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib or ametinib, especially resistance to gefitinib, afatinib, osimertinib or ametinib, more preferably resistance to gefitinib or osimertinib.

[0107] The present invention provides a method for treating or preventing a drug-resistant proliferative disease, comprising administering a therapeutically effective dose of a pharmaceutical composition of the present invention to a patient in need thereof; the drug-resistant proliferative disease is a disease associated with abnormal expression of HER2, HER3, or EGFR, including cancer. The drug-resistant cancer is preferably a drug-resistant cancer caused by a mutation in the HER2, HER3, or EGFR gene. The cancer is preferably breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer or lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma or relapsed anaplastic large cell lymphoma), more preferably colon cancer, rectal cancer, lung cancer or pancreatic cancer. The drug resistance is preferably resistance to receptor tyrosine kinase inhibitors, further preferably resistance to first-generation, second-generation or third-generation EGFR inhibitors, further preferably resistance to gefitinib, erlotinib, icotinib, afatinib, dacomitinib, osimertinib or ametinib, especially resistance to gefitinib, afatinib, osimertinib or ametinib, more preferably resistance to gefitinib or osimertinib.

[0108] The pharmaceutical composition of the present invention can be used alone or in combination with other anti-tumor agents.

[0109] The method for preparing the antibody drug conjugate of the present invention comprises the following steps:

[0110] S1: reduction of antibodies;

[0111] Specifically, the antibody medium was replaced with PBS 6.0 / EDTA to a concentration of 10 mg / mL. Appropriate amounts of 10 mM TCEP aqueous solution and 1 M potassium dihydrogen phosphate aqueous solution were added according to the reducing agent. After confirming the correct pH, the solution was incubated at 37°C for several hours to reduce the disulfide bonds within the antibody.

[0112] S2: Conjugation of the antibody to the linker-drug compound;

[0113] Specifically, 10 mM of the compound dissolved in an appropriate amount of dimethyl sulfoxide solution was added to the above solution at room temperature, mixed, and reacted at room temperature for 0.5-4 hours to attach the linker-drug compound to the antibody. Next, 100 mM NAC aqueous solution was added, and the reaction was terminated by stirring at room temperature.

[0114] S3: Purification of antibody-drug conjugates;

[0115] Specifically, the reaction solution was purified by ultrafiltration using an ultrafiltration centrifuge tube. 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added to the reaction solution as a purification buffer. The solution was concentrated to 1-2 mL, and the purification buffer was added again at a displacement factor greater than 1000. This removed unconjugated linker-drug compounds and other low molecular weight reagents, yielding a purified antibody-drug conjugate.

[0116] The antibody-drug conjugates (ADCs) contained in the pharmaceutical compositions of the present invention exhibit superior anti-cell proliferation and significant tumor suppression compared to prior art ADCs, such as DS-8201, or currently effective experimental samples at different targets, as controls. They exhibit superior inhibitory activity against drug-resistant tumors, such as those caused by HER2, HER3, or EGFR gene mutations, and particularly those resistant to receptor tyrosine kinase inhibitors, compared to DS-8201. They also exhibit improved stability, including plasma stability and buffer stability, lower toxicity, a reduced bystander effect, and a wider therapeutic window. The ADCs obtained by conjugating the modified antibodies to linker-drug compounds, which modify their pH dependence, exhibit comparable tumor suppression activity to the original ADCs in terms of efficacy and demonstrate reduced on-target toxicity to normal tissues in terms of safety. The ADCs obtained from the optimized antibodies further expand the therapeutic window.

[0117] Under the formulated conditions, the pharmaceutical composition of the present invention can be stored for long periods at 2-8°C, ensuring the stability of the physicochemical properties of the antibody component while minimizing the shedding of free drug. Long-term stability studies have shown no significant changes in the physicochemical properties of the antibody component, meeting the storage requirements for antibody preparations. The DAR value and DAR8 distribution of the ADC component have also remained unchanged, demonstrating that the conjugated drug maintains uniform quality during storage. While free drug shedding occurs, the amount is very low, posing no safety risk to clinical administration and allowing for long-term storage.

[0118] definition

[0119] Unless otherwise specified, the term "antibody-drug conjugate (ADC)" refers to an antibody (such as a monoclonal antibody) or antibody fragment linked to a biologically active cytotoxic drug via a stable chemical linker compound.

[0120] Unless otherwise specified, the term "linker-drug compound" refers to the partial structure of an antibody drug conjugate consisting of a linker compound and a drug compound.

[0121] The linker-drug compound of the present invention is linked to the antibody by conventional coupling methods in the art, including lysine coupling, inter-light and heavy chain reductive disulfide bond coupling, and directional coupling (Beck, Alain, and Janice M. Reichert. "Antibody-drug conjugates: present and future." MAbs. Vol. 6. No. 1. Taylor & Francis, 2014.; McCombs, Jessica R., and Shawn C. Owen. "Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry." The AAPS journal 17 (2015): 339-351.). The present invention preferably couples the linker-drug compound to the antibody by inter-light and heavy chain reductive disulfide bond coupling, i.e., one or more of the inter-light and heavy chain disulfide bond sites (two sites between the heavy chain and two sites between the heavy and light chains) are reduced to form a thiol group (sulfur atom of a cysteine ​​residue) for reaction.

[0122] Unless otherwise specified, the term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched chain group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C 1-10 Alkyl), further preferably containing 1 to 8 carbon atoms (C 1-8 Alkyl), more preferably containing 1-6 carbon atoms (ie C 1-6 alkyl). For example, "C 1-6 The term "alkyl" means that the group is an alkyl group and the number of carbon atoms in the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl and the like.

[0123] Unless otherwise specified, the term "about" refers to a numerical value that is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which value depends in part on how it is measured or determined (i.e., the limitations of the measurement system). For example, each occurrence of "about" in this art can mean a range of ±20%, ±15%, ±10%, ±5%, or ±1% of the particular value subsequently indicated. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] Figure 1 shows the DAR value test results of DP001-JSSW-001

[0125] Figure 2 shows the DAR value test results of DP001-JSSW-001

[0126] Figure 3 shows the DAR value test results of partitumab-JSSW-001

[0127] Figure 4 shows the DAR value test results of SWY2110-JSSW-001

[0128] Figure 5 shows the DAR value test results of SWY2111-JSSW-001

[0129] Figure 6 shows the DAR value test results of SWY2112-JSSW-001

[0130] Figure 7 shows the DAR value test results of SWY2113-JSSW-001

[0131] Figure 8 shows the DAR value test results of partitumab-Dxd

[0132] Figure 9 shows the DAR value test results of SWY2110-Dxd

[0133] Figure 10 shows the SWY2110-Vc MMAE DAR value test results

[0134] Figure 11 shows the effect of DP001-ADC on the tumor volume of HER2-expressing human breast cancer JIMT-1 cell transplanted tumors in NU / NU mice

[0135] Figure 12 shows the effect of patritumab-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice

[0136] Figure 13 shows the effect of SWY2110-ADC on the tumor volume of human colorectal cancer DiFi cell transplanted tumors in NU / NU mice

[0137] Figure 14 shows the effect of SWY2110-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice

[0138] Figure 15 shows the results of the inhibitory activity test of SWY2110-ADC on DiFi cells

[0139] Figure 16 shows the results of the inhibitory activity test of SWY2110-ADC on PC9-GR cells

[0140] Figure 17 shows the bystander effect detection results of DP001-ADC

[0141] Figure 18 shows the effect of SWY2110-ADC on the tumor volume of human lung adenocarcinoma PC9-AR (PC9-Del19 / T790M / C797S, Osimertinib-resistant cells) transplanted tumors in NU / NU mice

[0142] Figure 19 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma NCI-H1975 cell transplanted tumors in NU / NU mice.

[0143] Figure 20 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human breast cancer MDA-MB-468 cell transplanted tumors in NOD-SCID mice.

[0144] Figure 21 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human colorectal cancer DiFi cell transplanted tumors in NU / NU mice

[0145] Figure 22 shows the effects of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on the tumor volume of human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice

[0146] Figure 23 shows the effect of patritumab-ADC on the volume of human lung adenocarcinoma PC9-DTC (Del19 / T790M / C797S, Osimertinib-resistant cells) transplanted tumors in NOD-SCID mice

[0147] Figure 24 shows the effect of patritumab-ADC on the volume of human colon cancer SW620 cell transplanted tumors in NU / NU mice

[0148] Figure 25 shows the results of patritumab-ADC inhibitory activity test on DiFi cells DETAILED DESCRIPTION

[0149] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Experimental methods in the following examples where specific conditions are not specified are generally performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as those familiar to professionals in the field. In addition, any methods and materials similar or equivalent to those described herein can be applied to the present invention. The preferred embodiments and materials shown herein are for demonstration purposes only.

[0150] The structures of the compounds of the present invention are determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or liquid chromatography (HPLC). The NMR measurements were performed using a Bruker Avance III 400 MHz nuclear magnetic resonance spectrometer; the LC-MS measurements were performed using a SHIMADZU LC-20AD-PDA-LCMS-2020 instrument; and the HPLC measurements were performed using a SHIMADZU LC-20AD-PDA high performance liquid chromatograph.

[0151] The starting materials in the examples of the present invention are known and can be purchased commercially, or can be synthesized using or according to methods known in the art.

[0152] The antibodies of the present invention can be prepared using the hybridoma technique first described by Kohler et al., Nature (1975), or by recombinant DNA methods (US Pat. No. 4,816,567, etc.).

[0153] Preparation Example

[0154] The antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 9 and two light chains with the amino acid sequence shown in SEQ ID NO: 10 is defined as DP001;

[0155] An antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 19 and two light chains with the amino acid sequence shown in SEQ ID NO: 20 is defined as patritumab;

[0156] The antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 29 and two light chains with the amino acid sequence shown in SEQ ID NO: 30 is defined as SWY2110;

[0157] The antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 34 and two light chains with the amino acid sequence shown in SEQ ID NO: 30 is defined as SWY2111;

[0158] The antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 37 and two light chains with the amino acid sequence shown in SEQ ID NO: 30 is defined as SWY2112;

[0159] The antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 40 and two light chains with the amino acid sequence shown in SEQ ID NO: 30 was defined as SWY2113.

[0160] Antibody sequence description

[0161] 1. HER2 antibody DP001 (trastuzumab)

[0162] Light chain (SEQ ID NO: 10):

[0163] Heavy chain (SEQ ID NO: 9):

[0164] DP001 HCDR1:GFNIKDTYIH(SEQ ID NO:1)

[0165] DP001 HCDR2:RIYPTNGYTRYAD(SEQ ID NO:2)

[0166] DP001 HCDR3:WGGDGFYAMDY(SEQ ID NO:3)

[0167] DP001 LCDR1:RASQDVNTAVA(SEQ ID NO:4)

[0168] DP001 LCDR2:SASFLYS(SEQ ID NO:5)

[0169] DP001 LCDR3:QQHYTTPPT(SEQ ID NO:6)

[0170] DP001 HV: SEQ ID NO: 7

[0171] DP001 LV: SEQ ID NO: 8

[0172] 2. HER3 antibody patritumab

[0173] Light chain (SEQ ID NO: 20):

[0174] Heavy chain (SEQ ID NO: 19):

[0175] Patritumab HCDR1: GGSFSGYYWS SEQ ID NO:11

[0176] Patritumab HCDR2: EINHSGSTNYNPSLKS SEQ ID NO:12

[0177] Patritumab HCDR3: DKWTWYFDL SEQ ID NO:13

[0178] Patritumab LCDR1: RSSQSVLYSSSNRNYLA SEQ ID NO:14

[0179] Patritumab LCDR2: WASTRES SEQ ID NO:15

[0180] Patritumab LCDR3: QQYYSTPRT SEQ ID NO:16

[0181] Patritumab HV: SEQ ID NO:17

[0182] Patritumab LV: SEQ ID NO:18

[0183] 3. EGFR antibody SWY2110

[0184] Light chain (SEQ ID NO:30):

[0185] Heavy chain (SEQ ID NO:29):

[0186] SWY2110 HCDR1: NYDVH SEQ ID NO:21

[0187] SWY2110 HCDR2: VIWSGGNTDYNTPFTS SEQ ID NO:22

[0188] SWY2110 HCDR3: ALDYYDYEFAY SEQ ID NO:23

[0189] SWY2110 LCDR1: RASQSIGTNIH SEQ ID NO:24

[0190] SWY2110 LCDR2: YASESIS SEQ ID NO:25

[0191] SWY2110 LCDR3: QQNNEWPTS SEQ ID NO:26

[0192] SWY2110 HV: SEQ ID NO:27

[0193] SWY2110 LV: SEQ ID NO:28

[0194] 4. EGFR antibody SWY2111

[0195] Light chain (SEQ ID NO:30):

[0196] Heavy chain (SEQ ID NO:34):

[0197] SWY2111 HCDR1: DYDVH SEQ ID NO:31

[0198] SWY2111 HCDR2: VIWSGGNTDYNTPFTS SEQ ID NO:22

[0199] SWY2111 HCDR3: ALDDYDYEFAY SEQ ID NO:32

[0200] SWY2111 LCDR1: RASQSIGTNIH SEQ ID NO:24

[0201] SWY2111 LCDR2: YASESIS SEQ ID NO:25

[0202] SWY2111 LCDR3: QQNNEWPTS SEQ ID NO:26

[0203] SWY2111 HV: SEQ ID NO:33

[0204] SWY2111 LV: SEQ ID NO:28

[0205] 5. EGFR antibody SWY2112

[0206] Light chain (SEQ ID NO:30):

[0207] Heavy chain (SEQ ID NO:37):

[0208] SWY2112 HCDR1: EYDVH SEQ ID NO:35

[0209] SWY2112 HCDR2: VIWSGGNTDYNTPFTS SEQ ID NO:22

[0210] SWY2112 HCDR3: ALDDYDYEFAY SEQ ID NO:32

[0211] SWY2112 LCDR1: RASQSIGTNIH SEQ ID NO:24

[0212] SWY2112 LCDR2: YASESIS SEQ ID NO:25

[0213] SWY2112 LCDR3: QQNNEWPTS SEQ ID NO:26

[0214] SWY2112 HV: SEQ ID NO:36

[0215] SWY2112 LV: SEQ ID NO:28

[0216] 6. EGFR antibody SWY2113

[0217] Light chain (SEQ ID NO:30):

[0218] Heavy chain (SEQ ID NO:40):

[0219] SWY2113 HCDR1: HYDVH SEQ ID NO:38

[0220] SWY2113 HCDR2: VIWSGGNTDYNTPFTS SEQ ID NO:22

[0221] SWY2113 HCDR3: ALDDYDYEFAY SEQ ID NO:32

[0222] SWY2113 LCDR1: RASQSIGTNIH SEQ ID NO:24

[0223] SWY2113 LCDR2:YASESIS SEQ ID NO:25

[0224] SWY2113 LCDR3:QQNNEWPTS SEQ ID NO:26

[0225] SWY2113 HV: SEQ ID NO: 39

[0226] SWY2113 LV: SEQ ID NO: 28

[0227] Example 1

[0228] N-((2R,10S)-10-Benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-methyl-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (JSSW-001)

[0229] Option 1:

[0230] Step 1: Preparation of compound B2-1: B1-1 (1.0 g, 2.28 mmol), anhydrous tetrahydrofuran (30 mL), toluene (10 mL), pyridine (0.5 mL), and lead tetraacetate (1.83 g, 4.12 mmol) were added to a 100 mL three-necked flask. The solution turned orange and was heated under reflux. After one hour, the solution became colorless and a white solid was formed. After three hours of reaction, the solution was filtered through diatomaceous earth, the solid was rinsed with ethyl acetate, and the filtrate was dried using a rotary evaporator. The crude product was purified by column chromatography to obtain a solid (640 mg). LC-MS [M+Na] + :m / z 391.1.

[0231] Step 2: Preparation of Compound A2-1: To a 100ml three-necked flask, add A1-1 (2.7g, 30.0mmol) and N,N-dimethylformamide (30mL), followed by potassium carbonate (4.17g, 30.0mmol) and benzyl bromide (2.4mL, 20.0mmol). After reacting at room temperature for 14 hours, the reaction solution was added dropwise to water and extracted with ethyl acetate. The organic layers were combined and washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was purified by column chromatography (PE / EA = 3:1) to obtain 2.61g of the target compound as a colorless liquid.

[0232] 1 H NMR (400MHz, CDCl3): δ7.33-7.41(m,5H),5.21(s,2H),4.29-4.35(m,1H),2.77(d,J=5.6Hz,1H),1.44(d,J=6.8Hz,3H).

[0233] Step 3: Preparation of compound A3-1: A2-1 (720 mg, 4.0 mmol), B2-1 (368 mg, 1.0 mmol) and dichloromethane (5 mL) were added to a 100 ml three-necked flask, and 4-methylbenzenesulfonic acid pyridinium (75 mg, 0.30 mmol) was added. The reaction solution was heated under reflux overnight. After the reaction was completed, ethyl acetate (20 mL) was added to the reaction solution, washed three times with water, and dried over anhydrous magnesium sulfate. The anhydrous magnesium sulfate was removed by filtration, and the solution was concentrated. The crude product was purified by column chromatography (PE / EA=2:1) ​​to obtain A3-1 (290 mg) as a white solid.

[0234] LC-MS[M+Na] + :m / z 511.1. 1 H NMR (400MHz, CDCl3): δ7.77(d,J=7.2Hz,2H),7.58(d,J=7.6Hz,2H),7.40(t,J=7.6Hz,2H),7.26-7.37(m,6H),6.70(brs,1H ),5.12-5.30(m,3H),4.76-4.89(m,2H),4.45(d,J=6.8Hz,1H),4.19-4.27(m,2H),3.69-3.85(m,2H),1.41(d,J=6.8Hz,3H).

[0235] Step 4: Preparation of Compound A4-1: In a 100ml three-necked flask, A3-1 (291mg, 0.60mmol) was dissolved in tetrahydrofuran:ethyl acetate (4mL / 2mL). Pd / C (10% w / w, 60mg) was added. The atmosphere was replaced with hydrogen three times, then covered with a hydrogen balloon and stirred overnight. After the reaction, the reaction mixture was passed through celite and rinsed with methanol. The organic layer was concentrated to obtain product A4-1. LC-MS [M+Na] + :m / z 399.1.

[0236] Step 5: Preparation of compound A6-1: A4-1 (50 mg, 0.13 mmol, 1.3 eq), isotecan mesylate (A5-1) (50 mg, 0.049 mmol, 1.0 eq), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (48 mg, 0.13 mmol, 1.3 eq), N,N-diisopropylethylamine (36 mg, 0.28 mmol, 3 0eq), dissolved in N,N-dimethylformamide (2 ml), stirred at room temperature overnight, diluted with ethyl acetate, washed with half-saturated citric acid, then washed with brine, washed with sodium bicarbonate solution, washed with brine, the organic layer was dried over anhydrous sodium sulfate and filtered to remove anhydrous sodium sulfate, concentrated, and purified using a prep-TLC developing solvent system (DCM / MeOH=15:1) to obtain product A6-1 (56 mg), LC-MS [M+H] + :m / z 816.3.

[0237] Step 6: Preparation of compound A7-1: Add A6-1 (56 mg, 0.069 mmol), N,N-dimethylformamide (2 ml), and piperidine (12 mg, 0.14 mmol) to a 25 ml single-necked bottle, stir for 2 hours, and concentrate the solution to obtain a white solid. LC-MS [M+H] + :m / z 594.3.

[0238] Step 7: Preparation of compound JSSW-001: A7-1 (0.069 mmol), C7 (66 mg, 0.14 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (53 mg, 0.14 mmol), and a solution of N,N-diisopropylethylamine (36 mg, 0.28 mmol) in N,N-dimethylformamide (2 ml) were added to a 25 ml single-necked bottle and stirred at room temperature for 2 hours. The mixture was diluted with ethyl acetate (30 ml) and washed with half-saturated citric acid, followed by washing with brine, sodium bicarbonate solution, and brine. The organic layer was dried over anhydrous sodium sulfate and filtered to remove anhydrous sodium sulfate. The mixture was concentrated and purified using a prep-TLC developing solvent system (DCM / MeOH = 15:1) to obtain the product JSSW-001 (21 mg).

[0239] LC-MS[M+H] + :m / z 1048. 1 H NMR (400MHz, DMSO-d6): δ8.63(t,J=6.4Hz,1H),8.53(d,J=8.8Hz,1H),8.28(t,J=6.0Hz,1H),8.03-8.10(m,2H),7.98(t,J=6.0Hz,1H ),7.77(d,J=11.2Hz,1H),7.30(s,1H),7.14-7.25(m,5H),6.99(s,2H),6.51(s,1H),5.56-5.62(m,1H),5.40(s,2H),5.08-5.23(m,2H ),4.66-4.71(m,1H),4.42-4.56(m,2H),4.09-4.15(m,1H),3.54-3.76(m,7H),3.09-3.27(m,1H),2.97-3.02(m,1H),2.67-2.77(m,1H ),2.38(s,3H),2.16-2.21(m,2H),2.05-2.10(m,2H),1.83-1.89(m,2H),1.37-1.46(m,7H),1.14-1.33(m,4H),0.87(t,J=7.2Hz,3H).

[0240] Option 2:

[0241] Step 1: Compounds C1-1 (8 g, 88.86 mmol) and C2-1 (22.66 g, 133.33 mmol) were added to a 250 mL reaction flask. Dimethylacetamide (DMAc) (40 mL, 5 v / w) was added and stirred until dissolved. The temperature was maintained at 0-10°C, followed by the dropwise addition of N,N-diisopropylethylamine (DIEA) (34.45 g, 266.6 mmol). After the addition was complete, the mixture was allowed to warm naturally to approximately 25°C and stirred at this temperature for 16 hours. TLC was used to monitor the complete reaction of the starting materials. The reaction mixture was poured into 120 mL of ice water, and methyl tert-butyl ether (MTBE) (40 mL) was added and stirred. The mixture was allowed to stand for separation. The aqueous phase was extracted four times with MTBE (40 mL x 4). The organic phases were combined and washed sequentially with 0.5 M hydrochloric acid (40 mL), water (40 mL), and saturated brine (40 mL x 2). The mixture was dried over anhydrous sodium sulfate, filtered, concentrated, and wet loaded onto a column for purification using a mixture of petroleum ether:ethyl acetate = 30:1 → 20:1 → 10:1 to obtain a light yellow liquid C3-1. LC-MS [M+Na] + :m / z 203.

[0242] Step 2: Under nitrogen protection, C4-1 (6 g, 16.29 mmol, 1.0 eq) and C3-1 (3.23 g, 17.92 mmol, 1.1 eq) were dissolved in tetrahydrofuran (THF) (60 mL, 10 V / w), cooled to 10-15 ° C, and p-toluenesulfonic acid (TsOH) (281 mg, 1.63 mmol, 0.1 eq) was added. After the addition was complete, the reaction was carried out at 14-18 ° C for 4 h and monitored by TLC. After the reaction was completed. The reaction solution was added to ice water (60 mL), and then extracted with ethyl acetate (EA) (60 mL*3). The organic phases were combined, washed with saturated NaHCO3 aqueous solution (60 mL), washed with water (60 mL*2), washed with saturated brine (60 mL), dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness, mixed and purified by column chromatography. The system of n-hexane:ethyl acetate = 10:1 → 5:1 → 3:1 → 2:1 → 1:1 was used to obtain colorless oil C5-1. LC-MS [M+Na] + :m / z 511.

[0243] Step 3: Under nitrogen protection, C5-1 (2.8 g, 5.74 mmol, 1.0 eq) was dissolved in DMAc (28 mL, 10 V / w), cooled to 14-18 ° C, and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (436.6 mg, 2.87 mmol, 0.5 eq) was added dropwise. The mixture was stirred at this temperature for 1.5 h. The reaction of the raw materials was completed by TLC monitoring. The temperature was cooled to 0-10 ° C, and 4-methylbenzenesulfonic acid pyridinium (PPTS) was added. )(721.23 mg, 2.87 mmol, 0.5 eq), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI) (1.1 g, 5.74 mmol, 1.0 eq), 1-hydroxybenzotriazole (HOBT) (775 mg, 5.74 mmol, 1.0 eq) and C7-1 (2.45 g, 4.88 mmol, 0.85 eq) were added and reacted at 0-10 ° C for 3-4 h. After the reaction of C7-1 was completed, TLC was monitored. The reaction solution was added to ice water (100 mL), and then extracted with 2-methyltetrahydrofuran (2-MeTHF) (100 mL*3). The organic phases were combined, washed with 0.5 M hydrochloric acid (150 mL*2), saturated NaHCO3 aqueous solution (100 mL*3), water (100 mL), saturated brine (100 mL), dried over anhydrous sodium sulfate, filtered, concentrated and evaporated to dryness. The mixed sample was purified by column chromatography with a system of DCM→DCM:MeOH=80:1→60:1→50:1→40:1→30:1→20:1 to obtain C8-1 as a white foamy solid, 3.1 g, with a yield of 72%. LC-MS [M+Na] + :m / z 772.

[0244] Step 4: Under nitrogen, C8-1 (3.1 g, 4.14 mmol, 1.0 eq) was dissolved in dichloromethane (DCM) (46.5 mL, 15 V / w). DBU (314.6 mg, 2.07 mmol, 0.5 eq) was added dropwise at room temperature (25-30°C). The reaction was stirred at this temperature for 16 h. TLC monitored the complete reaction of the starting material. The reaction solution was directly wet-filtered through a column using a DCM→DCM:MeOH ratio of 50:1→30:1→20:1→10:1 ratio to obtain C9-1 as a white foamy solid. LC-MS [M+H] + :m / z 528.

[0245] Step 5: Under nitrogen, dissolve C9-1 (1.7 g, 3.2 mmol, 1.0 eq) in water (25.5 mL, 15 V / w) and tert-butanol (8.5 mL, 5 V / w). Stir and dissolve until clear. Replace the atmosphere with nitrogen once, then add Pd / C (0.34 g, 20% w / w). Replace the atmosphere with hydrogen three times. Hydrogenate at room temperature (25-30°C) for 12 h. LCMS monitors the reaction for complete reaction. The reaction mixture is filtered, and the filter cake is washed with water (25 mL x 2). The filtrate is filtered again and concentrated to remove the tert-butanol. The aqueous phase is then lyophilized to yield 1.5 g of C10-1 as a white powdery solid, with a yield of 100%. LC-MS [M+H] + :m / z 438.

[0246] Step 6: Under nitrogen, dissolve C10-1 (1.4 g, 3.2 mmol, 1.0 eq) in acetonitrile (14 mL, 10 V / w) and water (28 mL, 20 V / w). Add C11-1 (1.86 g, 6.097 mmol, 1.1 eq) and stir to dissolve. Cool to 0-10°C, then add DIEA (330.24 mg, 2.56 mmol) dropwise. Stir and react at this temperature for 16 h. LC-MS monitors the complete reaction. Add Na2HPO4 (260 mg) and NaH2PO4 (5.6 g) to 20 mL of water, stir to dissolve, and prepare a buffer solution. Cool the buffer solution to 0-5°C. Pour the reaction solution into chilled buffer and extract with DCM / isopropanol = 4 / 1 (50 mL x 4). Combine the organic phases, dry, filter, concentrate, evaporate, and purify by column chromatography. The system of DCM→DCM:MeOH=50:1→30:1→20:1→15:1 was passed through the column to obtain yellow solid C12-1.

[0247] Step 7: Under nitrogen, A5-1 (547.4 mg, 1.03 mmol, 1.0 eq) was dissolved in 5% anhydrous sodium sulfate in water (6.5 mL) and THF (7.8 mL). Stirring was not effective until the solution was dissolved. The mixture was cooled to 0-10°C and N-methylmorpholine (NMM) (104 mg, 1.03 mmol, 1.0 eq) was added. The mixture was reacted at this temperature for 1 h. C12-1 (650 mg, 1.03 mmol, 1.1 eq), EDCI (296.2 mg, 1.545 mmol, 1.5 eq), and ethyl 2-oximecyanoacetate (73.9 mg, 0.52 mmol, 0.5 eq) were added in sequence. The mixture was reacted at 0-10°C for 4-5 h. The reaction was monitored by TLC to confirm the complete reaction of the starting material. The reaction mixture was added to 0.1M hydrochloric acid (13 mL, 20 V / w) under an ice bath and extracted with 2-methyltetrahydrofuran (2-MeTHF) (13 mL*3). The organic phases were combined and washed once with 0.05M hydrochloric acid (13 mL, 20 V / w), washed with water (13 mL*2), dried, filtered, concentrated and evaporated to dryness, and purified by column chromatography. The column chromatography was performed using a DCM→DCM:MeOH ratio of 50:1→30:1→20:1→15:1→12:1 to obtain JSSW-001 as a yellow foamy solid. LC-MS [M+H] + :m / z 1048.

[0248] Example 2 Antibody-Drug Conjugate (DP001-JSSW-001 ADC, DAR=4.0)

[0249] The first step is the reduction of antibodies:

[0250] Using known techniques, the DP001 antibody was prepared by transiently transfecting HEK293 cells with plasmids encoding the light and heavy chain genes for expression and purification, followed by replacement of the medium with PBS 6.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, to which was added a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (17 μL; 2.5 equivalents per antibody molecule) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd., 50 μL). After confirming that the pH of the solution was within 7.4 ± 0.1, the solution was incubated at 37°C for 1 hour to reduce the disulfide bonds within the antibody.

[0251] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0252] To this solution, a 10 mM dimethyl sulfoxide solution of the JSSW-001 compound obtained in Example 1 (31.28 μL; 4.6 equivalents per antibody molecule) was added at room temperature. The mixture was mixed and allowed to react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0253] Step 3: Purification of Antibody-Drug Conjugate:

[0254] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cell μLose (30kDa MWCO, 15mL sample volume). 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added as a purification buffer to the reaction solution. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low molecular weight reagents, yielding the purified antibody-drug conjugate (DP001-JSSW-001ADC, DAR = 4.0).

[0255] Step 4: Determination of the Drug-Antibody Coupling Ratio (DAR) of the Antibody-Drug Conjugate:

[0256] Dithiothreitol was added to a final concentration of 20 mM to 2 mg / kg of the antibody-drug conjugate, and the sample, which had broken the interchain disulfide bonds of the antibody-drug conjugate, was incubated at 37°C in a water bath for 30 minutes. The sample was then used for HPLC analysis. The HPLC system used was an Agilent Technologies 1260 Infinity HPLC, and the column was a PLRP-S (5 μm particle size; 2.1 mm × 50 mm; Agilent Technologies). The column temperature was 80°C, and the mobile phase A consisted of 0.1% trifluoroacetic acid (TFA) in water, and the mobile phase B consisted of 0.1% trifluoroacetic acid (TFA) in acetonitrile. The sample volume was 10 μL, and the gradient program was 27%-27% from 0-3 minutes, 27%-35% from 3-8 minutes, 35%-43% from 8-25 minutes, 43%-95% from 25-26 minutes, 95%-95% from 26-31 minutes, 95%-27% from 31-32 minutes, and 27%-27% from 32-40 minutes. Compared to unlinked light chains (L0) and heavy chains (H0), the hydrophobicity of drug-linked light chains (light chains linked to one drug, L1) and heavy chains (heavy chains linked to one drug, H1; heavy chains linked to two drugs, H2; and heavy chains linked to three drugs, H3) increases with the number of linked drugs. Therefore, the elution order is L0, L1, H0, H1, H2, and H3. The DAR values ​​were calculated based on the peak areas at 280 nm. The DAR values ​​are shown in Figure 1.

[0257] Example 3 Antibody-Drug Conjugate (DP001-JSSW-001 ADC, DAR=7.32)

[0258] The first step is the reduction of antibodies:

[0259] Using known techniques, the DP001 antibody was prepared by transiently transfecting HEK293 cells with plasmids encoding the light and heavy chain genes for expression and purification, replacing the medium with PBS 6.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, to which was added 6.66 μL of a 100 mM TCEP (J&K Technology Co., Ltd.; 10 equivalents per antibody molecule) and 18 μL of a 1 M potassium dihydrogen phosphate solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0260] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0261] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0262] Step 3: Purification of Antibody-Drug Conjugates:

[0263] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cell μLose (30kDa MWCO), 15mL sample volume. 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added as a purification buffer to the reaction solution. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (DP001-JSSW-001ADC, DAR = 7.32). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 2.

[0264] Example 4 Antibody-Drug Conjugate (patritumab-JSSW-001 ADC, DAR=7.36)

[0265] The first step is the reduction of antibodies:

[0266] The patritumab antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 100 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (6.66 μL; 10 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd., 18 μL) were added thereto. After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37 ° C for 3 hours to reduce the disulfide bonds in the antibody.

[0267] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0268] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0269] Step 3: Purification of Antibody-Drug Conjugates:

[0270] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cell μLose (30kDa MWCO), 15mL sample volume. 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added to the reaction solution as a purification buffer. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (patritumab-JSSW-001ADC, DAR = 7.36). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 3.

[0271] Example 5 Antibody-Drug Conjugate (SWY2110-JSSW-001 ADC, DAR=7.12)

[0272] The first step is the reduction of antibodies:

[0273] The SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (66.6 uL; 10 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.) were added thereto. After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37 ° C for 3 hours to reduce the disulfide bonds in the antibody.

[0274] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0275] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0276] Step 3: Purification of Antibody-Drug Conjugate:

[0277] Ultrafiltration centrifuge tube (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30 kDa MWCO), 15 mL sample volume. A buffer solution consisting of 0.89 mg / mL L-histidine, 4.04 mg / mL L-histidine hydrochloride, 0.03% Tween 80, and 90 mg / mL sucrose (pH 5.5) was added to the reaction solution to remove unconjugated drug linkers and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (SWY2110-JSSW-001ADC, DAR = 7.12). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 4.

[0278] Example 6 Preparation of pH-dependent antibodies

[0279] Through computer simulation-assisted technology, SWY2110 and EGFR protein were modeled and docked to obtain the antigen-antibody complex. D, E, and H scans were performed on amino acids within 100 μg / mL, yielding several pH-dependent antibodies with reduced affinity at pH 7.4 and essentially unchanged or with a minimal decrease in affinity at pH 6.0. The resulting antibody sequences were cloned into mammalian expression vectors and expressed via HEK-293 cell transfection. After expression, the supernatant was collected and purified using an AKTA system equipped with a Protein A prepacked column to obtain the desired antibodies.

[0280] Example 7 Antibody Affinity Determination

[0281] MDA-MB-468 cells were digested into single cells using 0.25% trypsin-EDTA and divided into multiple aliquots with more than 10 cells per aliquot. 6300 μL of 5-fold serially diluted SWY2110 (control) and the antibody obtained in Example 9 (the antibodies were diluted with PBS buffer solutions of pH 7.4 and pH 6.0, respectively) were added to the cells, incubated at 4°C for 1 hour, and centrifuged at 1000 rpm for 4 minutes to remove the supernatant. Wash twice with PBS solution, then add 200 μL of 1:200 diluted FITC-labeled mouse anti-human Fc secondary antibody, incubate at 4°C for 1 hour, and centrifuge at 1000 rpm for 4 minutes after the incubation to remove the supernatant. Use flow cytometer to detect the fluorescence intensity, save the data, and perform data analysis using relevant software. The results of the antibody affinity determination after screening are shown in Table 1. The results of affinity measurements of the pH-dependent optimized antibodies with the target antigen showed that at pH 7.4, the affinity of the pH-optimized antibodies SWY2111, SWY2112, and SWY2113 relative to SWY2110 was significantly reduced (approximately 3.0-9.7 times). At pH 6.0, the affinity of SWY2111 and SWY2112 relative to SWY2110 remained basically unchanged (1.2-1.3 times), and the affinity of SWY2113 antibody decreased by 6.6 times.

[0282] Table 1. Antibody affinity determination results

[0283] Example 8 Antibody-Drug Conjugate (SWY2111-JSSW-001 ADC, DAR=7.48)

[0284] The first step is the reduction of antibodies:

[0285] The obtained SWY2111 antibody medium was replaced with PBS6.0 / EDTA to prepare an antibody concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (66.6 uL; 10 equivalents per one antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.) were added thereto. After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37°C for 3 hours to reduce the disulfide bonds in the antibody.

[0286] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0287] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0288] Step 3: Purification of Antibody-Drug Conjugate:

[0289] Ultrafiltration centrifuge tube (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30 kDa MWCO), 15 mL sample volume. A buffer solution consisting of 0.89 mg / mL L-histidine, 4.04 mg / mL L-histidine hydrochloride, 0.03% Tween 80, and 90 mg / mL sucrose (pH 5.5) was added to the reaction solution to remove unconjugated drug linkers and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (SWY2111-JSSW-001 ADC, DAR = 7.48). The DAR value was determined using the same method as in Step 4 of Example 2. The resulting DAR values ​​are shown in Figure 5.

[0290] Example 9 Antibody-Drug Conjugate (SWY2112-JSSW-001 ADC, DAR=7.51)

[0291] The first step is the reduction of antibodies:

[0292] The obtained SWY2112 antibody medium was replaced with PBS6.0 / EDTA to prepare an antibody concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, to which was added a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (66.6 uL; 10 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37°C for 3 hours to reduce the disulfide bonds in the antibody.

[0293] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0294] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0295] Step 3: Purification of Antibody-Drug Conjugate:

[0296] Ultrafiltration centrifuge tube (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30 kDa MWCO), 15 mL sample volume. A buffer solution consisting of 0.89 mg / mL L-histidine, 4.04 mg / mL L-histidine hydrochloride, 0.03% Tween 80, and 90 mg / mL sucrose (pH 5.5) was added to the reaction solution to remove unconjugated drug linkers and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (SWY2112-JSSW-001ADC, DAR = 7.51). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 6.

[0297] Example 10 Antibody-Drug Conjugate (SWY2113-JSSW-001 ADC, DAR=7.50)

[0298] The first step is the reduction of antibodies:

[0299] The obtained SWY2113 antibody medium was replaced with PBS6.0 / EDTA to prepare an antibody concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, to which was added a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (66.6 uL; 10 equivalents per antibody molecule) and a 1 M dipotassium hydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37°C for 3 hours to reduce the disulfide bonds in the antibody.

[0300] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0301] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of the compound JSSW-001 obtained in Example 1 was added at room temperature, mixed, and allowed to react at room temperature for 30 minutes to attach the drug linker to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added, and the reaction was terminated by stirring at room temperature for another 20 minutes.

[0302] Step 3: Purification of Antibody-Drug Conjugate:

[0303] Ultrafiltration centrifuge tube (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30kDa MWCO), 15mL sample volume. A buffer solution consisting of 0.89mg / ml L-histidine, 4.04mg / ml L-histidine hydrochloride, 0.03% Tween 80, and 90mg / ml sucrose (pH 5.5) was added to the reaction solution to remove unconjugated drug linkers and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (SWY2113-JSSW-001ADC, DAR = 7.50). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 7.

[0304] Reference Example 1 Antibody-Drug Conjugate (DP001-DXD ADC, DAR = 4.3)

[0305] The first step is the reduction of antibodies:

[0306] Using known techniques, the DP001 antibody was prepared by transiently transfecting HEK293 cells with plasmids encoding the light and heavy chain genes for expression and purification, followed by replacement of the medium with PBS 6.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, to which was added a 10 mM TCEP (J&K Technology Co., Ltd.) aqueous solution (17 μL; 2.5 equivalents per antibody molecule) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd., 50 μL). After confirming that the pH of the solution was within 7.4 ± 0.1, the solution was incubated at 37°C for 1 hour to reduce the disulfide bonds within the antibody.

[0307] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0308] To this solution, a 10 mM dimethyl sulfoxide solution (31.28 μL; 4.6 equivalents per antibody molecule) of commercially available Deruxtecan (CAS No.: 1599440-13-7) was added at room temperature. The mixture was mixed and allowed to react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technologies, Inc.) (10.2 μL) was added and stirred at room temperature for another 20 minutes to terminate the reaction.

[0309] Step 3: Purification of Antibody-Drug Conjugates:

[0310] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cell μLose (30 kDa MWCO, 15 mL sample volume). 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added to the reaction solution as a purification buffer. The solution was concentrated to 1-2 mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (DP001-DXD ADC, DAR = 4.3). The drug-antibody conjugate ratio (DAR) of the antibody-drug conjugate was determined using the same method as in Step 4 of Example 2.

[0311] Reference Example 2 Antibody-Drug Conjugate (DP001-DXD ADC, DAR=7.0)

[0312] The first step is the reduction of antibodies:

[0313] The DP001 antibody was transiently transfected into HEK293 cells with plasmids encoding the light and heavy chain genes using known techniques for expression and purification. The medium was replaced with PBS 6.0 / EDTA to a concentration of 10 mg / mL. This solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, to which was added 6.66 μL of a 100 mM TCEP (J&K Technology Co., Ltd.); 10 equivalents per antibody molecule) and 18 μL of a 1 M potassium dihydrogen phosphate solution (Tianjin Guangfu Technology Development Co., Ltd.). After confirming that the pH of the solution was within 7.0 ± 0.1, the solution was incubated at 37°C for 3 hours to reduce the disulfide bonds within the antibody.

[0314] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0315] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of commercially available Deruxtecan (CAS No.: 1599440-13-7) was added at room temperature. The mixture was mixed and allowed to react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technologies, Inc.) (10.2 μL) was added and stirred at room temperature for another 20 minutes to terminate the reaction.

[0316] Step 3: Purification of Antibody-Drug Conjugates:

[0317] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cell μLose (30 kDa MWCO, 15 mL sample volume). 25 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added to the reaction solution as a purification buffer. The solution was concentrated to 1-2 mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (DP001-DXD ADC, DAR = 7.0). The drug-antibody coupling ratio (DAR) of the antibody-drug conjugate was determined using the same method as in Step 4 of Example 2.

[0318] Reference Example 3 Antibody-Drug Conjugate (patritumab-DXD ADC, DAR = 7.3)

[0319] The first step is the reduction of antibodies:

[0320] The patritumab antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 100 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (6.66 μL; 10 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd., 18 μL) were added thereto. After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37 ° C for 3 hours to reduce the disulfide bonds in the antibody.

[0321] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0322] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of commercially available Deruxtecan (CAS No.: 1599440-13-7) was added at room temperature. The mixture was mixed and allowed to react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technologies, Inc.) (10.2 μL) was added and stirred at room temperature for another 20 minutes to terminate the reaction.

[0323] Step 3: Purification of Antibody-Drug Conjugates:

[0324] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cell μLose (30kDa MWCO), 15mL sample volume. 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added to the reaction solution as a purification buffer. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (patritumab-DXD ADC, DAR = 7.3). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 8.

[0325] Reference Example 4 Antibody-Drug Conjugate (SWY2110-DXD ADC, DAR = 6.93)

[0326] The first step is the reduction of antibodies:

[0327] The SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (66.6 uL; 10 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.) were added thereto. After confirming that the pH of the solution was within 7.0 ± 0.1, it was incubated at 37 ° C for 3 hours to reduce the disulfide bonds of the antibody.

[0328] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0329] To this solution, a 10 mM dimethyl sulfoxide solution (87 μL; 13 equivalents per antibody molecule) of a commercially available Deruxtecan (CAS No. 1599440-13-7) compound was added at room temperature. Mix thoroughly and allow to react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technologies, Inc.) (10.2 μL) was added and stirred at room temperature for another 20 minutes to terminate the reaction.

[0330] Step 3: Purification of Antibody-Drug Conjugate:

[0331] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration using Regenerated Cellulose (30kDa MWCO), 15mL sample volume. 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added to the reaction solution as a purification buffer. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low-molecular-weight reagents, yielding a purified antibody-drug conjugate (SWY2110-DXD ADC, DAR = 6.93). The DAR value was determined using the same method as in Step 4 of Example 2. The DAR values ​​are shown in Figure 9.

[0332] Reference Example 5 Antibody-Drug Conjugate (SWY2110-VC MMAE ADC, DAR = 4.53)

[0333] The first step is the reduction of antibodies:

[0334] The SWY2110 antibody medium obtained after transient transfection of HEK293 cells expressing and purifying the plasmid encoding the light and heavy chain genes was replaced with PBS6.0 / EDTA by known techniques to prepare an antibody concentration of 10 mg / mL. The solution (1.0 mL) was placed in a 1.5 mL polypropylene tube, and a 10 mM TCEP (B&K Technology Co., Ltd.) aqueous solution (17 uL; 2.5 equivalents relative to one molecule of antibody) and a 1 M potassium dihydrogen phosphate aqueous solution (Tianjin Guangfu Technology Development Co., Ltd.) were added thereto. After confirming that the pH of the solution was within 7.4 ± 0.1, it was incubated at 37 ° C for 1 hour to reduce the disulfide bonds of the antibody.

[0335] Step 2: Conjugation of Antibody to Linker-Drug Compound:

[0336] To the above solution, a 10 mM dimethyl sulfoxide solution (31.28 μL; 4.6 equivalents per antibody molecule) of commercially available mc-vc-PAB-MMAE (CAS No. 646502-53-6, abbreviated as Vc MMAE) was added at room temperature. Mix thoroughly and react at room temperature for 30 minutes to attach the linker-drug compound to the antibody. Next, a 100 mM aqueous solution of NAC (J&K Technology Co., Ltd.) (10.2 μL) was added and stirred at room temperature for 20 minutes to terminate the reaction.

[0337] Step 3: Purification of Antibody-Drug Conjugate:

[0338] Ultrafiltration centrifuge tubes (Merck, The reaction solution was purified by ultrafiltration through Regenerated Cellulose (30kDa MWCO), 15mL sample volume). 25mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, was added as a purification buffer to the reaction solution. The solution was concentrated to 1-2mL and the purification buffer was added again, with a displacement factor greater than 1000. This removed unconjugated linker-drug compound and other low molecular weight reagents, yielding a purified antibody-drug conjugate (SWY2110-VC MMAE ADC, DAR = 4.53).

[0339] Step 4: Determination of the Drug-Antibody Coupling Ratio (DAR) of the Antibody-Drug Conjugate:

[0340] A 10 μg sample of SWY2110-Vc MMAE ADC was loaded onto a chromatographic column (2.5 μm particle size; 4.6 mm × 10 cm; TSKgel Butyl-NPR). Mobile phase A consisted of 20 mM PB, pH 7.0, in a 1.5 M (NH₄)₂SO₄ aqueous solution, and mobile phase B consisted of 20 mM PB, pH 7.0, in a 25% isopropanol solution. The flow rate was controlled at 0.8 mL / min, the column temperature was 30°C, and the detection wavelength was 280 nm. The gradient program was 0-20 min, with mobile phase B increasing from 0% to 100%, then 20-25 min, 100% mobile phase B, and then 25-30 min, 100% mobile phase A. The unattached small molecules are designated DAR0, the attached small molecules are designated DAR2, the attached small molecules are designated DAR4, the attached small molecules are designated DAR6, and the attached small molecules are designated DAR8. Therefore, the elution order was DAR0, DAR2, DAR4, DAR6, and DAR8. The DAR values ​​were calculated based on the peak areas at 280 nm, where A represents the percentage of each peak area. The DAR values ​​are shown in Figure 10.

[0341] DAR=2×ADAR2+4×ADAR4+6×ADAR6+8×ADAR8

[0342] Biological Examples

[0343] Test 1 Antibody-drug in vitro cell activity

[0344] 1. Test objectives:

[0345] The purpose of this study is to detect the inhibitory activity of the antibody-drug conjugate of the present invention on the in vitro proliferation of SK-BR-3 (ATCC / HTB-30) tumor cells, human colorectal cancer cells DiFi, human lung cancer gefitinib-resistant cells PC9-GR, and human lung cancer mutant cells PC-9 (Del19-T790M-C797S). The cells were treated with different concentrations of the compound in vitro, cultured, and then resazurin was added to read the fluorescence values ​​at ex550nm / em610nm. The data were processed by four-parameter fitting to obtain the IC50 value, thereby calculating the biological activity of the compound.

[0346] 2. Test materials and equipment:

[0347] Material

[0348] equipment

[0349] 3. Test operation method 1:

[0350] 3.1 Culture medium: DMEM, 10% FBS, 1× double antibody

[0351] 3.2 Cell culture: Take out a frozen SK-BR-3 / MDA-MB-468 cell from liquid nitrogen and resuscitate to 75cm 2 Culture in culture flasks. Grow until the cells are >75% confluent and have been passaged at least 3 times.

[0352] 3.2.1 If cell passage is not performed, replace the culture medium every 3 to 4 days.

[0353] 3.2.2 If cell expansion is required, subculture the cells into larger culture flasks and allow the cells to reach a confluency of >75% before use in the assay.

[0354] 3.3 Cell collection: When the culture flask is nearly full, collect SK-BR-3 / MDA-MB-468 cells.

[0355] 3.3.1 Discard the culture medium and wash with PBS to remove dead cells and residual culture medium.

[0356] 3.3.2 Add 2-3 mL of 0.25% Trypsin-EDTA, gently shake the culture flask, and incubate at 37°C for 2-3 minutes to digest the cells.

[0357] 3.3.3 Immediately add 5 mL of culture medium to the culture flask and gently pipette up and down to disperse the cells.

[0358] 3.3.4 Transfer the cell suspension into a sterile centrifuge tube and centrifuge at 200×g for 3 minutes.

[0359] 3.3.5 Discard the culture medium in the tube and add 5-10 mL of fresh culture medium to resuspend the cells.

[0360] 3.4 Cell density determination: Count the cells using a cell counting plate under a microscope.

[0361] 3.5 Inoculation of assay plates

[0362] 3.5.1 Dilute the cells to 1×10 5 cells / mL, and 100 μL / well was seeded into the assay plate (except rows A and H).

[0363] 3.5.2 Add 120 μL of culture medium to each well in rows A and H as blank controls.

[0364] 3.5.3 Incubate at 37°C, 5% CO2 for 4-6 hours to allow cells to adhere.

[0365] 3.6 Prepare diluted compound samples

[0366] The compound was diluted to a starting concentration of 18 μg / ml and then diluted 3-fold to obtain a total of 11 gradient solutions, with the 12th column being a blank control.

[0367] 3.7 Dosing treatment

[0368] 3.7.1 Take 20 μL of each drug dilution in columns 1 to 11 of the dilution plate and add them to columns 1 to 11 of the assay plate.

[0369] 3.7.2 Add 20 μL of fresh culture medium to column 12 and rows A and H.

[0370] 3.7.4 Gently shake the plate on a plate shaker for 10-15 seconds, then incubate the plate at 37°C for 3 days.

[0371] 3.8 Analysis

[0372] 3.8.1 After incubation, add 20 μL of 0.03% resazurin (diluted with 1× PBS) to each well and gently shake for 10-15 seconds.

[0373] 3.8.2 After incubation at 37°C for 3-4 hours, read the plate reader. Set the parameters as follows:

[0374] Excitation light: 550nm

[0375] Emission light: 610nm

[0376] Points time: 50

[0377] Shake: 15 seconds, vortex

[0378] Reading: Top reading

[0379] Read times / well: 1

[0380] Gain: Set to optimize (should be between 35 and 42)

[0381] If you are reading multiple boards, make sure that the gain settings used are consistent across all boards.

[0382] 3.8.3 Use Excel data to draw a graph and fit the IC values ​​of the reference standard and sample 50 .

[0383] 3.8.3.1 Use Model 201 to plot data points.

[0384] 3.8.3.2 For the Fit parameter, use the following command:

[0385] aA: Provisioning

[0386] bB: Provisioning

[0387] cC: Provisioning

[0388] dD: Provisioning

[0389] e. No constraints for all

[0390] 3.8.3.3 Output parameter C is IC 50 , unit is ng / mL.

[0391] The results are shown in Table 2.

[0392] Test operation method 2:

[0393] This study validated the inhibitory effect of SWY2110 monoclonal antibody (SWY2110 mAb), SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001 ADC (DAR=7.12) on the proliferation of DiFi (human colorectal cancer cells) and PC9-GR (human lung cancer gefitinib-resistant cells). The specific concentrations were set as follows:

[0394] DiFi: SWY2110mAb, SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001 ADC (DAR=7.12) were diluted to 3000 ng / ml. Ten concentrations were set at 3× descending dilutions, namely 3000, 1000, 333.33, 111.11, 37.04, 12.35, 4.12, 1.37, 0.46, and 0.15 ng / ml, and the drug exposure lasted for 144 h.

[0395] PC9-GR: SWY2110mAb, SWY2110-DXD ADC (DAR=6.93), and SWY2110-JSSW-001ADC (DAR=7.12) were diluted to a starting concentration of 1000 ng / ml, and 5× decreasing dilutions were used to set a total of 8 concentrations: 1000, 200, 40, 8, 1.6, 0.32, 0.64, and 0.013 ng / ml. The drug action time was 144 h.

[0396] Logarithmically growing cells were seeded in 96-well plates (100 μL / well) at a specific density. After 24 hours of adherent attachment, 100 μL of culture medium containing varying concentrations of SWY2110 mAb, SWY2110-DXD ADC (DAR = 6.93), or SWY2110-JSSW-001 ADC (DAR = 7.12) was added to each well the same day. Each drug concentration was replicated in triplicate, along with corresponding blank wells (medium alone) and normal wells (drug concentration 0). After 144 hours of drug exposure, 20 μL of MTT working solution (5 mg / ml) was added to each well. The plates were incubated at 37°C for 4 hours, the supernatant was removed, and 150 μL of DMSO (analytical grade) was added. The plates were shaken and mixed thoroughly on a microplate shaker. The plates were wiped clean, and the optical density (OD) was measured at 550 nm on a microplate reader.

[0397] The cell growth inhibition rate was calculated using the following formula: Inhibition rate (%) = (OD value 正常孔 -OD value 给药孔 ) / (OD value 正常孔 -OD value 空白孔 )×100%

[0398] According to the inhibition rate of each concentration, the drug half-maximal inhibitory concentration IC was calculated using SPSS19.0 50 The results are shown in Table 3 and Figures 15 and 16.

[0399] Test data:

[0400] Table 2: IC of DP001-ADC against SK-BR-3 cells 50 value

[0401] As can be seen from the data recorded in Table 2, the antibody-drug DP001-JSSW-001 ADC (DAR=7.32) of the present invention has a lower IC50 value than DP001-DXD ADC (DAR=7.0) against SK-BR-3 cells, showing a better anti-cell proliferation effect.

[0402] Table 3: IC of SWY2110-ADC against two cell lines 50 Value (ng / ml)

[0403] “--” indicates that the results showed no anti-cell proliferation effect.

[0404] As can be seen from the data in Table 3, the antibody-drug conjugate SWY2110-JSSW-001 ADC (DAR=7.12) and SWY2110-DXD ADC (DAR=6.93) of the present invention have similar IC values ​​for the EGFR high-expressing cell line DiFi and the gefitinib-resistant cell line PC9-GR. 50 The results showed that SWY2110 mAb has a better anti-cell proliferation effect than SWY2110 mAb.

[0405] Experiment 2: Efficacy of Antibody-Drug Conjugates on HER2-Expressing Human Breast Cancer JIMT-1 Cell Xenografts in NU / NU Mice

[0406] 1. Experimental Animals

[0407] NU / NU mice, 5-6 weeks old, 24

[0408] 2. Purpose of the test

[0409] The anti-tumor effect of antibody-drug conjugates in vivo was investigated using a NU / NU mouse xenograft tumor model harboring HER2-expressing human breast cancer JIMT-1 cells.

[0410] 3. Drug dosage and grouping

[0411] Table 4 Animal grouping and dosage table Note: iv: intravenous injection.

[0412] 4. Test methods

[0413] In this study, human breast cancer cells JIMT-1 were used to construct a nude mouse tumor transplant model. 3At d0, the animals were divided into four groups (6 animals in each group) according to tumor volume according to Table 4. A single intravenous administration of 10 mL / kg was performed. The solvent control group (Vehicle) was given 0.9% sodium chloride injection. The animals were observed for 13 days after administration to compare the inhibitory effects of the three antibody-drug conjugates in the table on HER2-expressing cell line human breast cancer JIMT-1 nude mouse xenograft tumors.

[0414] 5. Observation indicators

[0415] 5.1 Evaluation Metrics

[0416] (1) Tumor volume: V = 1 / 2 × A × B 2

[0417] (2) Relative tumor volume:

[0418] (3) Relative tumor volume growth rate:

[0419] (4) Tumor inhibition rate:

[0420] Note: V: tumor volume

[0421] A: Tumor growth

[0422] B: Tumor width

[0423] RTV: relative tumor volume

[0424] TVnd: tumor volume on day n

[0425] TV0d: tumor volume on day 0

[0426] RTVxnd: average relative tumor volume on day n

[0427] TV X n: average tumor volume of the treatment group on day n

[0428] TV X0 : Average tumor volume on day 0 of the drug-treated group

[0429] TV M n: average tumor volume on day n in the solvent group

[0430] TV M0 : Average tumor volume on day 0 in the solvent group

[0431] 6 Results

[0432] 6.1 Tumor volume

[0433] At the end of the experiment, compared with the vehicle group, DP001-DXD ADC (DAR = 4.3) (P < 0.05), DP001-JSSW-001 ADC (DAR = 4.0) (P < 0.01), and DP001-ZW-002 ADC (DAR = 5.78) (P < 0.05) all significantly inhibited the volume of the transplanted tumor. See Figure 11 and Table 5 for details.

[0434] Table 5 Tumor parameters of each group after 13 days of antibody-drug conjugate administration *: P < 0.05 compared with the solvent group, **: P < 0.01 compared with the solvent group

[0435] Experiment 3: Efficacy of Antibody-Drug Conjugates on Human Lung Adenocarcinoma PC9-GR (Gefitinib-resistant Cells) Xenografts in NU / NU Mice

[0436] 1. Experimental Animals

[0437] NU / NU mice, 5-6 weeks old, 16

[0438] 2. Purpose of the test

[0439] The anti-tumor effects of two patritumab-ADC drugs in vivo were investigated using a NU / NU mouse xenograft tumor model of human lung adenocarcinoma PC-9 / GR (Gefitinib-resistant cells).

[0440] 3. Drug dosage and grouping

[0441] Table 6 Animal grouping and dosage table Note: iv: intravenous injection, qw*4w: once a week for four weeks.

[0442] 4. Test methods

[0443] In this study, human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) cells were used to construct a nude mouse tumor transplantation model. 3At d0, the animals were divided into three groups according to tumor volume according to Table 6: a vehicle group of 6 animals, a patritumab-DXD ADC (DAR=7.3) group, and a patritumab-JSSW-001 ADC (DAR=7.36) group of 5 animals each. The drugs were administered intravenously once a week for 4 consecutive weeks in a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to compare the inhibitory effects of patritumab-DXD ADC (DAR=7.3) and patritumab-JSSW-001 ADC (DAR=7.36) on human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) xenografts in nude mice.

[0444] 5. Observation indicators

[0445] 5.1 Evaluation Metrics

[0446] See Experiment 2, 5.1, Evaluation Indicators

[0447] 6 Results

[0448] 6.1 Tumor volume

[0449] At the end of the trial, the patritumab-JSSW-001 ADC group (DAR = 7.36) (P < 0.001) had a superior effect in inhibiting the volume of transplanted tumors compared to the patritumab-DXD ADC group (DAR = 7.3) (P < 0.01). See Figure 12 and Table 7 for details.

[0450] Table 7 Tumor parameters of each group after 28 days of patritumab-ADC administration **: P < 0.01 compared with the vehicle group, ***: P < 0.001 compared with the solvent group

[0451] Study 4: Efficacy of SWY2110-ADC against human colorectal cancer DiFi cell transplants in NU / NU mice

[0452] 1. Experimental Animals

[0453] NU / NU mice, 5-6 weeks old, 14

[0454] 2. Purpose of the test

[0455] The in vivo antitumor effects of two SWY2110-ADC drugs were investigated using a human colorectal cancer DiFi cell NU / NU mouse xenograft tumor model.

[0456] 3. Drug dosage and grouping

[0457] Table 8 Animal grouping and dosage table Note: iv: intravenous injection.

[0458] 4. Test methods

[0459] In this study, human colorectal cancer DiFi cells were used to construct a nude mouse tumor transplant model. 3 At d0, the animals were divided into three groups balanced by tumor volume according to Table 8: a vehicle group and a SWY2110-JSSW-001 ADC (DAR = 7.12) group of 5 animals each, and a SWY2110-Vc MMAE ADC (DAR = 4.53) group of 4 animals. Each group received a single intravenous administration of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The animals were observed for 27 days after administration to compare the inhibitory effects of the two SWY2110-ADCs listed in the table on human colorectal cancer xenografts in DiFi nude mice.

[0460] 5. Observation indicators

[0461] 5.1 Evaluation Metrics

[0462] See Experiment 2, 5.1, Evaluation Indicators

[0463] 6 Results

[0464] 6.1 Tumor volume

[0465] At the end of the trial, compared with the vehicle group, both SWY2110-Vc MMAE ADC (DAR = 4.53) (P < 0.05) and SWY2110-JSSW-001 ADC (DAR = 7.12) (P < 0.05) significantly inhibited the volume of transplanted tumors. The 27-day tumor inhibition rate in the SWY2110-JSSW-001 ADC (DAR = 7.12) group was as high as 74.6%, followed by the SWY2110-Vc MMAE ADC (DAR = 4.53) at 69.7%. Since the toxicity of the JSSW-001 small molecule is lower than that of Vc MMAE, it is expected that the SWY2110-JSSW-001 ADC (DAR = 7.12) can achieve a more effective and low-toxic effect. See Figure 13 and Table 9 for details.

[0466] Table 9 Tumor parameters of each group after 27 days of SWY2110-ADC administration *: P < 0.05 compared with the vehicle group

[0467] Experiment 5: Efficacy of SWY2110-ADC on human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) transplanted tumors in NU / NU mice

[0468] 1. Experimental Animals

[0469] NU / NU mice, 5-6 weeks old, 30

[0470] 2. Purpose of the test

[0471] The in vivo antitumor effects of SWY2110 monoclonal antibody and three SWY2110-ADC drugs were investigated using a NU / NU mouse xenograft tumor model of human lung adenocarcinoma PC-9-GR (Gefitinib-resistant cells).

[0472] 3. Drug dosage and grouping

[0473] Table 10 Animal grouping and dosage table Note: iv: intravenous injection, qw*4w: once a week for four weeks.

[0474] 4. Test methods

[0475] In this study, human lung adenocarcinoma PC9-GR (Gefitinib-resistant cells) cells were used to construct a nude mouse tumor transplantation model. 3 At 4 d, the animals were divided into 5 groups (d0) according to tumor volume according to Table 10, with 6 animals in each group. The drugs were administered intravenously once a week for 4 consecutive weeks at a dosage of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to compare the inhibitory effects of SWY2110 monoclonal antibody and three SWY2110-ADC drugs on human lung adenocarcinoma PC-9-GR (Gefitinib-resistant cells) xenografts in nude mice.

[0476] 5. Observation indicators

[0477] 5.1 Evaluation Metrics

[0478] See Experiment 2, 5.1, Evaluation Indicators

[0479] 6 Results

[0480] 6.1 Tumor volume

[0481] At the end of the experiment, compared with the vehicle group, SWY2110-Vc MMAE ADC (DAR=4.53) (P<0.05), SWY2110-DXD ADC (DAR=6.93) (P<0.05) and SWY2110-JSSW-001 ADC (DAR=7.12) (P<0.001) could inhibit the volume of transplanted tumors. The antibody SWY2110 had a poor tumor inhibition effect, with a tumor inhibition rate of only 20.3%. The SWY2110-JSSW-001 ADC (DAR = 7.12) group had the most significant inhibitory effect on PC9-GR transplanted tumor volume, with a tumor inhibition rate of up to 77.7%. The tumor inhibition rates of SWY2110-Vc MMAE ADC (DAR = 4.53) and SWY2110-DXD ADC (DAR = 6.93) were 40.4% and 50.2%, respectively. The tumor inhibition effect of SWY2110-JSSW-001 ADC (DAR = 7.12) on PC9-GR was 154% of that of SWY2110-DXD ADC (DAR = 6.93). The tumor inhibition effect of SWY2110-JSSW-001 ADC (DAR = 7.12) on PC9-GR was 154% of that of SWY2110-DXD ADC (DAR = 6.93). The tumor inhibition effect of ADC (DAR = 4.53) on PCR-GR was 192%, indicating that SWY2110-JSSW-001 ADC (DAR = 7.12) still has a significant inhibitory effect on gefitinib-resistant tumor cells. See Figure 14 and Table 11 for details.

[0482] Table 11 Tumor parameters of each group after 28 days of SWY2110-ADC administration *: P < 0.05 compared with the vehicle group, ***: P < 0.001 compared with the vehicle group

[0483] Test 6 Antibody-drug in vitro plasma stability

[0484] DP001-DXD ADC (DAR = 7.0) and DP001-JSSW-001 ADC (DAR = 7.32) were incubated with 0.5% BSA-PBS and ICR mouse plasma, respectively. The incubation concentrations were DP001-DXD ADC (DAR = 7.0) (91.7 μg / mL) and DP001-JSSW-001 ADC (DAR = 7.32) (80 μg / mL), respectively. Samples were collected after incubation at 37°C under sterile conditions for 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 336 h, and 504 h. The concentrations of shed molecules in the plasma samples were detected by LC-MS / MS. The results are as follows:

[0485] Table 12 In vitro plasma stability

[0486] Note: 1. Calculation of shedding rate: Based on the DAR value, calculate the mass ratio of DXD / ADC to obtain DP001-DXD ADC (DAR = 7.0) (91.7 μg / mL) and DP001-JSSW-001 ADC (DAR = 7.32) (80 μg / mL). Shedding rate (%) = C t / C total *100%, where Ctotal is the total small molecule concentration, C t The concentration of free small molecules at each time point. 2. “-” indicates no sampling.

[0487] The results showed that free small molecules were generated in both BSA-PBS and ICR mouse plasma at 0 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, 168 h, 336 h, and 504 h after incubation at 37°C. After 504 h of incubation, the shedding rates of 0.5% BSA-PBS and ICR mouse plasma were DP001-DXD ADC (DAR = 7.0) (3.90%), DP001-JSSW-001 ADC (DAR = 7.32) (1.09%), and DP001-DXD ADC (DAR = 7.0) (4.07%) and DP001-JSSW-001 ADC (DAR = 7.32) (2.75%), respectively. In 0.5% BSA-PBS, the shedding rate of small molecules from the antibody was less than 4.0% for all antibodies: DP001-DXD ADC (DAR = 7.0) > DP001-JSSW-001 ADC (DAR = 7.32), indicating that DP001-JSSW-001 ADC (DAR = 7.32) is more stable in 0.5% BSA-PBS. In ICR mouse plasma, the shedding rate of small molecules from the antibody was less than 5.0% for all antibodies: DP001-DXD ADC (DAR = 7.0) > DP001-JSSW-001 ADC (DAR = 7.32), indicating that DP001-JSSW-001 ADC (DAR = 7.32) is more stable than DP001-DXD ADC (DAR = 7.0) in ICR mouse plasma.

[0488] Experiment 7: Study of Antibody-Drug Bystander Effects in Vitro

[0489] For the bystander effect test, the positive cell line SK-BR-3 and the negative control cell MDA-MB-468 were first used and co-incubated with DP001-DXD ADC (DAR=7.0) and DP001-JSSW-001 ADC (DAR=7.32), respectively. The released small molecule toxins can cause apoptosis of the surrounding co-cultured negative cells, that is, the bystander effect. This experiment mainly uses the reporter gene cell HEK293-Luc to co-culture with the positive cell line SK-BR-3 and the negative control cell MDA-MB-468 for four days, and then judges the effect of the released small molecule toxins on the growth of HEK293-Luc by adding chemiluminescent substrate. The research results showed that DP001-DXD ADC (DAR=7.0) and DP001-JSSW-001 ADC (DAR=7.32) can bind to the HER2 receptor on the surface of tumor cells, enter the cells through endocytosis and release small molecule toxins; at the same time, DP001-JSSW-001 ADC (DAR=7.32) exhibits a stronger inhibitory effect on the proliferation of bystander cells.

[0490] 2. Experimental Preparation

[0491] 2.1 Test equipment

[0492] 2.2 Test consumables

[0493] 2.3 Test reagents

[0494] 2.4 Cell Information

[0495] 3. Test methods

[0496] 3.1 Reagent preparation

[0497] 3.1.1 Preparation of cell recovery medium

[0498] DMEM complete medium: Add 10% Fetal Bovine Serum and 1% Penicilin / Streptomycin to DMEM basal medium, mix thoroughly and store at 2-8°C.

[0499] 3.1.2 Preparation of cell culture medium (including selection agent)

[0500] Add 10% Fetal Bovine Serum, 1% Penicilin / Streptomycin, and 200 μg / ml G418 to the DMEM basal culture medium, mix thoroughly, and store at 2-8°C.

[0501] 3.2 Cell Preparation

[0502] 3.2.1 Cell recovery

[0503] Place the cryopreserved cell tube in a 37°C water bath and shake rapidly until completely thawed. Transfer the cell suspension to a clean bench or biosafety cabinet. Dilute the cell suspension with cell recovery medium and centrifuge. Discard the supernatant and resuspend the cells in cell recovery medium. Determine the cell density and viability. Adjust the cell density with cell culture medium. After mixing, transfer the cell suspension to a cell culture flask. After microscopic examination, transfer the suspension to an incubator and incubate at 37°C with 5% CO2.

[0504] 3.2.2 Cell passaging

[0505] Remove the cell culture flask from the CO2 incubator and transfer it to a clean bench or biosafety cabinet. Wash the cells with PBS, trypsinize them, and terminate the digestion with cell growth medium. Collect the cell suspension and measure cell density and viability. Add an appropriate amount of the cell suspension to an appropriate amount of fresh culture medium (with or without a selective agent) and subculture according to the cell growth characteristics and experimental requirements. After subculture, continue culturing the cells in the incubator.

[0506] 3.3 Bystander effect test method

[0507] 3.3.1 When the cell confluence reaches 90%, trypsinize and collect SK-BR-3, MDA-MB-468, and HEK293-Luc cells respectively, and resuspend the cells in the corresponding culture medium;

[0508] 3.3.2 Gently pipette and resuspend the target cells several times to a single-cell suspension. Use trypan blue staining to determine cell viability and cell count; adjust the cell density to 2.0 × 10 5 cells / ml;

[0509] 3.3.3 SK-BR-3 and MDA-MB-468 cells at adjusted density were mixed with HEK293-Luc cell suspension at a 1:1 ratio. 50 μl was added to each well of a 96-well clear flat-bottom culture plate. The positive cell group consisted of a mixture of SK-BR-3 and HEK293-Luc cells; the negative cell group consisted of a mixture of MDA-MB-468 and HEK293-Luc cells.

[0510] 3.3.4 DP001-DXD ADC (DAR = 7.0) and DP001-JSSW-001 ADC (DAR = 7.32) were administered at three concentrations, with final concentrations of 100 ng / ml, 500 ng / ml, and 1000 ng / ml, respectively. 50 μl per well was added to a 96-well clear flat-bottom culture plate seeded with cells and cultured in a 37°C CO2 incubator for 4 days.

[0511] 3.3.5 After incubation, remove the culture plate from the incubator and equilibrate the temperature to room temperature; add Bright-Glo TM Add the Luciferase Assay System to the culture plate and read the chemiluminescence for 5-15 minutes in the dark.

[0512] 4. Experimental Results: DP001-DXD (DAR = 7.0) and DP001-JSSW-001 ADC (DAR = 7.32) can bind to HER2 receptors on the surface of tumor cells, enter the cells through endocytosis, and release small molecule toxins. At the same time, DP001-JSSW-001 ADC (DAR = 7.32) exhibits a stronger inhibitory effect on bystander cells than DP001-DXD ADC (DAR = 7.0). The experimental results are shown in Table 13 and Figure 17.

[0513] Table 13: Inhibition rate of the antibody-drug of the present invention on fluorescent cells

[0514] Experimental octahedral linker-drug compound stability study

[0515] Different linker-drug compounds such as Deruxtecan and JSSW-001 were tested under high temperature (40°C, 60°C), high humidity (25°C / 75% RH and 25°C / 92.5% RH), light (illuminance 4500 Lux, near-ultraviolet energy 90 μw / cm 2 ) or 0.5% BSA-PBS, and collected samples at different times after being placed under different conditions. The concentration changes of the linker-drug compound in the samples were detected by LC-MS / MS. The experimental results showed that the JSSW-001 compound had better stability.

[0516] Experiment 9: Efficacy of SWY2110-ADC on human lung adenocarcinoma PC9-AR (PC9-Del19 / T790M / C797S, Osimertinib-resistant cells) cell transplants

[0517] 1. Experimental Animals

[0518] Balb / c nude mice, 5-6 weeks old, 24

[0519] 2. Purpose of the test

[0520] The anti-tumor effects of three SWY2110-ADC drugs in vivo were investigated using a NU / NU mouse xenograft tumor model derived from human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells, hereinafter referred to as PC9-AR).

[0521] 3. Drug dosage and grouping

[0522] Table 14 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: administration once.

[0523] 4. Test methods

[0524] In this study, human lung adenocarcinoma PC9-Del19 / T790M / C797S (Osimertinib-resistant cells) cells were used to construct a nude mouse tumor transplantation model. 3 At d0, the animals were divided into four groups (6 animals per group) based on tumor volume according to Table 14. The drugs were administered intravenously once at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 24 days after administration to compare the inhibitory effects of the three SWY2110-ADC drugs on nude mouse xenografts of human lung adenocarcinoma PC9-Del19 / T790M / C797S (osimertinib-resistant cells).

[0525] 5. Observation indicators

[0526] 5.1 Evaluation Metrics

[0527] See Experiment 2, 5.1, Evaluation Indicators

[0528] 6 Results

[0529] 6.1 Tumor volume

[0530] At the end of the experiment, compared with the vehicle group, SWY2110-Vc MMAE ADC (DAR=4.53) (P<0.001), SWY2110-Dxd ADC (DAR=6.93) (P<0.001) and SWY2110-JSSW-001 ADC (DAR=7.12) (P<0.001) could inhibit the volume of transplanted tumors. The SWY2110-JSSW-001ADC (DAR = 7.12) group had the most significant inhibitory effect on PC9-AR transplanted tumor volume, with a tumor inhibition rate of up to 97.5%. The tumor inhibition rates of SWY2110-Vc MMAE ADC (DAR = 4.53) and SWY2110-DXD ADC (DAR = 6.93) were 89.7% and 76.2%, respectively. The tumor inhibition effect of SWY2110-JSSW-001ADC (DAR = 7.12) was 128% of that of SWY2110-DXD ADC (DAR = 6.93), and the tumor inhibition rate of SWY2110-JSSW-001ADC (DAR = 7.12) was 128% of that of SWY2110-Vc MMAE. The tumor inhibition effect of ADC (DAR=4.53) was 109%, indicating that SWY2110-JSSW-001 ADC (DAR=7.12) still has a significant inhibitory effect on osimertinib-resistant tumor cells. See Figure 18 and Table 15 for details.

[0531] Table 15 Tumor parameters of each group after 24 days of SWY2110-ADC administration ***: Statistically significant p < 0.001 relative to vehicle group

[0532] Experiment 10: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human lung adenocarcinoma NCI-H1975 (human lung adenocarcinoma cell) transplanted tumors

[0533] 1. Experimental Animals

[0534] NU / NU mice, 5-6 weeks old, 30

[0535] 2. Purpose of the test

[0536] The human lung adenocarcinoma NCI-H1975 (human lung adenocarcinoma cell) NU / NU mouse transplant tumor model was used to investigate the in vivo anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 drugs.

[0537] 3. Drug dosage and grouping

[0538] Table 16 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: one administration;

[0539] 4. Test methods

[0540] In this study, human lung adenocarcinoma NCI-H1975 cells (human lung adenocarcinoma cells) were used to construct a nude mouse tumor transplantation model. 3 At 37 ℃, the animals were divided into 5 groups (d0) according to the tumor volume according to Table 16, with 6 animals in each group. Each dose group received a single dose of 1 mg / kg.

[0541] The drug was administered intravenously once at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 21 days after administration to compare the inhibitory effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 on human lung adenocarcinoma NCI-H1975 xenografts in nude mice.

[0542] 5. Observation indicators

[0543] 5.1 Evaluation Metrics

[0544] See Experiment 2, 5.1, Evaluation Indicators

[0545] 6 Results

[0546] 6.1 Tumor volume

[0547] At the endpoint of the trial, compared with the vehicle group, SWY2110-JSSW-001ADC (DAR = 7.12), SWY2112-JSSW-001ADC (DAR = 7.51), SWY2111-JSSW-001ADC (DAR = 7.48), and SWY2113-JSSW-001ADC (DAR = 7.50) (P < 0.001) all showed excellent tumor inhibition effects, with tumor inhibition rates reaching 84.1%, 83.0%, 80.9%, and 74.2% (P < 0.001), respectively. This indicates that all four ADCs had significant inhibitory effects on human lung adenocarcinoma NCI-H1975 tumor cells. See Figure 19 and Table 17 for details.

[0548] Table 17 Tumor parameters of each group after 21 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0549] Experiment 11: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human breast cancer MDA-MB-468 (human breast cancer cell) transplanted tumors

[0550] 1. Experimental Animals

[0551] NOD-SCID mice, 5-6 weeks old, 28

[0552] 2. Purpose of the test

[0553] The anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 were investigated in vivo using a NOD-SCID mouse xenograft tumor model of human breast cancer MDA-MB-468 (human breast cancer cells).

[0554] 3. Drug dosage and grouping

[0555] Table 18 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: one administration;

[0556] 4. Test methods

[0557] In this study, human breast cancer MDA-MB-468 cells (human breast cancer cells) were used to construct a mouse tumor transplant model. 3 According to Table 18, the animals were divided into 5 groups according to tumor volume (d0), with 8 animals in the control group and 5 animals in the experimental group. The drugs were intravenously administered once with a dosage of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to investigate the anti-tumor effects of the four ADC drugs in vivo.

[0558] 5. Observation indicators

[0559] 5.1 Evaluation Metrics

[0560] See Experiment 2, 5.1, Evaluation Indicators

[0561] 6 Results

[0562] 6.1 Tumor volume

[0563] At the endpoint, compared to the vehicle group, the tumor inhibition rates of SWY2110-JSSW-001 (DAR = 7.12), SWY2111-JSSW-001 ADC (DAR = 7.48), SWY2112-JSSW-001 ADC (DAR = 7.51), and SWY2113-JSSW-001 ADC (DAR = 7.50) reached 65.8%, 65.1%, 58.3%, and 47.1%, respectively (P < 0.001), demonstrating excellent tumor inhibitory activity. This suggests that the four ADCs have significant inhibitory effects on human breast cancer MDA-MB-468 tumor cells. See Figure 20 and Table 19 for details.

[0564] Table 19 Tumor parameters of each group after 28 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0565] Experiment 12: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human colorectal cancer DiFi (human colorectal cancer cell) transplanted tumors

[0566] 1. Experimental Animals

[0567] NU / NU mice, 5-6 weeks old, 35

[0568] 2. Purpose of the test

[0569] The human colorectal cancer DiFi (human colorectal cancer cells) NU / NU mouse transplant tumor model was used to investigate the in vivo anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001 drugs.

[0570] 3. Drug dosage and grouping

[0571] Table 20 Animal grouping and dosage table Note: iv: intravenous injection, iv / qw*2 times: administration twice a week;

[0572] 4. Test methods

[0573] In this study, human colorectal cancer DiFi (human colorectal cancer cell) cells were used to construct a mouse tumor transplant model. 3At 5 d, animals were divided into 5 groups (d0) according to tumor volume according to Table 20, with 7 animals in each group. The drugs were intravenously administered twice at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 21 days after administration to investigate the in vivo anti-tumor effects of the four ADC drugs.

[0574] 5. Observation indicators

[0575] 5.1 Evaluation Metrics

[0576] See Experiment 2, 5.1, Evaluation Indicators

[0577] 6 Results

[0578] 6.1 Tumor volume

[0579] At the endpoint, compared to the vehicle group, the tumor inhibition rates of SWY2110-JSSW-001 (DAR = 7.12), SWY2111-JSSW-001 ADC (DAR = 7.48), SWY2112-JSSW-001 ADC (DAR = 7.51), and SWY2113-JSSW-001 ADC (DAR = 7.50) reached 86.5%, 82.3%, 83.5%, and 76.8%, respectively (P < 0.001), demonstrating excellent tumor inhibitory activity. This suggests that the four ADCs have significant inhibitory effects on human colorectal cancer DiFi tumor cells. See Figure 21 and Table 21 for details.

[0580] Table 21 Tumor parameters of each group after 21 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0581] Experiment 13: Efficacy of SWY2110-ADC, SWY2111-ADC, SWY2112-ADC, and SWY2113-ADC on human lung adenocarcinoma PC9-GR (human lung adenocarcinoma gefitinib-resistant cell line) transplanted tumors

[0582] 1. Experimental Animals

[0583] NU / NU mice, 5-6 weeks old, 30

[0584] 2. Purpose of the test

[0585] The human lung adenocarcinoma cell line PC9-GR NU / NU mouse transplant tumor model was used to investigate the in vivo anti-tumor effects of SWY2110-JSSW-001, SWY2111-JSSW-001, SWY2112-JSSW-001, and SWY2113-JSSW-001.

[0586] 3. Drug dosage and grouping

[0587] Table 22 Animal grouping and dosage table Note: iv: intravenous injection, iv / 1 time: one administration;

[0588] 4. Test methods

[0589] In this study, human lung adenocarcinoma cell line PC9-GR was used to construct a mouse tumor transplant model. 3 At d0, the animals were divided into 5 groups according to tumor volume according to Table 22, with 6 animals in each group. The drugs were intravenously administered once at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to investigate the in vivo anti-tumor effects of the four ADC drugs.

[0590] 5. Observation indicators

[0591] 5.1 Evaluation Metrics

[0592] See Experiment 2, 5.1, Evaluation Indicators

[0593] 6 Results

[0594] 6.1 Tumor volume

[0595] At the endpoint, compared to the vehicle group, the tumor inhibition rates of SWY2110-JSSW-001 (DAR = 7.12), SWY2111-JSSW-001 ADC (DAR = 7.48), SWY2112-JSSW-001 ADC (DAR = 7.51), and SWY2113-JSSW-001 ADC (DAR = 7.50) were 99.0%, 100.1%, 92.4%, and 89.3%, respectively (P < 0.001), all demonstrating a positive tumor inhibition effect. This indicates that the four ADCs have a significant inhibitory effect on human lung adenocarcinoma cell lines PC9-GR. See Figure 22 and Table 23 for details.

[0596] Table 23 Tumor parameters of each group after 28 days of SWY2110-SWY2113-ADC administration ***: P < 0.001 compared with the vehicle group

[0597] Experiment 14: Efficacy of Antibody-Drug Conjugates on Human Lung Adenocarcinoma PC9-DTC (Del19 / T790M / C797S, Osimertinib-resistant Cells) Xenografts in NOD-SCID Mice

[0598] 1. Experimental Animals

[0599] NOD-SCID mice, 5-6 weeks old, 18

[0600] 2. Purpose of the test

[0601] The in vivo antitumor effects of two patritumab-ADC drugs were investigated using a NU / NU mouse xenograft tumor model of human lung adenocarcinoma PC9-DTC (Del19 / T790M / C797S, osimertinib-resistant cells).

[0602] 3. Drug dosage and grouping

[0603] Table 24 Animal grouping and dosage table Note: iv: intravenous injection, qw*3w: once a week for three weeks.

[0604] 4. Test methods

[0605] In this study, human lung adenocarcinoma PC9-DTC (Del19 / T790M / C797S, Osimertinib-resistant cells) cells were used to construct a nude mouse tumor xenograft model. 3 At 3 pm, the animals were divided into three groups according to tumor volume according to Table 24 (d0), namely, the vehicle group, the patritumab-DXD ADC (DAR=7.3) group, and the patritumab-JSSW-001 ADC (DAR=7.36) group, with 6 animals in each group. The drugs were administered intravenously once a week for 3 consecutive weeks at a dosage of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 26 days after administration to compare the inhibitory effects of patritumab-DXD ADC (DAR=7.3) and patritumab-JSSW-001 ADC (DAR=7.36) on nude mouse xenografts of human lung adenocarcinoma PC9-DTC (Del19 / T790M / C797S, osimertinib-resistant cells).

[0606] 5. Observation indicators

[0607] 5.1 Evaluation Metrics

[0608] See Experiment 2, 5.1, Evaluation Indicators

[0609] 6 Results

[0610] 6.1 Tumor volume

[0611] At the end point of the experiment, the patritumab-JSSW-001 ADC (DAR = 7.36) group (P < 0.001) had a better effect of inhibiting the volume of the transplanted tumor than the patritumab-DXD ADC (DAR = 7.3) (P < 0.001) group. See Figure 23 and Table 25 for details.

[0612] Table 25 Tumor parameters of each group after 26 days of patritumab-ADC administration ***: P < 0.001 compared with the vehicle group

[0613] Experiment 15: Efficacy of Antibody-Drug Conjugates on Human Colon Cancer SW620 Cell Transplanted Tumors in NU / NU Mice

[0614] 1. Experimental Animals

[0615] NU / NU mice, 5-6 weeks old, 18

[0616] 2. Purpose of the test

[0617] The in vivo antitumor effects of two patritumab-ADC drugs were investigated using a human colon cancer SW620 cell NU / NU mouse xenograft tumor model.

[0618] 3. Drug dosage and grouping

[0619] Table 26 Animal grouping and dosage table

[0620] 4. Test methods

[0621] In this study, a nude mouse tumor xenograft model was constructed using human colon cancer SW620 cells. When the tumor volume reached approximately 110 mm3, the animals were divided into three groups (d0) according to tumor volume according to Table 26: a vehicle group, a patritumab-DXD ADC (DAR = 7.3) group, and a patritumab-JSSW-001 ADC (DAR = 7.36) group, each consisting of 6 animals. Each group received a single intravenous dose of 10 mL / kg. A vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 26 days after administration to compare the inhibitory effects of patritumab-DXD ADC (DAR = 7.3) and patritumab-JSSW-001 ADC (DAR = 7.36) on human colon cancer SW620 xenograft tumors in nude mice.

[0622] 5. Observation indicators

[0623] 5.1 Evaluation Metrics

[0624] See Experiment 2, 5.1, Evaluation Indicators

[0625] 6 Results

[0626] 6.1 Tumor volume

[0627] At the end of the trial, the patritumab-JSSW-001 ADC group (DAR = 7.36) (P < 0.001) had a superior effect in inhibiting the volume of transplanted tumors compared to the patritumab-DXD ADC group (DAR = 7.3) (P < 0.001). See Figure 24 and Table 27 for details.

[0628] Table 27 Tumor parameters of each group after 21 days of patritumab-ADC administration ***: P < 0.001 compared with the solvent control group

[0629] Experiment 16: Efficacy of Antibody-Drug Conjugates on Human Colon Cancer DiFi Cell Transplanted Tumors in NU / NU Mice

[0630] 1. Experimental Animals

[0631] NU / NU mice, 5-6 weeks old, 18

[0632] 2. Purpose of the test

[0633] The in vivo anti-tumor effects of two patritumab-ADC drugs were investigated using a human colorectal cancer DiFi cell NU / NU mouse xenograft tumor model.

[0634] 3. Drug dosage and grouping

[0635] Table 28 Animal grouping and dosage table Note: iv: intravenous injection.

[0636] 4. Test methods

[0637] In this study, human colorectal cancer DiFi cells were used to construct a nude mouse tumor transplant model. 3 At d0, the animals were divided into three groups according to tumor volume according to Table 28: a vehicle group consisting of six animals, a patritumab-DXD ADC (DAR=7.3) group, and a patritumab-JSSW-001 ADC (DAR=7.36) group consisting of five animals each. The animals were administered intravenously as a single dose at a volume of 10 mL / kg. The vehicle control group (Vehicle) was given 0.9% sodium chloride injection. The experiment was terminated 28 days after administration to compare the inhibitory effects of patritumab-DXD ADC (DAR=7.3) and patritumab-JSSW-001 ADC (DAR=7.36) on human colorectal cancer xenografts in DiFi nude mice.

[0638] 5. Observation indicators

[0639] 5.1 Evaluation Metrics

[0640] See Experiment 2, 5.1, Evaluation Indicators

[0641] 6 Results

[0642] 6.1 Tumor volume

[0643] At the end of the trial, the patritumab-JSSW-001 ADC group (DAR = 7.36) (P < 0.01) had a superior effect in inhibiting the volume of transplanted tumors compared to the patritumab-DXD ADC group (DAR = 7.3) (P < 0.05). See Figure 25 and Table 29 for details.

[0644] Table 29 Tumor parameters of each group after 28 days of patritumab-ADC administration *: P < 0.05 compared with the vehicle group, **: P < 0.01 compared with the vehicle group

[0645] Experiment 17: Safety Assessment Study

[0646] This study enrolled six age-appropriate cynomolgus macaques, half male and half female, and administered SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001 intravenously. The dosage regimen is shown in the table below: Intravenous injections were administered once every three weeks for three consecutive doses. Patients were observed for seven consecutive days after administration, with general observations twice daily and detailed observations once daily. Body weight changes were observed before and after administration, and hematological tests were performed. The experimental results showed that ADCs prepared with pH-dependent antibodies (SWY2111-JSSW-001 and SWY2113-JSSW-001) exhibited reduced skin and gastrointestinal toxicity compared to SWY2110-JSSW-001, with no significant target-related toxicity (skin rupture, scabs, loose stools, etc.). This suggests that the pH-modified ADCs reduced toxicity to normal tissues, but there was no significant difference in the hematological toxicity caused by the three ADC small molecules.

[0647] Table 30 Pre-toxicological experimental design plan

[0648] Table 31 Pre-toxicity test results HGB: hemoglobin; WBC: white blood cell count; LYMP: absolute lymphocyte count.

[0649] Formulation Examples

[0650] Preparation Example 1

[0651] pH is a key factor affecting the stability of biological products. It regulates the charge distribution on the protein surface, thereby affecting the intermolecular and intramolecular forces, the protein's conformation and colloidal stability, and its chemical stability, such as degradation reactions like deamidation. An appropriate amount of buffer salt maintains the pH of the solution, and the type of buffer salt plays a significant role in the physicochemical properties and stability of biological products. Histidine-histidine hydrochloride and citric acid-sodium citrate were selected as screening buffer salts with a buffer ion concentration of 25 mM. The pH values ​​of the histidine-histidine hydrochloride buffer were adjusted to 5.0, 5.5, 6.0, and 6.5, and the pH values ​​of the citric acid-sodium citrate buffer were adjusted to 5.5, 6.0, 6.5, and 7.0. 5% sucrose was added to each buffer solution to prepare eight solutions with different pH values ​​and buffer salts. Antibody drug conjugate (SWY2110-JSSW-001) samples were then ultrafiltered into these eight solutions, with a final concentration of approximately 5 mg / ml. The appearance of the samples after ultrafiltration replacement was observed. The histidine-histidine hydrochloride (pH 6.0, 6.5) group and the citric acid-sodium citrate (pH 5.5, 6.0) group were obviously turbid, and the antibody drug conjugate was precipitated. The above four groups of samples were discarded. The remaining four groups of samples were placed under high temperature (40±2℃) and sampled on the 7th and 14th days; placed under light (5±3℃, white light illumination: 5000Lux, UV intensity: 90μw / cm 2 ) conditions, samples were taken on day 10, and the antibody drug conjugate protein concentration was determined by UV spectrophotometry. The molecular size variants of the antibody drug conjugate were detected by size exclusion chromatography (SEC-HPLC). The stability of the antibody drug conjugate in four groups of buffers after high temperature and light treatment was investigated. After 7 days of high temperature, protein precipitation occurred in the pH 5.5 test group, and the protein concentration and SEC monomer decreased significantly. After 10 days of light exposure, a white precipitate appeared in the citric acid test group, and the protein concentration and SEC monomer decreased significantly. In summary, pH 5.0 25mM histidine-histidine hydrochloride was selected as the target buffer.

[0652] Detection method:

[0653] Protein concentration detection: Based on the extinction coefficient of the antibody drug conjugate of 1.251, a placebo containing no protein was used for zero calibration and the protein concentration was determined using a Nanodrop 2000 micro-spectrophotometer.

[0654] SEC-HPLC: According to the high performance liquid chromatography method 0512 of the fourth part of the 2020 edition of the "Chinese Pharmacopoeia", the analysis and detection were carried out using a chromatographic column filled with a chromatographic gel suitable for separating proteins with a molecular weight of 10-500 kD. The protein monomer peak and the ratio of the high molecular weight substance (HMWS) peak to the low molecular weight substance (HMWS) peak were calculated according to the area normalization method.

[0655] The test results are as follows:

[0656] Table 31 Appearance results

[0657] Table 32 Protein concentration test results

[0658] Table 33 SEC-HPLC test results

[0659] After replacing SWY2110-JSSW-001 with antibody-drug conjugates SWY2111-JSSW-001 and SWY2113-JSSW-001, the stability of the antibody-drug conjugates in buffer was investigated using the same method as above. The test results were basically equivalent to those of SWY2110-JSSW-001, and pH 5.0 25mM histidine-histidine hydrochloride was selected as the target buffer.

[0660] Preparation Example 2

[0661] Sugars are widely used stabilizers in monoclonal antibodies and ADC products. They have the function of stabilizing monoclonal antibody drugs, and can also be used as excipients for freeze-dried products. They also have the function of regulating the osmotic pressure of the product. pH 5.0, 25mM histidine-histidine hydrochloride were selected as buffer salts, and 5% and 9% sucrose or trehalose were added respectively to prepare four groups of solutions with different sugars and sugar concentrations. The antibody drug conjugate (SWY2110-JSSW-001) samples were ultrafiltered and replaced into the above four groups of solutions. The final concentration of the antibody drug conjugate was about 10mg / ml. Microcalorimetric differential scanning calorimetry (DSC) was used to detect the thermal stability of the initial sample. The sample was placed under high temperature (40±2℃) and sampled at 7d and 14d; placed under light (5±3℃, white light illumination: 5000Lux, UV intensity: 90μw / cm 2 ) conditions, samples were taken on day 10 and size exclusion chromatography (SEC-HPLC) was used to detect molecular size variants of the antibody-drug conjugates, and ion exchange chromatography (CEX-HPLC) was used to detect charge variants of the antibody-drug conjugates. The stability of the antibody-drug conjugates in eight solution groups after high temperature and light treatment was investigated. Furthermore, the appearance of two lyophilized powders containing 9% sucrose and trehalose, freeze-dried under the same lyophilization formula, was compared.

[0662] The results of DSC test showed that the experimental group T onsetThe Tm2 and Tm2 values ​​of the sample were significantly higher than those in the 5% sugar group. No significant differences were observed between sucrose and trehalose. SEC results showed that increasing the sugar concentration from 5% to 9% significantly increased the SEC-HPLC monomer. CEX results showed that in the high-temperature test, the addition of sugars and their concentration had no significant effect on the purity of the main peak. However, in the illumination test, the acid-base variants of the sample changed significantly, and the chromatogram was no longer able to distinguish the acid peak, main peak, and base peak. CEX data were not included in the analysis. Comparing the morphology of the freeze-dried powders prepared with different stabilizers, the sucrose group showed significantly more severe shrinkage at the bottom than the trehalose group. Therefore, the addition of 9% trehalose was selected as the stabilizer.

[0663] Detection method

[0664] DSC: Dilute the sample to be tested with the same buffer to a protein concentration of 1 mg / ml. Add 350 μl of each sample and buffer to the corresponding positions in a 96-well plate. Place the 96-well plate in the sample well and set the sample well temperature to 15°C. Instrument parameters: Start temperature 20°C, End temperature 100°C, and ramp rate 90°C / min.

[0665] SEC-HPLC: See Preparation Example 1 for the detection method.

[0666] CEX-HPLC: According to the high performance liquid chromatography method 0512 of the fourth part of the 2020 edition of the "Chinese Pharmacopoeia", a cation exchange column was used as the chromatographic column for analysis and detection, and the ratio of the acidic peak, main peak and basic peak of the test sample was calculated according to the area normalization method.

[0667] Test results:

[0668] Table 34 DSC test results

[0669] Table 35 SEC-HPLC test results

[0670] Table 36 CEX-HPLC test results

[0671] Table 37 Test results of basic evaluation indexes of freeze-dried powder

[0672] After replacing SWY2110-JSSW-001 with antibody-drug conjugates SWY2111-JSSW-001 and SWY2113-JSSW-001, the same method as above was used to investigate the effect of stabilizers on the antibody-drug conjugates. The test results were basically equivalent to those of SWY2110-JSSW-001, and trehalose was selected as the surfactant.

[0673] Preparation Example 3

[0674] A sample of the antibody-drug conjugate (SWY2110-JSSW-001) was ultrafiltered into the aforementioned solution using 25 mM histidine-histidine hydrochloride as the buffer at pH 5.0 and 9% trehalose. Polysorbate 80 was then added to prepare a 10 mg / ml sample of the antibody-drug conjugate. The sample was dispensed into injection vials and stability studied under freeze-thaw conditions (freezing at -20°C and thawing at room temperature for six cycles). A microflow imaging system (Flowcam) was used to monitor changes in subvisible particles in the samples under different conditions. The results showed that the addition of the surfactant polysorbate 80 effectively controlled the growth of the particle count, with a significant inhibitory effect on particle formation at 0.01% polysorbate 80. The preferred surfactant was 0.03%.

[0675] Table 38 Effects of freeze-thaw on insoluble particles in samples with different polysorbate 80 addition amounts

[0676] After replacing SWY2110-JSSW-001 with antibody-drug conjugates SWY2111-JSSW-001 and SWY2113-JSSW-001, the same method as above was used to investigate the effect of surfactants on the stability of antibody-drug conjugates. The test results were basically equivalent to those of SWY2110-JSSW-001, and polysorbate 80 was selected as the surfactant.

[0677] Preparation Example 4

[0678] Using 10 mg / ml antibody-drug conjugate (SWY2110-JSSW-001), 25 mmol / L histidine-histidine hydrochloride, 0.03% polysorbate 80, 9% trehalose, pH 5.0 as the basic formulation, the glass transition temperature and collapse temperature of the samples composed of this formulation were tested. Low-temperature DSC testing showed that the glass transition temperature of the sample was -29.28°C, and freeze-drying microscopy testing showed that the collapse temperature of the sample was -25.7°C.

[0679] Pre-freezing: The pre-freezing temperature is set at -45°C according to the glass transition temperature. First, the plate temperature is quickly reduced to -5°C. After maintaining this temperature for 30 minutes, the product is cooled quickly (2°C / min) or slowly (0.5°C / min) to the pre-freezing temperature of -45°C. The pre-freezing temperature is maintained for no less than 180 minutes. The freeze-drying process is completed under the same primary drying and analytical drying conditions. There is no significant difference in the moisture content of the freeze-dried powder between the two pre-freezing methods. Small ice crystals are formed during the quick freezing process, but their moisture content is no higher than that of the slow freezing process. Therefore, the quick freezing method is preferred for pre-freezing.

[0680] Table 39 Effects of quick freezing and slow freezing at different positions on moisture content

[0681] Note: C is the center of the plate layer, A is a corner of the plate layer near the door, E is the position of the diagonal of A near the septum valve, B and D correspond to the center of AC and CE respectively

[0682] Primary drying: Set the plate temperature to above the product's allowable disintegration temperature to ensure that the temperature of the unsublimated portion of the product does not exceed its disintegration temperature. A pre-freezing and quick-freezing process is preferred. When the primary drying temperature is -20°C, the freeze-drying effects of freeze-drying chamber vacuum values ​​of 0.20mbar and 0.25mbar are compared. Both batches of freeze-dried powder are white, cake-like, loose bodies with no collapse or significant shrinkage. The moisture content and reconstitution time are basically the same. When the moisture content of the freeze-dried powder is basically the same, the primary drying time at -20°C and 0.20mbar is 200 minutes shorter than that at -20°C and 0.25mbar. Primary drying at -20°C and 0.20mbar is preferred.

[0683] Table 40 Freeze-dried powder morphology, moisture content and reconstitution time under different primary drying pressures

[0684] Note: C is the center of the board layer, A is the corner of the board layer near the door, and B corresponds to the center of AC

[0685] Desorption drying: After primary drying, the board temperature is set below the maximum temperature allowed by the product. 30°C is selected as the desorption drying temperature, and the front box pressure is set to 0.20mbar, the same as the primary drying.

[0686] The key qualities of the freeze-dried samples were compared before and after freeze-drying. The results showed that the product quality did not change significantly after freeze-drying, and the freeze-drying process did not have a significant impact on the product quality.

[0687] Table 41 Effect of freeze-drying process on product quality

[0688] After replacing SWY2110-JSSW-001 with antibody-drug conjugates SWY2111-JSSW-001 and SWY2113-JSSW-001, the same method as above was used to investigate the effect of the freeze-drying process on product quality. The test results were basically equivalent to those of SWY2110-JSSW-001, and the freeze-drying process did not have a significant impact on product quality.

[0689] Preparation Example 5

[0690] Stability studies were conducted at 5±3°C on both the pre-lyophilized stock solution of the antibody-drug conjugate (SWY2110-JSSW-001) and the finished product after lyophilization. The results showed that the lyophilized product was more stable than the liquid state, with a significant increase in high-molecular-weight fragments in the liquid state compared to the lyophilized state. Furthermore, the liquid state was more prone to the production of free small molecule toxins, with a more pronounced shedding rate than the lyophilized powder. Therefore, the lyophilized formulation is more conducive to the long-term storage of this product.

[0691] Detection method:

[0692] SEC-HPLC: According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0512, High Performance Liquid Chromatography, a chromatographic gel suitable for separating proteins with a molecular weight of 10 to 500 kD is used as a filler (e.g. 7.8×300mm, 3.5 μm or other suitable chromatographic column) with a mobile phase consisting of 100 mmol / L phosphate buffer, 100 mmol / L NaCl, 10% isopropanol, pH 6.7 ± 0.1, at a flow rate of 0.8 ml / min. Detection wavelength: 280 nm. Inject an appropriate amount of the test solution into the liquid chromatograph and calculate the protein monomer peak and the ratio of the high molecular weight species (HMWS) peak to the low molecular weight species (LMWS) peak using the area normalization method.

[0693] CEX-HPLC: According to the 2020 edition of the Chinese Pharmacopoeia, Part IV, General Chapter 0512, high-performance liquid chromatography, use an ion exchange column (e.g., BioPro IEX SF, 4.6×100 mm or other suitable chromatographic column) as the chromatographic column, mobile phase A is 20 mmol / L MES (morpholineethanesulfonic acid), pH 6.1, mobile phase B is 20 mmol / L MES, 500 mmol / L NaCl, pH 6.1, the flow rate is 1 ml / min, and the detection wavelength is 280 nm. An appropriate amount of the test solution is injected into the liquid chromatograph for gradient elution. The ratio of the acidic peak, main peak, and basic peak of the test sample is calculated by the area normalization method.

[0694] NR-CE-SDS: The test was performed according to the monoclonal antibody size variant determination method in 3127 of the General Chapter 4 of the 2020 edition of the Chinese Pharmacopoeia. The monomer peak ratio and the low molecular weight substance (LMWS) peak ratio were calculated by the area normalization method.

[0695] icIEF: Detection is performed according to the monoclonal antibody charge variant determination method 3129 in the 2020 edition of the Chinese Pharmacopoeia, Part IV. Different charge variants are separated according to their isoelectric point (PI) characteristics using capillary electrophoresis (General Chapter 0542). The isoelectric points of the charge variants are determined and the relative percentages are calculated.

[0696] DAR distribution: According to the HPLC method in Part IV of the 2020 edition of the Chinese Pharmacopoeia, a hydrophobic column (e.g., TSKgel Butyl-NPR, 4.6×100 mm or other suitable column) was used as the chromatographic column. Mobile phase A consisted of 1.0 M ammonium sulfate, 25 mM phosphate buffer (pH 7.0), and mobile phase B consisted of 25% isopropanol, 75% 25 mM phosphate buffer (pH 7.0). The flow rate was 0.8 ml / min, and the detection wavelength was 280 nm. An appropriate amount of the test solution was injected into the liquid chromatograph for gradient elution, and the DAR value was calculated.

[0697] Free small molecule toxin content: According to the 0512 high performance liquid chromatography method and 0431 mass spectrometry method of the 2020 edition of the Chinese Pharmacopoeia, octadecyl bonded silica gel is used as the filler (such as Waters ACQUITY A chromatographic column (BEH-C18, 2.1×50mm, particle size 1.7μm or other suitable chromatographic column) was used. Mobile phase A was an aqueous solution containing 0.1% FA, and mobile phase B was an acetonitrile solution containing 0.1% FA. The column temperature was 40°C and the flow rate was 0.4ml / min. 50μL (10mg / mL) of the test sample was added to the internal standard working solution and precipitated with acetonitrile in a ratio of 1:3. After being placed in a -20°C ice bath for 30min, the supernatant was collected and loaded. Different concentrations of small molecule toxins were loaded as reference substances. The sample was subjected to liquid chromatography gradient elution and then entered into the mass spectrometer detector. After setting the mass spectrometer capillary and cone voltages, ion source temperature, collision energy, nebulizer flow rate and other parameters, the free small molecule toxin content in the test sample was calculated according to the standard curve.

[0698] Free small molecule toxin content (%) = free small molecule toxin detection concentration / ADC concentration × 100%

[0699] Antigen binding activity: determined by enzyme-linked immunosorbent assay (ELISA).

[0700] Biological activity: Determined by cell proliferation inhibition method.

[0701] Test results:

[0702] Table 42 Stability results of key quality indicators of GMP batch 1

[0703] Table 43 Stability results of key quality indicators of GMP batch 2

[0704] After replacing SWY2110-JSSW-001 with the antibody-drug conjugates SWY2111-JSSW-001 and SWY2113-JSSW-001, stability studies were conducted at 5±3°C using the same method described above on both the pre-lyophilized stock solution and the lyophilized finished product. The test results and conclusions were essentially equivalent to those for SWY2110-JSSW-001, demonstrating that the lyophilized product was more stable than in the liquid state, that the high-molecular-weight fragments in the liquid state were significantly increased compared to the lyophilized state, and that free small molecule toxins were more readily generated in the liquid state, with a more pronounced shedding rate compared to the lyophilized powder. Therefore, the lyophilized formulation is more suitable for long-term storage of this product.

[0705] Preparation Example 6

[0706] The antibody drug conjugate (patritumab-JSSW-001) was replaced with 25 mM histidine-histidine hydrochloride buffer, citric acid-sodium citrate buffer, and succinic acid-sodium hydroxide buffer at pH 5.0, 5.5, 6.0, and 6.5, respectively, and the protein concentration was diluted to about 20 mg / ml. The reaction was carried out under high temperature (40 ± 2 ° C, 75% ± 5% RH) and light (5 ± 3 ° C, white light: 5000 ± 500 Lux, UV: 90 μW / cm 2 ) screening test. Table 44 shows the changes in the appearance of antibody drug conjugates in high temperature and light tests. After 3 days of light exposure, the appearance of the 12 test groups changed significantly, among which the sodium citrate test group and the sodium succinate test groups at pH 5.5, 6.0, and 6.5 showed phase separation or turbidity. The histidine test group and the sodium succinate test group at pH 5.0 were slightly yellow. After 5 days of high temperature, except for the sodium citrate test groups at pH 6.0 and 6.5, which showed turbidity, the appearance of the other test groups was clear. The pH 5.0, 5.5, and 6.0 histidine test groups and the pH 5.0 sodium succinate test group were selected to measure the SEC data of the light and high temperature tests. Table 45 shows the SEC changes in pH and buffer salt type screening. After 5 days of high temperature, there was no significant difference in the main SEC peaks of the four test groups, while after 3 days of light exposure, the main SEC peak of the pH 5.0 histidine test group was significantly higher than that of the other test groups. Compared with high temperature, antibody drug conjugates are more sensitive to light. The optimal buffer system is histidine-histidine hydrochloride at pH 5.0.

[0707] Detection method:

[0708] SEC-HPLC: According to the high performance liquid chromatography method 0512 of the fourth part of the 2020 edition of the "Chinese Pharmacopoeia", the analysis and detection were carried out using a chromatographic column filled with a chromatographic gel suitable for separating proteins with a molecular weight of 10-500 kD. The protein monomer peak and the ratio of the high molecular weight substance (HMWS) peak to the low molecular weight substance (HMWS) peak were calculated according to the area normalization method.

[0709] Test results:

[0710] Table 44 Appearance results Note: H: histidine, C: sodium citrate, S: sodium succinate

[0711] Table 45 SEC-HPLC test results Note: H: histidine, S: sodium succinate

[0712] Preparation Example 7

[0713] The antibody-drug conjugate (patritumab-JSSW-001) was displaced into histidine-histidine hydrochloride buffer at pH 5.0 with different concentrations (15mM, 25mM, and 35mM) containing 6% sucrose. The protein concentration was diluted to approximately 20mg / ml. High-temperature and light-irradiation screening experiments were performed, and SEC and CEX were measured to screen for the optimal histidine concentration. Table 46 shows the DSC data for the histidine concentration screening. Table 47 shows the SEC data for the histidine concentration screening. In the light-irradiation experiment, the SEC main peak decreased significantly with increasing histidine concentration; in the high-temperature experiment, the SEC main peak decreased slightly with increasing histidine concentration. Due to the strong photosensitivity of the antibody-drug conjugate, the CEX chromatogram of the light-irradiation experiment could no longer distinguish between acidic peaks, basic peaks, and the main peak. Therefore, only the CEX data of the high-temperature experiment were counted. Table 48 shows the CEX main peak data for the high-temperature experiment of histidine concentration screening. The results show that the histidine concentration has little effect on the CEX main peak. Based on the SEC and CEX data, 15mM is the optimal histidine concentration.

[0714] Detection method

[0715] DSC: Dilute the sample to be tested with the same buffer to a protein concentration of 1 mg / ml. Add 350 μl of each sample and buffer to the corresponding positions in a 96-well plate. Place the 96-well plate in the sample well and set the sample well temperature to 15°C. Instrument parameters: Start temperature 20°C, End temperature 100°C, and ramp rate 90°C / min.

[0716] SEC-HPLC: See Preparation Example 6 for the detection method.

[0717] CEX-HPLC: According to the high performance liquid chromatography method 0512 of the fourth part of the 2020 edition of the "Chinese Pharmacopoeia", a cation exchange column was used as the chromatographic column for analysis and detection, and the ratio of the acidic peak, main peak and basic peak of the test sample was calculated according to the area normalization method.

[0718] Test results:

[0719] Table 46 DSC test results

[0720] Table 47 SEC-HPLC test results

[0721] Table 48 CEX-HPLC test results

[0722] Preparation Example 8

[0723] A 15 mM histidine-histidine hydrochloride buffer (pH 5.0) was prepared, and 4%, 6%, 8%, and 10% sucrose or trehalose were added, respectively. The antibody-drug conjugate (patritumab-JSSW-001) was ultrafiltered into each of the eight buffers, and the protein concentration was diluted to approximately 20 mg / ml. After filtration through a 0.22 μm microporous membrane, the antibody-drug conjugate was tested for thermal stability by DSC, and high-temperature and light stability tests were performed.

[0724] Table 49 shows the thermal stability data of antibody drug conjugates in different concentrations of sucrose and trehalose buffer systems. onset (℃) showed no significant effect, while the test group with the highest Tm1 value was 8% sucrose. Table 50 shows the effects of high temperature and light on the SEC monomer of antibody-drug conjugates in different types and concentrations of stabilizer buffer systems. After 14 days of high temperature, the SEC monomer of the 4% test group was significantly lower than that of other test groups. Under the same sugar concentration, there was no significant difference in SEC between the sucrose group and the trehalose group in the high temperature test. Table 51 shows the effects of high temperature on the main CEX peak of antibody-drug conjugates in different types and concentrations of stabilizer buffer systems. After the high temperature test, there was no significant difference in the main CEX peaks of the 8 test groups. In summary, 8% sucrose or trehalose can be added as a stabilizer. Considering that the cost of trehalose is higher than that of sucrose, this product chooses to add 8% sucrose.

[0725] Table 49 DSC test results

[0726] Table 50 SEC test results

[0727] Table 51 CEX test results

[0728] Preparation Example 9

[0729] A 15 mM histidine-histidine hydrochloride buffer (pH 5.0) was prepared, and 8% sucrose was added. An antibody-drug conjugate (patritumab-JSSW-001) sample was ultrafiltered into this solution. Various concentrations of polysorbate 80 were added to prepare samples with a 20 mg / ml concentration of the antibody-drug conjugate. The samples were dispensed into injection vials and stability studied under freeze-thaw conditions (freezing at -20°C and thawing at room temperature for 6 cycles) and shaking (5°C, 200 rpm for 10 days). A microflow imaging system (Flowcam) was used to detect changes in subvisible particles in the samples under different conditions. Table 52 shows the effects of freeze-thaw and shaking on insoluble particulate matter in samples with different polysorbate 80 addition levels. This shows that the addition of 0.005% polysorbate 80 significantly inhibits particle formation, with 0.02% polysorbate 80 preferably inhibiting aggregate formation during long-term storage of the antibody-drug conjugate.

[0730] Table 52 Effects of freeze-thaw and shaking on insoluble particles in samples with different amounts of polysorbate 80

[0731] Formulation Example 10: Lyophilization Process Development

[0732] Using 20 mg / ml antibody-drug conjugate (patritumab-JSSW-001), 15 mmol / L histidine-histidine hydrochloride, 0.02% polysorbate 80, 8% sucrose, pH 5.0 as the basic formulation, the glass transition temperature and collapse temperature of the samples composed of this formulation were tested. Low-temperature DSC testing showed that the glass transition temperature of the sample was -28.79°C, and freeze-drying microscopy testing showed that the collapse temperature of the sample was -28.1°C.

[0733] The proposed pre-freezing temperature is -40°C. Before the pre-freezing temperature reaches -40°C, first use 30 minutes to make the plate layer reach 5°C, and maintain it at 5°C for 30 minutes, so that all samples can be cooled starting from 5°C. Pre-freezing is performed at three cooling rates of 0.5°C / min, 2°C / min and 1°C / min respectively. The freeze-dried powder obtained at different pre-freezing cooling rates is a white cake-like loose body. After injecting 5.0 ml of injection water, all three batches of freeze-dried powder can be re-dissolved within 3 minutes to obtain a clear liquid. Table 53 shows the moisture content of 5 freeze-dried powders containing antibody drug conjugates at different positions. Among them, the freeze-dried powder obtained at a cooling rate of 0.5°C / min has the lowest average moisture value.

[0734] Table 53 Comparison of moisture content of freeze-dried powder at different pre-freezing cooling rates

[0735] Note: C is the center of the plate layer, A is a corner of the plate layer near the door, E is the position of the diagonal of A near the septum valve, B and D correspond to the center of AC and CE respectively

[0736] Primary Drying: The plate temperature was set above the product's permissible disintegration temperature, but the temperature of the unsublimed portion of the product should not exceed the disintegration temperature. Pre-freezing was performed at a cooling rate of 0.5°C / min, followed by desorption drying at 35°C and 0.2 mbar for 900 minutes. Five freeze-drying experiments were conducted at different plate temperatures (-15°C, -12°C, -8°C, -5°C, and 0°C). The temperature curves of the samples with the probe added during the initial primary drying phase (before the temperature inflection point) showed that the temperature of the frozen portion of the probe contacting the sample was between -28°C and -30°C, not exceeding the disintegration temperature (Tc: -28.1°C). All five freeze-dried batches yielded a white, fluffy freeze-dried powder with no significant collapse. After injection of 5.0 ml of injection water, all five freeze-dried powders reconstituted within 3 minutes to yield a clear liquid. Table 54 shows the primary drying time and freeze-dried powder moisture content under different primary drying conditions. The primary drying time decreased with increasing plate temperature settings, with the shortest time occurring when the plate temperature was set at 0°C. Under the same analytical drying conditions (35°C, 0.2mbar, 900min), the moisture values ​​of the freeze-dried powder obtained by different primary drying processes were compared. The moisture value of the freeze-dried powder obtained by primary drying at 0°C (1.52%) was significantly higher than that at -5°C (1.26%). Therefore, the plate layer temperature was set to -5°C for primary drying. Under the primary drying setting of -5°C, the freeze-drying effects of 0.15mbar, 0.20mbar, and 0.25mbar were compared. The moisture value of the batch of freeze-dried powder was the lowest when the primary drying vacuum value was set to 0.25mbar. Therefore, the vacuum value of the primary drying freeze drying chamber was set to 0.25mbar.

[0737] Table 54 Primary drying time and moisture value of freeze-dried powder under different primary drying conditions

[0738] Drying: The plate temperature is set according to the thermal stability of the product. The thermal denaturation onset temperature (T onset ) is around 50°C. The setpoint for the desorption drying plate layer should be below 50°C. Pre-freezing uses a cooling rate of 0.5°C / min, primary drying is at -5°C, and the vacuum value is 0.25 mbar. Table 55 compares the moisture content and purity of the lyophilized powders obtained by the two desorption drying methods. Compared with the original solution, the purity of the antibody-drug conjugate lyophilized powders obtained at the two desorption drying temperatures did not show significant differences, but the moisture content of the lyophilized powder dried at 35°C for 900 minutes was lower. In summary, the desorption drying process is set at 35°C and a vacuum value of 0.25 mbar.

[0739] Table 55 Comparison of moisture and purity of freeze-dried powder obtained by two analytical drying methods

[0740] Freeze-drying pilot process transfer:

[0741] The freeze-drying pilot transfer determines the primary drying time based on the product's temperature curve and pressure rise test (the pressure rise standard at the end of primary drying is <0.01mbar / min), and the analytical drying conditions are consistent with the small-scale test. A total of four batches of freeze-drying process transfer tests were carried out. The first pilot used the small-scale test to optimize the freeze-drying formula. There were serious pits on the side walls of the freeze-dried powder in the middle of the plate layer, while the phenomenon was not serious on the samples at the edge of the plate layer. The second pilot set the plate temperature to -20°C for primary drying. There was no significant improvement in the pits on the side walls of the freeze-dried powder in the middle of the plate layer. The third batch of freeze-drying added a -10°C annealing process during the pre-freezing process to allow the parts that were not completely crystallized during the pre-freezing process to recrystallize, thereby uniformly distributing the crystal shape and size, reducing the resistance of moisture to escape from the drying layer, and slightly improving the pits on the side walls of the freeze-dried powder in the middle of the plate layer. For the fourth batch of freeze-dried powder, a -10°C annealing step was added during the pre-freezing process. The vacuum level during the primary drying process was reduced to 0.06 mbar. This enhanced the ability of sublimated water vapor to escape, reducing water accumulation in the drying layer and preventing it from melting. This significantly improved the pitting phenomenon in samples located in the middle of the sheet. Table 56 summarizes the freeze-drying pilot test data. Table 57 compares the purity of the pilot freeze-dried powder reconstitution solution with the pre-freeze-drying stock solution. The purity of the reconstitution solution remained unchanged compared to the pre-freeze-drying stock solution.

[0742] Table 56 Summary of freeze-drying pilot transfer data

[0743] Table 57 Purity comparison data of pilot freeze-dried powder reconstitution solution and original solution before freeze-drying

[0744] Detection method: SEC and CEX are the same as in Preparation Example 7.

[0745] DAR distribution: According to the HPLC method in Part IV of the 2020 edition of the Chinese Pharmacopoeia, a hydrophobic column (e.g., TSKgel Butyl-NPR, 4.6×100 mm or other suitable column) was used as the chromatographic column. Mobile phase A consisted of 1.0 M ammonium sulfate, 25 mM phosphate buffer (pH 7.0). Mobile phase B consisted of 25% isopropanol, 75% 25 mM phosphate buffer (pH 7.0). The column temperature was 30°C, the flow rate was 0.5 ml / min, and the detection wavelength was 280 nm. An appropriate amount of the test solution was injected into the liquid chromatograph for gradient elution, and the DAR value was calculated.

[0746] Formulation Example 11

[0747] Stability studies were conducted at 5±3°C on both the pre-lyophilized stock solution and the finished lyophilized patritumab-JSSW-001 product. The results showed that the lyophilized product was more stable than the liquid state. The liquid state contained significantly more high-molecular-weight fragments and a higher content of charge variant acid peaks than the lyophilized state. Furthermore, the liquid state was more susceptible to the production of free small molecule toxins, with a more pronounced shedding rate than the lyophilized powder. Therefore, the lyophilized formulation is more suitable for long-term storage of this product.

[0748] Detection method:

[0749] SEC-HPLC: Same as Preparation Example 6.

[0750] NR-CE-SDS: Same as Preparation Example 5.

[0751] CZE: Refer to the capillary electrophoresis method (General Rule 0542) and use the capillary zone electrophoresis separation mode. The components in the test sample are separated according to their respective electrophoretic and electroosmotic velocities. After separation, the ratios of the acid peak, main peak, and base peak are calculated by the peak area normalization method.

[0752] DAR distribution: Same as Preparation Example 10.

[0753] Free small molecule toxin content: According to the 0512 high performance liquid chromatography method and 0431 mass spectrometry method of the 2020 edition of the Chinese Pharmacopoeia, octadecyl bonded silica gel is used as the filler (such as Waters ACQUITY A BEH-C18 column (2.1×50 mm, particle size 1.7 μm or other suitable chromatographic columns) was used. Mobile phase A was an aqueous solution containing 0.1% FA, and mobile phase B was an acetonitrile solution containing 0.1% FA. The column temperature was 40°C, and the flow rate was 0.4 ml / min. 50 μL of the test sample was added to the internal standard working solution and precipitated with acetonitrile in a 1:3 ratio. After placing in a -20°C ice bath for 30 minutes, the supernatant was collected and loaded. Small molecule toxins of varying concentrations were loaded as reference substances. The sample was subjected to liquid chromatography gradient elution and then entered into the mass spectrometer detector. After analysis, parameters such as the mass spectrometer capillary and cone voltages, ion source temperature, collision energy, and nebulizer flow rate were set. The content of free small molecule toxins in the test sample was calculated according to the standard curve.

[0754] Free small molecule toxin content (%) = free small molecule toxin detection concentration / ADC concentration × 100%.

[0755] Table 58 Stability results of key quality indicators of GMP batch 1

[0756] Table 59 GMP batch 2 key quality indicator stability results

[0757] Formulation Example 12.1: Example of Formulation Preparation

[0758] (1) Ultrafiltration fluid exchange: Prepare a solution of 25 mM histidine-histidine hydrochloride, 9% trehalose, pH 5.0, and use a 30 kD ultrafiltration membrane to exchange the solution. Replace the antibody-drug conjugate with a solution of 25 mM histidine-histidine hydrochloride, 9% trehalose, pH 5.0. The replacement multiple should be no less than 100 times, and the ADC concentration in the replacement solution should be controlled to be no less than 1.2 times the target concentration.

[0759] (2) Preparation of semi-finished product: According to the formula quantity, the corresponding amount of polysorbate 80 was added to the ultrafiltration replacement fluid, and the ADC was diluted to the target concentration with a solution of 25 mM histidine-histidine hydrochloride, 9% trehalose, pH 5.0. After stirring and mixing, a target solution of 10 mg / ml ADC, 25 mM histidine-histidine hydrochloride, 9% trehalose, and 0.03% polysorbate 80 was obtained. After filtering through two stages of 0.22 μm sterilizing filters, an aqueous solution of the SWY2110-JSSW001 formulation was obtained, which contained approximately 10 mg / ml of the antibody drug conjugate SWY2110-JSSW-001, approximately 0.33 mg / ml of histidine, approximately 4.79 mg / ml of histidine hydrochloride, approximately 0.3 mg / ml of polysorbate 80, approximately 90 mg / ml of trehalose, and the remainder of water.

[0760] (3) Filling and freeze drying: Based on the filling volume of this product, the sample is filled and half-stoppered on the fully automatic filling line. The half-stoppered product is transferred to the freeze dryer via an automatic track for freeze drying. The pre-freezing temperature is controlled at -45°C and the maintenance time is not less than 180 minutes; the primary drying temperature is -20°C, 0.20mbar, the maintenance time is not less than 4000 minutes, and the secondary drying temperature is 30°C, 0.20mbar, and the maintenance time is not less than 1200 minutes. After the freeze drying is completed, the freeze dryer is stoppered and transferred to the capping station via an automatic track for capping. After passing the light inspection, the finished product - SWY2110-JSSW001 preparation is obtained, which contains about 10 parts by weight of antibody drug conjugate SWY2110-JSSW-001, about 0.33 parts by weight of histidine, about 4.79 parts by weight of histidine hydrochloride, about 0.3 parts by weight of polysorbate 80, and about 90 parts by weight of trehalose.

[0761] Formulation Example 12.2: Example of Formulation Preparation

[0762] (1) Ultrafiltration fluid exchange: Prepare a solution of 15 mM histidine-histidine hydrochloride, 8% sucrose, pH 5.0, and use a 30 kD ultrafiltration membrane to exchange the antibody-drug conjugate into a solution of 15 mM histidine-histidine hydrochloride, 8% sucrose, pH 5.0. The exchange multiple should be no less than 100 times, and the ADC concentration in the exchange fluid should be controlled to be no less than 1.2 times the target concentration.

[0763] (2) Preparation of semi-finished product: According to the formula quantity, the corresponding amount of polysorbate 80 is added to the ultrafiltration replacement fluid, and the ADC is diluted to the target concentration with a solution of 15 mM histidine-histidine hydrochloride, 8% sucrose, pH 5.0. After stirring and mixing, a target solution of 20 mg / ml ADC, 15 mM histidine-histidine hydrochloride, 8% sucrose, and 0.02% polysorbate 80 is obtained. After filtering through two stages of 0.22 μm sterilizing filters, an aqueous solution of the Patritumab-JSSW001 formulation is obtained, which contains approximately 20 mg / ml of patritumab-JSSW-001 antibody drug conjugate, approximately 0.21 mg / ml of histidine, approximately 2.86 mg / ml of histidine hydrochloride, approximately 0.2 mg / ml of polysorbate 80, approximately 80 mg / ml of sucrose, and the remainder of water.

[0764] (3) Filling and freeze drying: Based on the filling volume of this product, the sample is filled and half-stoppered on the fully automatic filling line. The half-stoppered product is transferred to the freeze dryer via an automatic track for freeze drying. The pre-freezing temperature is controlled at -40°C and the maintenance time is not less than 180 minutes; the primary drying temperature is -5°C, 0.06mbar, the maintenance time is not less than 3000 minutes, and the secondary drying temperature is 35°C, 0.25mbar, and the maintenance time is not less than 900 minutes. After the freeze drying is completed, the freeze dryer is stoppered and transferred to the capping station via an automatic track for capping. After passing the light inspection, the finished product - Patritumab-JSSW001 preparation is obtained, which contains about 20 parts by weight of patritumab-JSSW-001 antibody drug conjugate, about 0.21 parts by weight of histidine, about 2.86 parts by weight of histidine hydrochloride, about 0.2 parts by weight of polysorbate 80, and about 80 parts by weight of sucrose.

[0765] Formulation Example 13: Biological Activity Assay

[0766] The aqueous solutions of the SWY2110-JSSW001 formulations and the aqueous solutions of the Patritumab-JSSW001 formulations obtained in step (2) of Formulation Examples 12.1 and 12.2 were used, respectively, to determine the biological activities of the two formulations according to the method for determining biological activity described in the aforementioned Biological Examples section. The results showed that the biological activities of the two formulations were comparable to those of the ADC alone.

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate of formula I: Wherein Ab is an antibody or antigen-binding fragment thereof targeting HER2, HER3 or EGFR, and R is selected from C 1-6 Alkyl, n is selected from an integer of 1-8 or a decimal of 1-8, preferably, n is 8; The pharmaceutical composition is an injection, preferably an injection or a lyophilized injection; Preferably, the antibody-drug conjugate of Formula I has the structure shown in Formula Ia and Formula Ib: More preferably, the antibody drug conjugates of Formula I have the structures shown in Formulas I-1, Ia-1 and Ib-1:

2. The pharmaceutical composition of claim 1, wherein When Ab is an antibody or an antigen-binding fragment thereof targeting HER2, it includes a heavy chain variable region (HV) and a light chain variable region (LV), in, The heavy chain variable region sequence includes a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3), and the light chain variable region includes a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:1, the amino acid sequence of HCDR2 is shown in SEQ ID NO:2, the amino acid sequence of HCDR3 is shown in SEQ ID NO:3, and / or the amino acid sequence of LCDR1 is shown in SEQ ID NO:4, the amino acid sequence of LCDR2 is shown in SEQ ID NO:5, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:6; Further preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting HER2, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 7, and the amino acid sequence of LV is as shown in SEQ ID NO: 8; More preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting HER2, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 9, and the amino acid sequence of LC is as shown in SEQ ID NO: 10; When the Ab is an antibody or an antigen-binding fragment thereof targeting HER3, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain variable region sequence comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3), and the light chain variable region comprises a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 11, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 12, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 13, and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 14, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 15, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 16; Further preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting HER3, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 17, and the amino acid sequence of LV is as shown in SEQ ID NO: 18; More preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting HER3, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 19, and the amino acid sequence of LC is as shown in SEQ ID NO: 20; When the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the heavy chain variable region sequence comprises a heavy chain complementary determining region 1 (HCDR1), a heavy chain complementary determining region 2 (HCDR2) and a heavy chain complementary determining region 3 (HCDR3), and the light chain variable region comprises a light chain complementary determining region 1 (LCDR1), a light chain complementary determining region 2 (LCDR2) and a light chain complementary determining region 3 (LCDR3), wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO: 21, the amino acid sequence of HCDR2 is as shown in SEQ ID NO: 22, the amino acid sequence of HCDR3 is as shown in SEQ ID NO: 23, and / or the amino acid sequence of LCDR1 is as shown in SEQ ID NO: 24, the amino acid sequence of LCDR2 is as shown in SEQ ID NO: 25, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO: 26; When the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain variable region (HV) and a light chain a variable region (LV), wherein the amino acid sequence of HCDR1 is shown in SEQ ID NO:31 or SEQ ID NO:35 or SEQ ID NO:38, the amino acid sequence of HCDR2 is shown in SEQ ID NO:22, the amino acid sequence of HCDR3 is shown in SEQ ID NO:23 or SEQ ID NO:32, and / or the amino acid sequence of LCDR1 is shown in SEQ ID NO:24, the amino acid sequence of LCDR2 is shown in SEQ ID NO:25, and the amino acid sequence of LCDR3 is shown in SEQ ID NO:26; When the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HCDR1 is as shown in SEQ ID NO:31 or SEQ ID NO:35 or SEQ ID NO:38, the amino acid sequence of HCDR2 is as shown in SEQ ID NO:22, the amino acid sequence of HCDR3 is as shown in SEQ ID NO:32, and the amino acid sequence of LCDR1 is as shown in SEQ ID NO:24, the amino acid sequence of LCDR2 is as shown in SEQ ID NO:25, and the amino acid sequence of LCDR3 is as shown in SEQ ID NO:26; Further preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 27, and the amino acid sequence of LV is as shown in SEQ ID NO: 28; Further preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain variable region (HV) and a light chain variable region (LV), wherein the amino acid sequence of HV is as shown in SEQ ID NO: 33 or SEQ ID NO: 36 or SEQ ID NO: 39, and the amino acid sequence of LV is as shown in SEQ ID NO: 28; More preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 29, and the amino acid sequence of LC is as shown in SEQ ID NO: 30; More preferably, when the Ab is an antibody or an antigen-binding fragment thereof targeting EGFR, it comprises a heavy chain (HC) and a light chain (LC), wherein the amino acid sequence of HC is as shown in SEQ ID NO: 34 or SEQ ID NO: 37 or SEQ ID NO: 40, and the amino acid sequence of LC is as shown in SEQ ID NO:

30.

3. The pharmaceutical composition according to any one of claims 1 to 2, wherein the antibody drug conjugate represented by formula I has the following structure: in: DP001 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:9 and two light chains with the amino acid sequence shown in SEQ ID NO:10; Patritumab is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO: 19 and two light chains with the amino acid sequence shown in SEQ ID NO: 20; SWY2110 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:29 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2111 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:34 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2112 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:37 and two light chains with the amino acid sequence shown in SEQ ID NO:30; SWY2113 is an antibody having two heavy chains with the amino acid sequence shown in SEQ ID NO:40 and two light chains with the amino acid sequence shown in SEQ ID NO:30; n is selected from an integer of 1-8 or a decimal of 1-8, and preferably, n is 8.

4. The pharmaceutical composition of any one of claims 1 to 3, wherein the weight percentage of the antibody-drug conjugate of Formula I is 1-30% (e.g., about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%), preferably 5-25%, 8-20%, 8-15%, 8-10%, 15-25%, wherein 100wt% of the pharmaceutical composition is taken as the benchmark; Alternatively, the content of the antibody-drug conjugate of Formula I is 1-30 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 10.5 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml), preferably 5-25 mg / ml, further preferably 5-20 mg / ml, 5-15 mg / ml, 15-25 mg / ml.

5. The pharmaceutical composition of any one of claims 1 to 4, further comprising a buffer; Preferably, the buffer is selected from acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, tromethamine (Tris), morpholineethanesulfonic acid (MES) and other organic acid buffers, preferably a histidine salt buffer, further preferably a histidine-histidine hydrochloride buffer. More preferably, the mass percentage of the buffer is 1-10% (such as about 1%, about 2%, about 2.8%, about 2.9%, about 3%, about 3.1%, about 3.2%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, about 10%), preferably 1-9.9%, 2-6%, 2-5%, 4-6%, wherein 100wt% of the pharmaceutical composition is taken as the benchmark; Alternatively, the content of the buffer is 1-10 mg / ml (such as about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml, about 5 mg / ml, about 5.1 mg / ml, about 5.2 mg / ml, about 5.3 mg / ml, about 5.4 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml), preferably 2-6 mg / ml, 2-4 mg / ml, 3-6 mg / ml, 4-6 mg / ml; Most preferably, the buffer is histidine-histidine hydrochloride buffer, Wherein the mass percentage of histidine is 0.1-1% (such as about 0.1%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%), preferably 0.1-0.5%, 0.2-0.4%, wherein 100wt% of the pharmaceutical composition is taken as the benchmark; Alternatively, the content of histidine is 0.1-1 mg / ml (e.g., about 0.1 mg / ml, about 0.2 mg / ml, about 0.21 mg / ml, about 0.22 mg / ml, about 0.23 mg / ml, about 0.24 mg / ml, about 0.25 mg / ml, about 0.26 mg / ml, about 0.27 mg / ml, about 0.28 mg / ml, about 0.29 mg / ml, about 0.3 mg / ml, about 0.31 mg / ml, about 0.32 1-0.5 mg / ml, about 0.1-0.5 mg / ml, about 0.1-0.3 mg / ml, about 0.2-0.4 mg / ml; about 0.2-0.5 mg / ml, about 0.33 mg / ml, about 0.34 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml), preferably 0.1-0.5 mg / ml, 0.1-0.3 mg / ml, 0.2-0.4 mg / ml; Wherein the mass percentage of said histidine hydrochloride is 1-10% (such as about 1%, about 2%, about 2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, about 3%, about 3.5%, about 4%, about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 6%, about 6.5%, about 7%, about 8%, about 9%, about 9.9%, about 10%), preferably 1-9.9%, 2-6%, 2-4%, 3-6%, 3.5-5.5%, 4-5%, wherein 100wt% of said pharmaceutical composition is taken as the benchmark; Alternatively, the content of histidine hydrochloride is 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 2.6 mg / ml, about 2.7 mg / ml, about 2.8 mg / ml, about 2.9 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 4.6 mg / ml, about 4.7 mg / ml, about 4.8 mg / ml, about 4.9 mg / ml). g / ml, about 5mg / ml, about 5.1mg / ml, about 5.2mg / ml, about 5.3mg / ml, about 5.4mg / ml, about 5.5mg / ml, about 6mg / ml, about 6.5mg / ml, about 7mg / ml, about 8mg / ml, about 9mg / ml, about 10mg / ml), preferably 2-6mg / ml, 2-4mg / ml, 3-6mg / ml, 4-5.5mg / ml, 4.5-5mg / ml.

6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a stabilizer; Preferably, the stabilizer is selected from sugars (such as sucrose, trehalose), polyols (such as mannitol, sorbitol), amino acids (L-serine, sodium glutamate, alanine, glycine, sarcosine, etc.), preferably sugars, more preferably sucrose, and / or trehalose; More preferably, the mass percentage of the stabilizer is 50-90% (such as about 50%, about 60%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%), preferably 60-90%, 70-90%, 80-90%, wherein 100wt% of the pharmaceutical composition is taken as the benchmark; Alternatively, the content of the stabilizer is 50-110 mg / ml (such as about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 75 mg / ml, about 80 mg / ml, about 81 mg / ml, about 82 mg / ml, about 83 mg / ml, about 84 mg / ml, about 85 mg / ml, about 86 mg / ml, about 87 mg / ml, about 88 mg / ml, about 89 mg / ml, about 90 mg / ml, about 91 mg / ml, about 92 mg / ml, about 93 mg / ml, about 94 mg / ml, about 95 mg / ml, about 100 mg / ml, about 105 mg / ml, about 110 mg / ml), preferably 70-100 mg / ml, 80-100 mg / ml, 75-95 mg / ml.

7. The pharmaceutical composition according to any one of claims 1 to 6, further comprising a surfactant; Preferably, the surfactant is selected from polysorbates, preferably polysorbate 20 and / or polysorbate 80; More preferably, the mass percentage of the surfactant is 0.1-1% (e.g., about 0.1%, about 0.15%, about 0.16%, about 0.17%, about 0.18%, about 0.19%, about 0.2%, about 0.25%, about 0.26%, about 0.27%, about 0.28%, about 0.29%, about 0.3%, about 0.31%, about 0.32%, about 0.33%, about 0.34%, about 0.35%, about 0.4%, about 0.45%, about 0.5%, about 0.55%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%), preferably 0.2-0.4%, further preferably 0.15-0.4%, 0.15-0.35%, 0.25-0.35%, wherein 100wt% of the pharmaceutical composition is taken as the benchmark; Alternatively, the content of the surfactant is 0.1-1 mg / ml (e.g., about 0.1 mg / ml, about 0.15 mg / ml, about 0.16 mg / ml, about 0.17 mg / ml, about 0.18 mg / ml, about 0.19 mg / ml, about 0.2 mg / ml, about 0.25 mg / ml, about 0.3 mg / ml, about 0.35 mg / ml, about 0.4 mg / ml, about 0.45 mg / ml, about 0.5 mg / ml, about 0.55 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, about 0.8 mg / ml, about 0.9 mg / ml, about 1 mg / ml), preferably 0.1-0.4 mg / ml, 0.15-0.25 mg / ml, 0.2-0.4 mg / ml, 0.25-0.35 mg / ml.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pH of the pharmaceutical composition is 4.0-7.5 (e.g., about 4.0, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.5, about 7.0), preferably 4.5-7.0, further preferably 4.5-5.5, further preferably 5.0-5.5, and most preferably about 5.

0.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein The pharmaceutical composition is a lyophilized injection. When the lyophilized injection is diluted with water for injection, the diluted pharmaceutical composition comprises about 10 mg / ml of the antibody drug conjugate of formula I, about 0.33 mg / ml of histidine, about 4.79 mg / ml of histidine hydrochloride, about 0.3 mg / ml of polysorbate 80, and about 90 mg / ml of trehalose. Based on the total volume of the diluted pharmaceutical composition, the pH of the diluted pharmaceutical composition is about 5.

0. Preferably, the antibody of formula I The drug conjugate is selected from the group consisting of SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably SWY2110-JSSW-001; or The pharmaceutical composition is an injection, comprising about 10 mg / ml of the antibody-drug conjugate of formula I, about 0.33 mg / ml of histidine, about 4.79 mg / ml of histidine hydrochloride, about 0.3 mg / ml of polysorbate 80, and about 90 mg / ml of trehalose, and the pH of the injection is about 5.

0. Preferably, the antibody-drug conjugate of formula I is selected from the group consisting of SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably SWY2110-JSSW-001; or The pharmaceutical composition comprises about 10 mg / ml of the antibody-drug conjugate of formula I, about 0.33 mg / ml histidine, about 4.79 mg / ml histidine hydrochloride, about 0.3 mg / ml polysorbate 80, about 90 mg / ml trehalose, and the remainder of water, and the pH of the pharmaceutical composition is about 5.

0. Preferably, the antibody-drug conjugate of formula I is selected from: SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably SWY2110-JSSW-001; or The pharmaceutical composition comprises or consists of the following composition: About 10 parts by weight of the antibody drug conjugate of formula I, about 0.33 parts by weight of histidine, about 4.79 parts by weight of histidine hydrochloride, about 0.3 parts by weight of polysorbate 80, and about 90 parts by weight of trehalose. Preferably, the antibody drug conjugate of formula I is selected from: SWY2110-JSSW-001, SWY2111-JSSW-001, and SWY2113-JSSW-001, preferably SWY2110-JSSW-001. or The pharmaceutical composition is a lyophilized injection. When the lyophilized injection is diluted with water for injection, the diluted pharmaceutical composition comprises about 20 mg / ml of the antibody drug conjugate of formula I, about 0.21 mg / ml of histidine, about 2.86 mg / ml of histidine hydrochloride, about 0.2 mg / ml of polysorbate 80, and about 80 mg / ml of sucrose. Based on the total volume of the diluted pharmaceutical composition, the pH of the diluted pharmaceutical composition is about 5.

0. Preferably, the antibody drug conjugate of formula I is patritumab-JSSW-001; or The pharmaceutical composition is an injection, comprising about 20 mg / ml of the antibody-drug conjugate of formula I, about 0.21 mg / ml of histidine, about 2.86 mg / ml of histidine hydrochloride, about 0.2 mg / ml of polysorbate 80, and about 80 mg / ml of sucrose, and the pH of the injection is about 5.

0. Preferably, the antibody-drug conjugate of formula I is patritumab-JSSW-001; or The pharmaceutical composition comprises about 20 mg / ml of the antibody drug conjugate of formula I, about 0.21 mg / ml histidine, about 2.86 mg / ml histidine hydrochloride, about 0.2 mg / ml polysorbate 80, about 80 mg / ml sucrose, and the balance water, and the pH of the pharmaceutical composition is about 5.

0. Preferably, the antibody drug conjugate of formula I is patritumab-JSSW-001; or The pharmaceutical composition comprises or consists of: about 20 parts by weight of an antibody-drug conjugate of formula I, about 0.21 parts by weight of histidine, about 2.86 parts by weight of histidine hydrochloride, about 0.2 parts by weight of polysorbate 80, and about 80 parts by weight of sucrose. Preferably, the antibody-drug conjugate of formula I is patritumab-JSSW-001.

10. A method for preparing the pharmaceutical composition according to any one of claims 1 to 9, comprising the following steps: S1) preparing the antibody-drug conjugate of formula I; S2) preparing ultrafiltration replacement buffer; S3) exchanging the antibody-drug conjugate prepared in S1 with the buffer prepared in S2); Preferably, the step S2) comprises weighing a buffer and a stabilizer according to the prescribed amount, diluting with water for injection to the target preparation volume, stirring and mixing evenly to obtain an ultrafiltration replacement buffer; the step S3) comprises using an ultrafiltration membrane to ultrafiltration replace the antibody-drug conjugate obtained in S1 into the ultrafiltration replacement buffer obtained in S2) according to a certain volume, diluting the antibody-drug conjugate with water for injection to the target concentration, adding a surfactant according to the mass fraction or volume fraction to obtain a stock solution; optionally, freeze-drying the stock solution, preferably, freeze-drying include: (1) pre-freezing, (2) primary drying, and (3) secondary drying, wherein (1) The pre-freezing conditions are: -60 to -30°C, time 18-1800 minutes, for example, -45°C, time 180 minutes; (2) The conditions for primary drying are: -25 to 0°C, pressure 0.02-0.5 mbar (absolute pressure), and time 300 to 40,000 minutes, for example, -5°C, 0.06 mbar, 3,000 minutes, or -20°C, 0.20 mbar, 4,000 minutes; (3) The conditions for secondary drying are: 15-45° C., pressure 0.1-0.5 mbar (absolute pressure), and time 90-12000 minutes, for example, 30° C., 0.20 mbar, 1200 minutes, or 35° C., 0.25 mbar, 900 minutes.

11. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for treating a proliferative disease, preferably, the proliferative disease is preferably a disease associated with abnormal expression of HER2, HER3 or EGFR, including cancer, preferably, the cancer is selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer), Colon cancer, rectal cancer, colorectal cancer, leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, or relapsed anaplastic large cell lymphoma); optionally, the cancer also includes triple-negative breast cancer, esophageal cancer, pancreatic cancer, biliary tract cancer, HR-positive breast cancer, squamous cell carcinoma of the lung, non-squamous non-small cell lung cancer, squamous cell carcinoma of the head and neck (including nasopharyngeal carcinoma), and urothelial carcinoma.

12. Use of the pharmaceutical composition according to any one of claims 1 to 9 in the preparation of a medicament for treating a proliferative disease that develops drug resistance; preferably, the proliferative disease that develops drug resistance is a disease that develops drug resistance and is associated with abnormal expression of HER2, HER3 or EGFR, including cancer; more preferably, the cancer that develops drug resistance is a resistant cancer caused by a mutation in the HER2, HER3 or EGFR gene, and the cancer is preferably selected from breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (such as small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia The invention relates to a cancer comprising: a cancer of the lung, a lung, a lung cancer ...

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