Pharmaceutical composition of antibody-drug conjugate

Abstract

Provided are a pharmaceutical composition of an antibody-drug conjugate, a preparation method therefor and a use thereof.

Description

A pharmaceutical composition of an antibody-drug conjugate

[0001] Cross-references

[0002] This invention claims priority to an earlier application filed on October 30, 2024, with patent application number 202411531308.9 and entitled "A Pharmaceutical Composition for an Antibody-Drug Conjugate," submitted to the China National Intellectual Property Administration. The entire contents of that earlier application are incorporated herein by reference. Technical Field

[0003] This invention relates to a pharmaceutical composition of an antibody-drug conjugate and a method for preparing the composition. Background Technology

[0004] Antibody-drug conjugates (ADCs) consist of three distinct components: antibody, linker, and drug. ADC technology uses a linker to conjugate antibody and drug molecules together, utilizing the antibody's specific targeting to deliver drug molecules to the target tissue to exert their effects, reducing systemic toxicity of drugs, expanding the therapeutic window of drugs, and enhancing the therapeutic potential of antibodies.

[0005] In the study of antibody and antibody-drug conjugate (ADC) formulations, the formation of aggregates and the generation of degradation products can cause pharmaceutically undesirable side effects, leading to increased immunogenicity or intravenous disease-related risks in patients receiving drug treatment. For these reasons, it is necessary to inhibit aggregate formation and degradation product generation when formulating related formulations. Particularly when studying drug formulations of antibody-drug conjugates, more technical challenges arise, requiring consideration not only of the specific properties of the antibody moiety but also of the drug-connector moiety. For example, small molecule toxins may detach during storage of antibody-drug conjugates, which can affect the efficacy and toxicity of the related drugs.

[0006] Developing stable drug compositions and addressing the aggregation and degradation issues of ADC molecules in antibody-drug conjugate (ADC) compositions are our research directions. Summary of the Invention

[0007] This invention provides a pharmaceutical composition comprising an antibody-drug conjugate of Formula I:

[0008] Where A is an antibody targeting DLL3 or its antigen-binding fragment, and R is selected from... Among them, R a Selected from C1-C3 alkylene groups, such as methylene, ethylene, n-propylene, and isopropylene; R bSelected from C1-C3 alkyl groups, such as methyl, ethyl, n-propyl, and isopropyl; r, t, and u are each independently selected from integers from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50); m is an integer or decimal from 1 to 12.

[0009] Furthermore, m is selected from integers of 4 to 8 or decimals of 4 to 8.

[0010] Furthermore, when m is an integer selected from 1 to 12, it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12.

[0011] Furthermore, when m is a decimal, it refers to the average number of linker-drug molecules conjugated to each antibody unit.

[0012] Furthermore, the antibody-drug conjugate shown in Formula I has the following structure:

[0013] The definitions of A, r, u, t, and m are as shown in Equation I.

[0014] Furthermore, the antibody-drug conjugate shown in Formula I has the following structure:

[0015] The definitions of A and m are as shown in Equation I.

[0016] In a preferred embodiment of the present invention, in the above-mentioned antibody-drug conjugate, A is an antibody or antigen-binding fragment targeting DLL3, the antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the heavy chain comprising three complementarity-determining regions (CDRs), wherein the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:3; the light chain comprising three complementarity-determining regions (CDRs), wherein the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID NO:6, wherein the CDRs are determined according to the Kabat numbering rules.

[0017] In some embodiments, A is an antibody or antigen-binding fragment targeting DLL3, the anti-DLL3 antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain being as shown in SEQ ID NO:9, and the variable region (LV) sequence of the light chain being as shown in SEQ ID NO:10.

[0018] In some embodiments, A is an antibody targeting DLL3, and the anti-DLL3 antibody further includes a heavy chain constant region sequence or a variant thereof, and / or a light chain constant region sequence or a variant thereof.

[0019] In some implementations, A is an antigen-binding fragment targeting DLL3, wherein the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, Fv, scFv, or F(ab')2.

[0020] In some embodiments, A is an antibody or antigen-binding fragment thereof targeting DLL3, wherein the anti-FRα antibody or antigen-binding fragment thereof is a humanized or fully human antibody or antigen-binding fragment thereof.

[0021] In some embodiments, A is an antibody targeting DLL3 or an antigen-binding fragment thereof, the anti-DLL3 antibody comprising a heavy chain and / or a light chain, the amino acid sequence of the heavy chain being as shown in SEQ ID NO:11, and the amino acid sequence of the light chain being as shown in SEQ ID NO:12.

[0022] In this invention, the numbering rule for the antibody amino acid sequence adopts the Kabat numbering rule.

[0023] In some implementations, the antibody for the ADC represented by any of the aforementioned structural formulas is Hab33B10E8.

[0024] Furthermore, the antibody-drug conjugate shown in Formula I has the following structure:

[0025] Where m is selected from 2, 4, 6, 8, 10;

[0026] or:

[0027] Where y is the average number of linker-drug molecules conjugated to each antibody unit, which is an integer or decimal of 1-12, preferably an integer or decimal of 4-8.

[0028] Furthermore, the pharmaceutical composition is a liquid formulation, a lyophilized formulation, or a powder for injection, preferably a liquid formulation or a lyophilized formulation, preferably an injection solution or a lyophilized injection, and more preferably a lyophilized injection.

[0029] The lyophilized injection is a solid injection (i.e., lyophilized powder), which can be used by dissolving it in a solvent (preferably water, more preferably water for injection) during use. It is obtained by lyophilizing a stock solution containing a predetermined amount of drug components in a solvent (preferably ethanol, methanol, or water).

[0030] In this invention, water for injection refers to water that conforms to the requirements of the Chinese Pharmacopoeia (2020) under the category of water for injection.

[0031] Furthermore, the pharmaceutical composition comprises an antibody-drug conjugate of Formula I and a buffer.

[0032] Furthermore, the pharmaceutical composition also contains a stabilizer.

[0033] Furthermore, the pharmaceutical composition also contains a surfactant.

[0034] Furthermore, the pharmaceutical composition may also contain water.

[0035] In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of Formula I, a buffer, a stabilizer, and a surfactant.

[0036] In some embodiments, the pharmaceutical composition further comprises water.

[0037] In some embodiments, the antibody-drug conjugate of Formula I in the pharmaceutical composition is Hab33B10E8-045.

[0038] In some embodiments, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, tromethamine (Tris), morpholine ethanesulfonic acid (MES), and other organic acid buffers.

[0039] In some embodiments, the histidine buffer is a buffer containing histidine ions. Examples of histidine buffers are selected from histidine-histidine hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., wherein the histidine-histidine hydrochloride buffer is prepared by reacting histidine with histidine hydrochloride, and the histidine-acetate buffer is prepared by reacting histidine with acetic acid. Similarly, the succinate buffer can be succinic acid-sodium succinate, and the citrate buffer can be citric acid-sodium citrate.

[0040] In some implementations, histidine-histidine hydrochloride buffer is preferred.

[0041] In some embodiments, the stabilizer is selected from: sugars (such as sucrose, trehalose), polyols (such as mannitol, sorbitol), amino acids (L-serine, monosodium glutamate, alanine, glycine, sarcosine, etc.); preferably, the stabilizer is selected from two of the above stabilizers, preferably mannitol and sucrose.

[0042] In some embodiments, the surfactant is selected from polysorbates (such as polysorbate 20, polysorbate 80).

[0043] In the embodiments described below regarding pH, mass percentage (in mass %), and content (in mass / volume, such as mg / ml), unless otherwise taught, the pH of the pharmaceutical composition is the pH of an aqueous solution of the pharmaceutical composition (wherein the water is water for injection); unless otherwise taught, when describing mass percentage, it is based on a total weight of 100 wt% of the pharmaceutical composition and is premised on the pharmaceutical composition excluding water for injection; unless otherwise taught, when describing content, the pharmaceutical composition includes water for injection and is based on the volume of the pharmaceutical composition.

[0044] 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, more preferably 4.5-6.0, more preferably 5.0-5.5, and most preferably about 5.3.

[0045] In the above implementation scheme and similar implementation schemes below, the "about" before the point value includes the value itself as well as various values ​​within the error range that can be understood by those skilled in the art.

[0046] In some embodiments, the pharmaceutical composition is a lyophilized injection, optionally containing residual moisture.

[0047] In some embodiments, the pharmaceutical composition contains, by mass percentage, the antibody-drug conjugate of Formula I in the range of 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%, 10-25%, or about 19.4%.

[0048] In some embodiments, the buffer (such as histidine-histidine hydrochloride) in the pharmaceutical composition comprises 1-10% by mass (e.g., 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%, 1-5%, or about 2.6%.

[0049] In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine content is 0.01-2% by mass (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%, about 1.5%, about 2%), preferably 0.1-0.5%, 0.2-0.5%, or about 0.3%.

[0050] In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine hydrochloride is present in a mass percentage of 1-10% (e.g., about 1%, about 2%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, 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%, 1-5%, or about 2.3%.

[0051] In some embodiments, the pharmaceutical composition comprises two stabilizers (such as mannitol and sucrose), each stabilizer comprising 1-50% by mass (e.g., about 1%, about 5%, about 6%, about 7%, about 8%, about 10%, about 15%, about 20%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%), preferably 20%-50%, 20%-40%, about 38.9%, based on 100 wt% of the pharmaceutical composition;

[0052] In some embodiments, the surfactant (such as polysorbate 80) in the pharmaceutical composition comprises 0.01-1% by mass (e.g., about 0.01%, about 0.02%, about 0.05%, 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%). Approximately 0.27%, 0.28%, 0.29%, 0.3%, 0.31%, 0.32%, 0.33%, 0.34%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.7%, 0.8%, 0.9%, and 1%), preferably 0.05-0.4% and 0.2%.

[0053] In some embodiments, the pharmaceutical composition, by mass percentage, comprises 1-30%, preferably 5-25%, 10-25%, about 19.4% of the antibody-drug conjugate of Formula I; 1-10%, preferably 1-9.9%, 1-5%, about 2.6% of a buffer (e.g., histidine-histidine hydrochloride), wherein the histidine content is 0.1-0.5%, preferably 0.25-0.5%, about 0.3%; the histidine hydrochloride content is 1-10%, preferably 1-9.9%, 1-5%, about 2.3%; two stabilizers (e.g., mannitol, sucrose), each stabilizer content is 1-50%, preferably 20%-50%, 20%-40%, about 38.9%; and the surfactant (e.g., polysorbate 80) content is 0.01-1%, preferably 0.05-0.4%, about 0.2%.

[0054] In some embodiments, the pharmaceutical composition, by weight percentage, comprises 5-25%, preferably 10-25%, or 19.4% of the antibody-drug conjugate of Formula I; 2-6%, preferably 1-5%, or about 2.6% of a buffer (e.g., histidine-histidine hydrochloride), wherein the histidine content is 0.25-0.5%, preferably 0.3%; the histidine hydrochloride content is 1-9.9%, preferably 1-5%, or 2.3% by weight; two stabilizers (e.g., mannitol, sucrose), each stabilizer content being 20%-50%, preferably about 38.9% by weight; and the surfactant (e.g., polysorbate 80) content being 0.05-0.4%, preferably 0.2% by weight.

[0055] In some embodiments, the pharmaceutical composition, by weight percentage, comprises about 19.4% of the antibody-drug conjugate of Formula I; about 2.6% of a buffer (e.g., histidine-histidine hydrochloride), wherein the buffer is histidine-histidine hydrochloride, and wherein the histidine is about 0.3%; about 2.3% of the histidine hydrochloride by weight; two stabilizers (e.g., mannitol, sucrose), each by weight percentage of about 38.9%; and about 0.2% of the surfactant (e.g., polysorbate 80).

[0056] In one embodiment, the composition includes two stabilizers, such as mannitol and sucrose.

[0057] In some embodiments, the pharmaceutical composition, by weight percentage, comprises about 19.4% of the antibody-drug conjugate of Formula I; about 2.6% of histidine-histidine hydrochloride; about 38.9% of mannitol; about 38.9% of sucrose; and about 0.2% of polysorbate 80.

[0058] In some embodiments, the pharmaceutical composition is an injectable preparation, wherein the antibody-drug conjugate of Formula I in the aforementioned solution, stock solution, and / or liquid formulation is present in a concentration of 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 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, more preferably 10-20mg / ml, and more preferably 15mg / ml.

[0059] In some embodiments, the buffer (such as histidine-histidine hydrochloride) in the pharmaceutical composition is present in a concentration of 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.1 mg / ml, about 2.2 mg / ml, about 2.3 mg / ml, about 2.4 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 4 mg / ml, about 4.5 mg / ml). (mg / ml, about 4.6mg / ml, about 4.7mg / ml, about 4.8mg / ml, about 4.9mg / 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 1-5mg / ml, about 2.01mg / ml. Alternatively, based on histidine, it may be 5-50 mM (e.g., about 5 mM, about 10 mM, about 10.5 mM, about 11 mM, about 11.5 mM, about 12 mM, about 12.5 mM, about 13 mM, about 13.5 mM, about 14 mM, about 14.5 mM, about 15 mM, about 20 mM, about 22.5 mM, about 23 mM, about 23.5 mM, about 24 mM, about 24.5 mM, about 25 mM, about 25.5 mM, about 26 mM, about 26.5 mM, about 27 mM, about 27.5 mM, about 30 mM, about 32.5 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM), preferably 5-25 mM, about 10 mM.

[0060] In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine content 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). (approximately 0.31 mg / ml, approximately 0.32 mg / ml, approximately 0.33 mg / ml, approximately 0.34 mg / ml, approximately 0.35 mg / ml, approximately 0.4 mg / ml, approximately 0.45 mg / ml, approximately 0.5 mg / ml, approximately 0.55 mg / ml, approximately 0.6 mg / ml, approximately 0.7 mg / ml, approximately 0.8 mg / ml, approximately 0.9 mg / ml, approximately 1 mg / ml), preferably 0.1-0.5 mg / ml, 0.2-0.5 mg / ml, and approximately 0.24 mg / ml.

[0061] In some embodiments, the buffer in the pharmaceutical composition is histidine-histidine hydrochloride, wherein the histidine hydrochloride content is 1-10 mg / ml (e.g., about 1 mg / ml, about 2 mg / ml, about 2.1 mg / ml, about 2.2 mg / ml, about 2.3 mg / ml, about 2.4 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 3.6 mg / ml, about 4 mg / ml). (approximately g / 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 1-6 mg / ml, about 1-4 mg / ml, about 1.77 mg / ml.

[0062] In some embodiments, the pharmaceutical composition comprises two stabilizers (such as mannitol and sucrose), each stabilizer being present in a concentration of 10-50 mg / ml (e.g., about 10 mg / ml, about 20 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 30 mg / ml, about 31 mg / ml, about 32 mg / ml, about 33 mg / ml, about 34 mg / ml, about 35 mg / ml, about 36 mg / ml, about 37 mg / ml, about 38 mg / ml, about 39 mg / ml, about 40 mg / ml, about 45 mg / ml, about 50 mg / ml), preferably 10-40 mg / ml, 20-40 mg / ml, or about 30 mg / ml.

[0063] In some embodiments, the surfactant (such as polysorbate 80) in the pharmaceutical composition is present in an amount of 0.005-1 mg / ml (e.g., about 0.05 mg / ml, 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 0.05-0.4 mg / ml, about 0.2 mg / ml.

[0064] In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of Formula I at a concentration of 1-30 mg / ml, more preferably 5-25 mg / ml, 10-20 mg / ml, or 15 mg / ml, and a buffer (such as histidine-histidine hydrochloride) at a concentration of 1-10 mg / ml, preferably 1-5 mg / ml, 1-4 mg / ml, or about 2.01 mg / ml, wherein the histidine content is 0.1-1 mg / ml, preferably 0.1-0.5 mg / ml or 0.2-0.5 mg / ml. The content of histidine hydrochloride is 1-10 mg / ml, preferably 1-6 mg / ml, about 1-4 mg / ml, about 1.77 mg / ml; two stabilizers (such as mannitol and sucrose), each of which is 10-50 mg / ml, preferably 10-40 mg / ml, 20-40 mg / ml, about 30 mg / ml; and the content of surfactant (such as polysorbate 80) is 0.005-1 mg / ml, preferably 0.05-0.4 mg / ml, 0.2 mg / ml.

[0065] In some embodiments, the pharmaceutical composition comprises an antibody-drug conjugate of Formula I at a concentration of 5-25 mg / ml, preferably 10-20 mg / ml, or 15 mg / ml; a buffer (such as histidine-histidine hydrochloride) at a concentration of 1-5 mg / ml, preferably 1-4 mg / ml, or 2.01 mg / ml, or 5-25 mM, preferably 5-20 mM, or 10 mM, calculated as histidine; wherein the histidine concentration is 0.1-0.5 mg / ml, preferably 0.2-0.5 mg / ml, or 0.24 mg / ml, and the histidine hydrochloride concentration is 1-6 mg / ml, preferably 1-4 mg / ml, or about 1.77 mg / ml; two stabilizers (such as mannitol and sucrose), each at a concentration of 20-40 mg / ml, preferably 30 mg / ml; and a surfactant (such as polysorbate 80) at a concentration of 0.05-0.4 mg / ml, preferably 0.2 mg / ml.

[0066] In one embodiment, the composition includes two stabilizers, such as mannitol and sucrose.

[0067] In some embodiments, the pharmaceutical composition comprises 15 mg / ml of the antibody-drug conjugate of Formula I, histidine-histidine hydrochloride at a concentration of 2.01 mg / ml or 10 mM based on histidine, wherein the histidine concentration is 0.24 mg / ml and the histidine hydrochloride concentration is about 1.77 mg / ml; mannitol at a concentration of 30 mg / ml; sucrose at a concentration of 30 mg / ml; and polysorbate 80 at a concentration of 0.2 mg / ml.

[0068] In some embodiments, the pharmaceutical composition comprises about 15 mg / ml of an antibody-drug conjugate of Formula I, about 0.24 mg / ml of histidine, about 1.77 mg / ml of histidine hydrochloride, 0.2 mg / ml of polysorbate 80, 30 mg / ml of mannitol, and 30 mg / ml of sucrose, and the pH of the injection solution is about 5.3.

[0069] In some embodiments, the pharmaceutical composition comprises: 1-30 (preferably 5-25, 10-20 mg / ml, about 15) parts by weight of the antibody-drug conjugate of Formula I; 1-10 (preferably 1-6, about 2.01) parts by weight of a buffer, wherein when the buffer is histidine-histidine hydrochloride, histidine is 0.1-1 (preferably 0.1-0.7, 0.1-0.5, about 0.24) parts by weight and histidine hydrochloride is 1-10 (preferably 1-6, about 1-4, about 1.77) parts by weight; two stabilizers (e.g., mannitol, sucrose), each stabilizer being 10-50 (preferably 20-50, 20-40, about 30) parts by weight; and 0.005-1 (preferably 0.05-0.4, about 0.2) parts by weight of a surfactant (e.g., polysorbate 80).

[0070] In one embodiment, the composition includes two stabilizers, such as mannitol and sucrose.

[0071] In some embodiments, the pharmaceutical composition is a lyophilized injection, and when the lyophilized injection is diluted with water for injection, the diluted pharmaceutical composition contains about 15 mg / ml of the antibody-drug conjugate of Formula I, about 0.24 mg / ml of histidine, about 1.77 mg / ml of histidine hydrochloride, 0.2 mg / ml of polysorbate 80, 30 mg / ml of sucrose, and 30 mg / ml of mannitol, and the pH of the injection solution is about 5.3.

[0072] In some embodiments, the pharmaceutical composition is a lyophilized injection, the stock solution of which comprises about 15 mg / ml of an antibody-drug conjugate of Formula I, about 0.24 mg / ml of histidine, about 1.77 mg / ml of histidine hydrochloride, 0.2 mg / ml of polysorbate 80, 30 mg / ml of sucrose, and 30 mg / ml of mannitol, and the pH of the injection solution is about 5.3.

[0073] In some embodiments, the pharmaceutical composition is an injection containing about 15 mg / ml of an antibody-drug conjugate of Formula I, about 0.24 mg / ml of histidine, about 1.77 mg / ml of histidine hydrochloride, 0.2 mg / ml of polysorbate 80, 30 mg / ml of sucrose, and 30 mg / ml of mannitol, wherein the pH of the injection is about 5.3.

[0074] In the aforementioned drug combination, the antibody-drug conjugate is any compound shown in Formula I, preferably Hab33B10E8-045, with the following structure:

[0075] The average number of linker-drug molecules conjugated to each antibody unit is approximately 8.

[0076] The present invention also relates to a method for preparing the pharmaceutical composition, the method comprising the following steps:

[0077] S1) Prepare the antibody-drug conjugate solution shown in Formula I;

[0078] S2) Prepare ultrafiltration replacement buffer;

[0079] S3) Replace the antibody-drug conjugate prepared in S1 with the buffer solution prepared in S2).

[0080] In some embodiments, the ultrafiltration replacement buffer comprises a buffer and a stabilizer, wherein the buffer (e.g., histidine-histidine hydrochloride) is present at a concentration of 1-5 mg / ml, preferably 1-4 mg / ml, or 2.01 mg / ml; the histidine concentration is present at a concentration of 0.1-0.5 mg / ml, preferably 0.2-0.5 mg / ml, or 0.24 mg / ml; and the histidine hydrochloride concentration is present at a concentration of 1-6 mg / ml, preferably 1-4 mg / ml, or approximately 1.77 mg / ml; and two stabilizers (e.g., mannitol, sucrose), each present at a concentration of 20-40 mg / ml, preferably 30 mg / ml.

[0081] In some embodiments, the method further includes step S4): adding a surfactant to the antibody-drug conjugate solution obtained in step S3); the surfactant (such as polysorbate 80) is present in a concentration of 0.05-0.4 mg / ml, preferably 0.2 mg / ml.

[0082] This invention provides the use of the pharmaceutical composition of this invention in the preparation of a medicament for treating or preventing individual tumors; preferably, the tumor is a tumor whose tumor cells express DLL3 (DLL3+); more preferably, the tumor is a solid tumor or hematologic malignancy associated with high DLL3 expression; even more preferably, the tumor is lung cancer; more preferably, the tumor is small cell lung cancer.

[0083] The present invention provides a method for treating or preventing an individual tumor, the method comprising administering a therapeutically effective dose of the pharmaceutical composition of the present invention to a patient in need of it; the tumor being a tumor on which tumor cells express DLL3 (DLL3+); more preferably, the tumor being a solid tumor or hematologic malignancy associated with high DLL3 expression; even more preferably, the tumor being lung cancer; and more preferably, the tumor being small cell lung cancer.

[0084] The pharmaceutical composition of the present invention can be used alone or in combination with other antitumor agents.

[0085] The pharmaceutical composition of the present invention, under the formulated conditions, is stably stored, ensuring the stability of the physicochemical properties of the antibody portion while minimizing the shedding of free drug. Long-term stability studies showed no significant changes in the physicochemical properties of the antibody portion, meeting the storage requirements for antibody preparations. The DAR value and DAR8 distribution of the ADC portion also showed no significant changes, indicating that the conjugate drug exhibits uniform quality during storage and can be stored for extended periods.

[0086] definition

[0087] Unless otherwise specified, the term "antibody-drug conjugate (ADC)" refers to the linking of an antibody (such as a monoclonal antibody) or antibody fragment to a biologically active cytotoxic drug via a stable chemical linker compound.

[0088] 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.

[0089] The linker-drug compound and the antibody described in this invention are linked by conventional coupling methods in the art, including: lysine coupling, inter-chain reducing 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.). This invention preferably uses inter-chain reducing disulfide bond coupling, i.e., the linkage is achieved by the reaction of one or more thiol groups (sulfur atoms of cysteine ​​residues) formed after reduction at the disulfide bond sites between the light and heavy chains (two sites between the heavy chain and two sites between the heavy and light chains).

[0090] Unless otherwise specified, the term "about" means a value within an acceptable margin of error for a specific value determined by a person skilled in the art, the value depending in part on how it is measured or determined (i.e., the limits of the measurement system). For example, each occurrence of "about" in the art may mean a range of ±20%, ±15%, ±10%, ±5%, ±1% for the specific value subsequently indicated.

[0091] Abbreviation Table Attached Figure Description

[0092] Figure 1. Single crystal diffraction pattern of compound 82.

[0093] Figure 2. Effects of high temperature and light on ADC product SEC monomers in different pH and buffer salts. Detailed Implementation

[0094] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as are familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of the present invention. The preferred embodiments and materials shown herein are for illustrative purposes only.

[0095] The structures of the compounds in this invention were determined by nuclear magnetic resonance (NMR) and / or liquid chromatography-mass spectrometry (LC-MS) and / or high-performance liquid chromatography (HPLC). The NMR determination was performed using a Bruker Avance III 400MHz NMR spectrometer; the LC-MS was performed using a SHIMADZU LC-20AD-PDA-LCMS-2020 instrument; and the HPLC was performed using a SHIMADZU LC-20AD-PDA high-performance liquid chromatograph.

[0096] The starting materials used in the embodiments of the present invention are known and commercially available, or can be synthesized using or in accordance with methods known in the art.

[0097] The antibodies in this invention can be prepared using the hybridoma technique first described by Kohler et al., Nature (1975), or using recombinant DNA methods (US Patent 4,816,567, etc.).

[0098] Preparation Examples

[0099] Example 1: Preparation of Compound 045

[0100] 1. Preparation of intermediate compound 18

[0101] Compound 11 (47.5 g, 189 mmol) and methanol (250 mL) were added to a reaction flask. 80% hydrazine hydrate (35.4 g, 567 mmol) was slowly added at room temperature. The mixture was heated to 70 °C and refluxed for 6 hours. After cooling, white crystals precipitated. The crystals were filtered, and the remaining solid was washed with methanol (20 mL × 3) to give white solid 12 (47.47 g, 100% yield). MS (ESI): (M + H) + Calculated value: 252.1, Experimental value: 252.2.

[0102] Compound 12 (47.4 g, 189 mmol), potassium hydroxide (12.7 g, 227 mmol), and ethanol (400 mL) were added to a reaction flask and dissolved by stirring at room temperature. Carbon disulfide (17 g, 283 mmol) was slowly added, and the mixture was heated to 100 °C and refluxed for 5 hours. After removing the solvent under reduced pressure, water (50 mL) was added, and the pH was adjusted to 6 with dilute hydrochloric acid. The mixture was extracted with dichloromethane (100 mL × 3), and the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 13 (49.9 g, 90% yield). MS (ESI): (M+H) + The calculated value is 294.1, and the experimental value is 294.3.

[0103] Compound 13 (5.86 g, 20 mmol), triethylamine (2.42 g, 24 mmol), and tetrahydrofuran (36 mL) were added to a reaction flask. Iodomethane (3.12 g, 22 mmol) was then added to the reaction mixture. The reaction was stirred at 25 °C for 1.5 hours until the reaction was complete. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography to give compound 14 (4.05 g, 66% yield). MS (ESI): (M+H) + Calculated value: 308.1, experimental value: 308.2.

[0104] Compound 14 (3.3 g, 10.6 mmol) and ethyl acetate (20 mL) were added to a reaction flask, and ethyl hydrochloride solution (2.7 mL, 4 M) was added dropwise at 25 °C. The reaction was continued for 6 hours. After removing the solvent under reduced pressure, crude product 15 was obtained and directly added to the next reaction.

[0105] Crude product 15, diethylene glycol anhydride 6 (1.35 g, 11.7 mmol), triethylamine (2.14 g, 21.2 mmol), and tetrahydrofuran (30 mL) were added to a reaction flask and reacted at 25 °C for 1.5 h. The solvent was removed under reduced pressure, and the residue was added to diethyl ether. After filtration, the residue was washed with water and diethyl ether to give compound 17 (3.4 g, 99% yield). MS (ESI): (M+H) +Calculated value 324.1, experimental value 324.2. Compound 17 (3.4 g, 10.5 mmol) and glacial acetic acid (20 mL) were added to a reaction flask. After dissolution, potassium permanganate (2.48 g, 15.7 mmol) was added at 0 °C, and the reaction was brought back to 25 °C for 1 hour. Saturated sodium sulfite solution was added until the solution became colorless. Extraction was performed with ethyl acetate (100 mL × 3). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel chromatography to give compound 18 (2.4 g, 65% yield). MS (ESI): (M+H) + Calculated value: 356.0, Experimental value: 356.0.

[0106] 2. Preparation of Compound 045

[0107] At 0°C, 1-(4-aminophenyl)-3-butyn-1-ol (4997 mg, 31 mmol), N-fluorenylmethoxycarbonyl-L-alanine (9651 mg, 31 mmol), EEDQ (11498 mg, 46.5 mmol), and ultradry DCM (90 mL) were added to a reaction flask. After reacting for 3 h, the solvent was removed under reduced pressure, and 500 mL of MTBE was added to the residue for slurry mixing to give compound 72 (9007 mg, yield 64%, dr = 1:1). MS (ESI): (M+H) + Calculated value: 455.2, Experimental value: 455.2.

[0108] Chiral resolution of intermediate 72 (retention times 13.55 min, 16.29 min): Compound 72 (50 g) was resolved by SFC to obtain 81 (19 g, retention time 13.55 min) and 82 (20 g, retention time 16.29 min). SFC resolution method: Column type: DAICL CHIRALCEL OD (250 mm - 50 mm, 10 μm); Mobile phase: A: CO2, B: CO2-ACN / i-PrOH (0.1% NH3H2O); Isocratic elution: B in A for 50%; Flow rate: 200 mL / min; Detector: PDA; Column temperature: 25 °C; Back pressure: 100 Bar;

[0109] HPLC Method: Instrument Information: Thermo Liquid Chromatograph (ADC-U3000-01); Column: ID (4.6*150mm, 5μm); Column temperature: 25℃; Injection plate temperature: 25℃; Mobile phase: A: 10mM NH4FA; B: ACN; Flow rate: 0.8mL / min; Detection wavelength: 254nm; Injection volume: 2μl;

[0110] Table 1 Gradient conditions

[0111] X-ray confirmation revealed the structure and configuration of compound 82 as follows: its single-crystal diffraction pattern is shown in Figure 1.

[0112] At 0°C, 82 (3632 mg, 8 mmol) and 90 mL of commercially available THF were added to a reaction flask. DBU (1215 mg, 8 mmol) was slowly added with stirring, and the mixture was reacted for half an hour before being moved to room temperature. After the starting material disappeared under TLC monitoring, the solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to give compound 83 (1707 mg, 92% yield). MS (ESI): (M+H) + Calculated value: 233.1, Experimental value: 233.2.

[0113] Compound 83 (1624 mg, 7 mmol), Fmoc-Val-OSu (362 mg, 8.4 mmol), and N,N-dimethylformamide (150 mL) were added to a reaction flask. The mixture was stirred, and DIEA (1158 μL, 7 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 12 hours. The solvent was removed under reduced pressure, and 50 mL of EA and 50 mL of PE were added and stirred. A white solid precipitated. This process was repeated three times to obtain compound 84. MS (ESI): (M+H) + Calculated value: 554.3, experimental value: 554.4.

[0114] Compound 84 (1661 mg, 3 mmol), p-nitrobenzene chloroformate (1206 mg, 6 mmol), and THF (150 mL) were added to a reaction flask. The mixture was stirred, and Py (474 ​​μL, 6 mmol) was added dropwise. The reaction was carried out at 65 °C for 1 hour. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 98:2) to give compound 85 (1831 mg, 85% yield). MS (ESI): (M+H) + Calculated value: 719.8, experimental value: 719.9.

[0115] Compound 85 (1436 mg, 2 mmol), eczemab mesylate 86 (1168.2 mg, 2.2 mmol), and N,N-dimethylformamide (100 mL) were added to a reaction flask. The mixture was stirred, and DIEA (695 μL, 4 mmol) was slowly added dropwise. The reaction was carried out at 25 °C for 20 hours. The solvent was removed under reduced pressure, and the residue was purified by silica gel chromatography (DCM / MeOH 95:5) to give compound 87 (1664 mg, 82% yield). MS (ESI): (M+H) + Calculated value: 1015.4, experimental value: 1015.1.

[0116] At 0°C, 87 (1522 mg, 1.5 mmol) and 40 mL of commercially available THF were added to a reaction flask. DBU (228 mg, 1.5 mmol) was added slowly with stirring. After reacting for half an hour, the mixture was moved to room temperature and reacted for another 40 minutes until the starting material disappeared by TLC. The reaction solution was concentrated under vacuum to remove THF, and 15 mL of DCM and 300 mL of PE were added, producing a large amount of yellow-green solid. Solid 88 (1177 mg, 99% yield) was filtered out using a Buchner funnel. MS (ESI): (M+H) + Calculated value: 793.3, experimental value: 793.6.

[0117] Compounds 88 (1031 mg, 1.3 mmol), 18 (554 mg, 1.56 mmol), EDCI (498 mg, 2.6 mmol), HOBT (263 mg, 1.95 mmol), and N,N-dimethylformamide (20 mL) were added to a reaction flask and reacted at 25 °C for 1 h. The solvent was removed under reduced pressure, and the residue was purified by C18 chromatography (60% ACN / 0.05% formic acid in H2O). The residue was lyophilized to give compound 89 (1322 mg, 90% yield). MS (ESI): (M+H) + Calculated value: 1130.4; Experimental value: 1130.7.

[0118] Compound 89 (903 mg, 0.8 mmol), compound 80 (702 mg, 1.2 mmol), tris(3-hydroxypropyltriazolylmethyl)amine (34.7 mg, 0.08 mmol), and cuprous bromide (11.4 mg, 0.08 mmol) were added to a reaction flask, and the mixture was purged with nitrogen three times. THF / DMF / H₂O (3.5 mL:0.6 mL:0.4 mL) was added, and the reaction was carried out at 25 °C for 0.5 h. The residue was purified by C18 preparative chromatography (62% ACN / 0.05% formic acid in H₂O) to give compound 045 (1276 mg, 93% yield). MS (ESI): (M+H) + Calculated value: 1715.7, experimental value: 1716.2. 1H NMR (600MHz, DMSO) δ10.71 (s, 1H), 10.08 (s, 1H), 8.48 (d, J = 6.7Hz, 1H), 8.17 ( d,J=8.6Hz,3H),8.09(d,J=8.8Hz,1H),8.03(d,J=8.7Hz,2H),7.89(s,1H),7. 81(d,J=10.8Hz,1H),7.66(d,J=8.4Hz,2H),7.42–7.36(m,3H),6.61(s,1H),6 .02(t,J=6.8Hz,1H),5.55(s,2H),5.38-5.26(m,3H),4.55–4.47(m,3H),4.46– 4.41(m,1H),4.36(q,J=15.6Hz,2H),4.28(s,2H),3.80(s,5H),3.62–3.55(m, 36H),3.52–3.47(m,9H),3.44–3.36(m,2H),3.30-2.29(m,1H),3.19–3.09(m, 1H),2.42(s,3H),2.28–2.19(m,1H),2.17(s,3H),2.17–2.10(m,1H),2.04–1. 89 (m, 2H), 1.42 (d, J = 7.0Hz, 3H), 1.02 (d, J = 6.7Hz, 3H), 0.98 (t, J = 7.2Hz, 6H).

[0119] Example 2: General Method for ADC Sample Preparation and DAR Measurement

[0120] 1. Obtaining anti-human DLL3 monoclonal antibodies

[0121] Healthy BALB / c mice were immunized with recombinant human DLL3 protein (purchased from Acro, catalog number DL3-H52H4). After the initial immunization, booster immunizations were administered every 14 days for a total of four immunizations. Serum titers were measured by flow cytometry, and mice with high serum antibody titers were selected for cell fusion. Three days prior to fusion, a pulse immunization of recombinant human DLL3 protein was administered via tail vein injection. On the day of fusion, mice were euthanized, and spleens were aseptically removed and prepared into single-cell suspensions. SP2 / 0 cells were mixed with spleen cells at a 1:1 ratio, and fusion was performed using electrofusion. The fused cells were seeded in 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator. Selection was performed using HAT medium.

[0122] Based on cell growth, hybridoma supernatant was analyzed by ELISA after approximately 10 days. Initial screening was performed using recombinant human DLL3 protein. Positive clones underwent secondary screening using human DLL3 (purchased from Acro, catalog number DL3-H525x), cynomolgus monkey DLL3 (purchased from Acro, catalog number DL3-C52H3), mouse DLL3 (purchased from Acro, catalog number DL3-M52H9), human DLL3-B (purchased from Acro, catalog number DL3-H52Hy), or human DLL3-C (purchased from Acro, catalog number DL3-H52Hz) proteins. Five hybridoma cell lines (22B12E5, 25G10A9, 31B6G8, 33B10E8, and 44B8H2) with high affinity were selected for sequencing. RNA was extracted from these five cell lines, reverse transcribed, and amplified by PCR to obtain the amino acid sequences of the variable regions of the mouse antibody heavy and light chains. CDRs were determined using Kabat numbering. The results are as follows:

[0123] 33B10E8

[0124] HCDR1: DYYMK SEQ ID NO.1

[0125] HCDR2:AFNLNNGDTFYNQKFKG SEQ ID NO.2

[0126] HCDR3: DVYGYGDY SEQ ID NO.3

[0127] Heavy chain variable region (HV)

[0128] LCDR1:RASKSVSTSGYSYMH SEQ ID NO.4

[0129] LCDR2: LASNLDS SEQ ID NO.5

[0130] LCDR3: QHSRELPYT SEQ ID NO.6

[0131] Light chain variable region (LV)

[0132] 2. Humanization of DLL3 antibody

[0133] Using Discovery Studio and Homology modeling was performed using Antibody Modeling. Through structural simulation and rational design, a human frame region most closely resembling the murine antibody frame region was obtained. The CDRs of the light and heavy chains were then transplanted into the frame sequences of the matching human light and heavy chain genes, respectively, to obtain the humanized antibody Hab33B10E8. This was then analyzed using Discovery Studio and... Antibody Modeling was used to construct 3D models and analyze whether any sites where mouse amino acids were substituted for human amino acids at framework positions would affect binding and / or CDR conformation. Reverse mutations were performed, and the resulting humanized antibody sequences are shown below. CDRs were determined using Kabat numbering:

[0134] Hab33B10E8

[0135] HCDR1: DYYMK SEQ ID NO.1

[0136] HCDR2:AFNLNNGDTFYNQKFKG SEQ ID NO.2

[0137] HCDR3: DVYGYGDY SEQ ID NO.3

[0138] Heavy chain variable region (HV):

[0139] Heavy chain (HC):

[0140] LCDR1:RASKSVSTSGYSYMH SEQ ID NO.4

[0141] LCDR2: LASNLDS SEQ ID NO.5

[0142] LCDR3: QHSRELPYT SEQ ID NO.6

[0143] Light chain variable region (LV):

[0144] Light Chain (LC):

[0145] The obtained antibody fermentation broth was subjected to affinity chromatography, ion chromatography and other steps to obtain a sample with high purity.

[0146] 3. ADC fabrication

[0147] The concentration of antibody Hab33B10E8 was adjusted to 10 g / L using phosphate buffer (pH 7.0). After buffer replacement, the antibody solution was placed in a centrifuge tube, and 10.0 equivalents of 5 mM TCEP (Adamas-beta, Ltd.) were added. The reaction was carried out at 37°C for 1 hour to reduce the interchain disulfide bonds to thiol groups. Subsequently, 20 mM of compound O45 dissolved in DMSO (12 equivalents of O45 per antibody) was added. The reaction was carried out at 25°C for 1 hour. After coupling, the solution was subjected to ultrafiltration (Cobetter, Ltd.) to remove residual linker toxins and stored in PBS solution at pH 7.4. The average drug loading of each antibody was determined to be approximately 8 g / L by reverse chromatography.

[0148] Refer to the example:

[0149] BATLD was synthesized according to the method in reference CN116333135A, and D-BATADC was synthesized using BATLD as the linker drug and Hab33B10E8 as the antibody, following the aforementioned ADC preparation method. The DAR value was determined to be 7.7 by reverse phase chromatography.

[0150] Example 3: Enzyme-linked immunosorbent assay (ELISA) to determine the binding activity of Hab33B10E8-045

[0151] The affinity of the antibody for recombinant human DLL3 protein was determined using ELISA. Human DLL3 protein (KaiKa Biotechnology, catalog number: DLL-HM103) was diluted to a final concentration of 0.1 μg / ml and plated into 96-well microplates. The plates were incubated overnight at 4°C. The supernatant was discarded the next day. After blocking, a four-fold serial dilution of the antibody (0-100 nM) was added. After incubation with secondary antibody, the plates were developed with TMB chromogenic buffer for 10 min. The reaction was terminated by adding 2 M HCl. The absorbance at 450 nm was read using a microplate reader, and the EC50 was calculated. 50 The results are shown in Table 2. The affinity of Hab33B10E8-045 for human DLL3 protein is basically equivalent to the binding ability of Hab33B10E8 before coupling.

[0152] Table 2. Enzyme-linked immunosorbent assay (ELISA) for determining the binding activity of Hab33B10E8-045.

[0153] Example 4: In vitro cytotoxicity of Hab33B10E8-045

[0154] Hab33B10E8-045-mediated in vitro cytotoxicity was evaluated using DLL3-positive cell lines NCI-H82, SHP77, NCI-H526, and NCI-H209, and DLL3-negative cell lines A431 and NCI-H69. Cells were harvested, cultured with serially diluted Hab33B10E8-045, and then incubated at 37°C. Viability was assessed after 6 days using CTL Plus. Cells were read and analyzed on an EnVision 2105 (PerkinElmer) to determine the IC50. 50 (Half-maximum inhibition concentration) value.

[0155] Experimental procedure: The cell density of the cells to be tested was adjusted to 6.7 × 10⁻⁶. 4 / mL, and seeded 75μL per well into 96-well plates, incubated overnight at 37℃ in a 5% CO2 incubator. Add 75μL of Hab33B10E8-045 (0-100nM) at 5-fold serial dilutions (9 concentrations) to each well. The blank control was the corresponding cell culture medium. Two replicates were set for each concentration. After culturing the cells at 37℃ in a 5% CO2 incubator for 6 days, 50μL of CTL Plus (manufacturer: Beyotime, catalog number: C0068XL) chemiluminescent reagent was added, and incubated at room temperature in the dark for 10 min. Chemiluminescence detection was performed using an EnVision 2105 microplate reader. Data analysis: Using the blank control as a zero-kill control, the inhibition rate was calculated using the following formula: Inhibition rate (%) = (1 - experimental group / blank control group) × 100%. The IC50 was calculated using GraphPad Prism to process and analyze the data. 50 The results are shown in Table 3. The results indicate that Hab33B10E8-045 exhibits good in vitro cytotoxicity against DLL3-positive cell lines.

[0156] Table 3. In vitro cytotoxicity of Hab33B10E8-045

[0157] Example 5: Pharmacodynamic evaluation of Hab33B10E8-045 in a mouse model of human small cell lung cancer SHP77 xenograft tumor

[0158] 10 cells were taken from the DLL3-positive cell line SHP77. 7 Seven cells were inoculated into the right forelimb axilla of null / null nude mice (n=7 per group). The cells were inoculated when the average tumor volume of the mice reached 100 mm². 3Mice were randomly divided into groups and administered the drug via a single intravenous injection. The day of grouping was designated as day 0, and drug administration began on day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior, specifically monitoring the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²). 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis), the pharmacodynamic evaluation of the test drug is shown in Table 4.

[0159] Table 4. Pharmacodynamic evaluation of the test drugs in the SHP-P77 mouse model of lung cancer xenograft. Note: a. Data are expressed as mean ± standard error; b. TGI% = [1 - (Ti - T0) / (Ci - C0)] × 100, where T0 and C0 are the mean tumor volumes on the day of grouping (Day 0) for the drug administration group and the solvent control group, respectively, and Ti and Ci are the mean tumor volumes on Day 22 for the drug administration group and the solvent control group, respectively; c. Compared with the tumor volume of the solvent control group.

[0160] Compared with the solvent control group, the ADC of the present invention significantly inhibited tumor growth in the SHP-77 model at a dose of 1 mg / kg, with a tumor growth inhibition rate (TGI) of 98.1%. There were no animal deaths or significant weight loss in the treatment group, and no obvious drug toxicity was observed. Mice tolerated the ADC of the present invention well during the treatment period.

[0161] Example 6: Pharmacodynamic evaluation of Hab33B10E8-045 in mouse models of human small cell lung cancer NCI-H526 and NCI-H69 xenograft tumors

[0162] 10 cells were taken from the DLL3-positive cell line NCI-H526. 7 Three cells were inoculated into the right forelimb axilla of null / null mice (n=3 per group). The cells were inoculated when the average tumor volume of the mice reached 160 mm². 3 Approximately 1-2 groups of mice were randomly assigned to receive the drug via a single intravenous injection. The day of group assignment was designated as day 0, and drug administration began on day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities observed in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²). 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). The pharmacodynamic evaluation of the test drug is shown in Table 5.

[0163] Table 5. Pharmacodynamic evaluation of the test drugs in the NCI-H526 mouse model of human small cell lung cancer xenograft.

[0164] 10 cells were taken from the DLL3-positive cell line NCI-H69. 7 Three cells were inoculated into the right forelimb axilla of null / null mice (n=3 per group). The cells were inoculated when the average tumor volume of the mice reached 140 mm². 3 Approximately 1-2 groups of mice were randomly assigned to receive the drug via a single intravenous injection. The day of group assignment was designated as day 0, and drug administration began on day 0. Post-tumor inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss (measured twice weekly), and any abnormalities observed in the eyes, fur, or other areas. Tumor volume was calculated using the formula: Tumor volume (mm²). 3 )=1 / 2×(a×b 2 (where a represents the major axis and b represents the minor axis). The pharmacodynamic evaluation of the test drug is shown in Table 6.

[0165] Table 6. Pharmacodynamic evaluation of the test drugs in the NCI-H69 mouse model of human small cell lung cancer xenograft.

[0166] In the NCI-H526 and NCI-H69 models, compared with the solvent control group, the ADC of the present invention significantly inhibited tumor growth in both models at a dose of 3 mg / kg, with tumor growth inhibition rates (TGI) of 113.0% and 76.5%, respectively. There were no animal deaths or significant weight loss in any treatment group, and no obvious drug toxicity was observed. Mice tolerated the ADC of the present invention well during the treatment period.

[0167] Example 7: Evaluation of toxicokinetics and tolerability of ADC in cynomolgus monkeys

[0168] Two female cynomolgus monkeys were selected and administered Hab33B10E8-045 intravenously at dose levels of 10, 30, and 30 mg / kg every two weeks (a total of three doses). Parameters assessed during the study included general observation, body weight, food intake, body temperature, electrocardiogram (lead II), clinicopathology (hematology, blood biochemistry, coagulation), and macroscopic and microscopic examination of numerous tissues. Toxicokinetic samples were collected at 0, 24, 72, 120, 336, and 504 hours after each administration. TK samples were analyzed using the MesoScale Discovery (MSD) electrochemiluminescence platform (total monoclonal antibody, total ADC) and LC-MS / MS (free eczema). The results showed that Hab33B10E8-045 was well-tolerated with an MTD of 30 mg / kg, and higher MTDs are possible. In the dose-response study, after injections of 10, 30, and 30 mg / kg Hab33B10E8-045, no abnormalities were observed in general cynomolgus monkeys. No toxicological abnormalities were observed in weight, body temperature, coagulation, or urine analysis. All changes were reversible before each administration. Hab33B10E8-045 was well tolerated in cynomolgus monkeys and exhibited stable pharmacokinetic characteristics.

[0169] Table 7. Toxicokinetics and Tolerance of Hab33B10E8-045 in Cynomolgus Monkeys

[0170] Formulation Examples

[0171] Formulation Example 1: pH and Buffer System Screening

[0172] pH is a crucial factor affecting the stability of biological products. It regulates the charge distribution on the protein surface, thereby influencing intermolecular and intramolecular forces, conformation, and colloidal stability. It also affects the chemical stability of proteins, such as degradation reactions like deamidation. Appropriate buffer salts play a role in maintaining a stable pH level in a solution, and the type of buffer salt significantly impacts the physicochemical properties and stability of biological products.

[0173] 1. Changes in appearance and particle size

[0174] Prepare 20mM histidine-histidine hydrochloride buffer, citrate-sodium citrate buffer, and succinate-sodium hydroxide buffer at pH 5.0, 5.5, 6.0, and 6.5. Displace Hab33B10E8-045 into these 12 buffer solutions via ultrafiltration, ultimately diluting the Hab33B10E8-045 (ADC product) concentration to approximately 10 mg / ml. After filtration through a 0.22 μm microporous membrane, the thermal stability and particle size distribution of the ADC product under different pH values ​​and buffer conditions were determined using DSC and DLS. The product was also subjected to high temperature (40±2℃) and light irradiation (5±3℃, white light: 5000±500 Lux, UV: 90 μW / cm²). 2 Stability tests. Table 7 shows the test data of ADC products under the influence of high temperature and light in different types of buffer salts and pH. Under the three buffer systems, the thermal denaturation initiation temperature (Tonset) increases with increasing pH. Except for the histidine-histidine hydrochloride test group, the thermal accumulation temperature (Tagg) increases with increasing pH. At the same pH, the histidine-histidine hydrochloride test group has a higher thermal accumulation temperature.

[0175] T0 particle size data showed that, at the same pH, the particle size of the histidine-histidine hydrochloride test group was significantly lower than that of the other two buffer salt test groups. After 7 days of high temperature, the particle size of the pH 5.0 succinic acid-sodium hydroxide test group increased significantly, while no significant changes were observed in the particle size of the other test groups. After 5 days of light exposure, no significant changes were observed in the particle size of the histidine-histidine hydrochloride test group and the pH 5.5 succinic acid-sodium hydroxide test group, while the particle size of the other test groups increased significantly. pH 5.0 and 5.5 histidine-histidine hydrochloride are the preferred buffer solutions for ADC products.

[0176] All T012 test groups were colorless and clear solutions. After 7 days of high temperature, protein precipitation occurred in the pH 5.0 citric acid-sodium citrate test group, and the protein content decreased significantly. No significant changes were observed in the appearance of the other test groups. After 5 days of light exposure, the pH 5.5, 6.0, and 6.5 histidine-histidine hydrochloride test groups turned yellow, and the color deepened with increasing pH. Protein precipitation occurred in the pH 6.5 succinic acid-sodium hydroxide test group, and the protein content decreased significantly. The other test groups remained colorless and clear solutions.

[0177] 2. The effects of high temperature and light on ADC products

[0178] Figure 2 shows the effects of high temperature and light exposure on the SEC monomer content of the ADC product in different pH and buffer salts. In the histidine-histidine hydrochloride buffer, the pH 5.5 test group showed the highest SEC content. In the other two buffer types, the SEC monomer content decreased with increasing pH. The pH 5.5 histidine-histidine hydrochloride, pH 5.0 citric acid-sodium citrate, and pH 5.0 succinic acid-sodium hydroxide buffers showed the best high-temperature stability for the three buffer types, respectively. After 5 days of light exposure, the SEC monomer content showed a significant decreasing trend with increasing pH in all three buffer types. At pH 5.0, there was no significant difference among the three buffer types.

[0179] Because light exposure significantly affects the charge heterogeneity of ADC products, it was impossible to distinguish between acidic, main, and basic peaks. Therefore, only CEX data were measured after 7 days at high temperature. T0 CEX main peak data showed that the proportion of the ADC product's main CEX peak decreased with increasing pH, with pH 5.0 and 5.5 being the preferred buffers. For the pH 5.0 and 5.5 buffers, the proportion of the main CEX peak in the histidine-histidine hydrochloride buffer did not decrease significantly, while the proportions in the other two buffers decreased significantly. pH 5.0 and 5.5 histidine-histidine hydrochloride buffers are the preferred buffers for ADC products.

[0180] Based on data on heat aggregation temperature (Tagg), high-temperature stability of the CEX main peak, particle size under light and high-temperature stability, and changes in appearance and protein content after high-temperature and light exposure experiments, histidine-histidine hydrochloride was selected as the preferred buffer solution. In the histidine-histidine hydrochloride experimental groups, the heat denaturation initiation temperature (Tonset) increased with increasing pH; however, after 7 days of high-temperature exposure, the SEC monomer content was higher in the low-pH groups of 5.0 and 5.5; after 5 days of light exposure, the SEC monomer content decreased significantly with increasing pH. Based on comprehensive analysis, histidine-histidine hydrochloride at pH 5.3 was selected as the target buffer system as a compromise.

[0181] Formulation Example 2: DOE Screening of Buffer Salt Concentration and ADC Product Concentration in the Formulation

[0182] 1. DOE scheme

[0183] The central composite design (CCD) of DOE response surface methodology was used to optimize histidine concentration and ADC product concentration. Five levels were set for each experimental factor, denoted by the codes -α, -1, 0, 1, and α (α = 1.414). Factors and levels are shown in Table 9. The experimental setup is shown in Table 10. The ADC product was ultrafiltered into histidine-histidine hydrochloride buffer solutions of different concentrations at pH 5.3, and the ADC product concentration was adjusted to the target level. The thermal aggregation temperature (T0) and particle size data were measured, and the product was subjected to high temperature (40±2℃) and light irradiation (5±3℃, white light: 5000±500 Lux, UV: 90 μW / cm²).2 Stability tests were conducted, measuring SEC, CEX, and particle size stability data. Minitab software was used to analyze the effects of histidine concentration and ADC product concentration on each indicator, establishing regression equations. The significance of the regression model was tested, and the lack-of-fit p-value was evaluated. Items with model p-values ​​less than 0.05 and lack-of-fit p-values ​​greater than 0.05 were selected, and response surface methodology was used to optimize the two experimental factors to obtain the optimal experimental level.

[0184] Table 9 Factors and Levels of Response Surface Design Note: α = 1.414.

[0185] Table 10 Experimental Arrangements for Response Surface Design

[0186] 2. DOE Test Results and Analysis

[0187] The buffering capacity of the buffer solution increases with increasing buffer salt concentration, and formulations with low ionic strength will effectively reduce the aggregation and fragmentation of ADCs. Therefore, the screening range for histidine concentration was set at 10–30 mM. Considering the later development of lyophilized formulations and the impact of filling volume on liquid level thickness at a specification of 100 mg / vial, the screening range for ADC product concentration was set at 10–20 mg / ml.

[0188] Table 11 shows the data for heat accumulation temperature (Tagg) and particle size, SEC monomers, and CEX main peak in the high-temperature and light-induced tests during the response surface design. Table 12 shows the P-values ​​and R-values ​​of the regression models of the two experimental factors on each test item after response surface analysis using Minitab software. 2 Statistical analysis of p-values ​​for lack of fit. For thermal aggregation temperature (Tagg), high-temperature 7-day particle size, SEC monomers, and 5-day light-irradiated SEC monomers, the regression model p-value was less than 0.05, and the p-value for lack of fit was greater than 0.05. 2 The regression models for these four items, all above 89%, fit the experiment well. Table 13 shows the statistical significance of the regression equations, linear terms, quadratic terms, and interactions on the response values ​​for these four items.

[0189] The effects of histidine concentration and ADC product concentration on Tagg (the heat accumulation temperature) from most significant to least significant are: histidine concentration, ADC product concentration, interaction between the two factors, and the quadratic term has no significant effect. The main influencing factor on Tagg is histidine concentration. When the ADC product concentration remains constant, Tagg decreases with increasing histidine concentration. When the histidine concentration is below 10 mM, the effect of ADC product concentration on Tagg has a small impact. When the histidine concentration is above 20 mM, Tagg shows a slight decreasing trend with increasing ADC product concentration. After optimization using a response surface methodology, within the target screening range, the optimal histidine concentration is 10 mM, and the optimal ADC product concentration is 10 mg / ml.

[0190] The effects of high-temperature 7-day particle size on particle size, ranked from most significant to least significant, are: ADC product concentration, quadratic term of ADC product concentration, histidine concentration, interaction between the two factors, and quadratic term of histidine concentration. The particle size at high temperature 7 days shows a significant increasing trend with increasing histidine concentration. At higher histidine concentrations (>20 mM), the particle size at high temperature 7 days also shows an increasing trend with increasing ADC product concentration. Within the target screening range, a histidine concentration of 10 mM is preferred, as the effect of ADC product concentration on particle size at high temperature 7 days is relatively small at this concentration.

[0191] The effects of high-temperature 7-day SEC monomer concentration on the following order of significance, from largest to smallest, are: ADC product concentration and histidine concentration. The interaction between these two factors and the quadratic term effect are not significant. The main influencing factor on high-temperature 7-day SEC monomer concentration is ADC product concentration. When the histidine concentration remains constant, the concentration of high-temperature 7-day SEC monomer decreases with increasing ADC product concentration. After optimization using a response surface methodology, within the target screening range, the optimal histidine concentration is 10 mM, and the optimal ADC product concentration is 10 mg / ml.

[0192] Histidine concentration had a significant impact on SEC monomers after 5 days of illumination. The effects of ADC product concentration, the interaction between the two factors, and the quadratic term were not significant. The main influencing factor on SEC monomers after 5 days of illumination was histidine concentration. After optimization using a response surface methodology, within the target screening range, the optimal histidine concentration was 10 mM, and the optimal ADC product concentration was 13.6 mg / ml.

[0193] Based on the comprehensive analysis of the above results, the ADC product exhibits better high-temperature and light stability at lower histidine concentrations. Within the screening target range, a histidine concentration of 10 mM is preferred. Except for SEC exposed to light for 5 days, the stability of the ADC product deteriorates with increasing ADC concentration. Considering that the dosage form for the later development of the ADC product is lyophilized powder, compared to liquid dosage forms, lyophilized powder will mitigate the adverse effects of increased ADC concentration on stability. Within the screening target range, the ADC product concentration can be appropriately increased to 15 mg / ml.

[0194] Table 12 Significance Analysis of Response Surface Design Regression Model

[0195] Table 13. Significance of the effects of linear terms, quadratic terms, and interaction on response values, and regression equations. Note: X1: Histidine concentration (mM), X2: ADC product concentration (mg / ml)

[0196] Formulation Example 3: Screening of Stabilizer Types

[0197] A pH 5.3 10 mM histidine-histidine hydrochloride buffer was prepared. Different types and amounts of sugar or mannitol were added as stabilizers (also serving as lyophilization protectants) according to Table 14. The ADC product was ultrafiltered and transferred to different stabilizer buffers, and the ADC product concentration was adjusted to approximately 15 mg / ml. The DSC, particle size, and Tagg value of the ADC product in different stabilizers were measured. ADC product samples with 1 ml / vial were lyophilized according to the platform process. The appearance of the lyophilized powder was observed. The lyophilized powder was reconstituted with 0.95 ml of ultrapure water and subjected to high temperature (40±2℃) and light irradiation (5±3℃, white light: 5000±500 Lux, UV: 90 μW / cm²). 2 Stability tests were conducted to measure data such as particle size, SEC, CEX, and free toxin Exatecan, and to compare the protective effects of different stabilizers on ADC products.

[0198] Table 14 Screening Test Design for Stabilizers (Lyophilization Protectants) Note: In Table 14, % represents w / v, g / 100ml.

[0199] Results and Analysis of Stabilizer Selection

[0200] Table 15 shows the DSC and DLS data of 15 ADC product samples containing different stabilizers. Under the same buffer solution, there were no significant differences in the thermal denaturation onset temperature (Tonset), thermal aggregation temperature (Tagg), and particle size of different stabilizers. For the same stabilizer, the thermal aggregation temperature (Tagg) and particle size showed a slight increasing trend with the addition of stabilizer.

[0201] Table 16 shows the evaluation data on the shrinkage of pressed powder and the measurement data on the bottom diameter of the pressed powder. No significant shrinkage was observed in the bottom of the pressed powder in the mannitol test group and the mixed group with a mannitol content of not less than 3%, and the bottom diameter of the pressed powder was the largest. The bottom diameter of the pressed powder in the sucrose and trehalose groups decreased with increasing dosage; at the same dosage, the bottom shrinkage of the pressed powder in the sucrose group was more severe than that in the trehalose group. The mixed groups with mannitol contents of 1% and 2% showed the most severe shrinkage in the bottom. From the perspective of pressed powder appearance, the mixed group and the mannitol test group with a mannitol content of not less than 3% are preferred.

[0202] Table 17 shows the purity data of ADC product samples with different lyophilization protectants before and after lyophilization. Compared with the original solution: the particle size of the lyophilized powder reconstituted solution did not increase significantly, the proportion of the CEX main peak did not increase significantly, and there were no significant differences among the experimental groups; the free toxin Exatecan remained at extremely low values, with no significant increase, and there were no significant differences among the experimental groups. The SEC monomer content in the sucrose group, trehalose group, and mixed group containing no less than 2% mannitol did not decrease significantly, while the SEC monomer content in the mannitol group and the mixed group containing 1% mannitol decreased, and the decrease rate increased with the increase of mannitol dosage. High concentrations of mannitol have an adverse effect on the purity of SEC after lyophilization.

[0203] Table 18 shows the high-temperature and light-induced stability data of samples with different freeze-drying protectants. After 7 days of high-temperature exposure, no significant change in particle size was observed in any of the experimental groups. SEC monomers all showed a slight decrease, with the mannitol group showing the largest decrease. CEX values ​​all showed a significant decrease, with no significant differences among the experimental groups. After 5 days of light exposure, the particle size of all experimental groups decreased slightly, and the CEX values ​​could not be analyzed for acid, main, and alkali peaks. SEC monomers all showed a significant decrease, with the mannitol group showing the largest decrease. Based on the appearance of the freeze-dried powder, changes in purity before and after freeze-drying, and the high-temperature and light-induced stability data, 3% mannitol and 3% sucrose were selected as the preferred stabilizers for the ADC product.

[0204] Table 15 DSC and DLS data of ADC product samples with different stabilizers Note: In Table 15, % represents w / v, g / 100ml.

[0205] Table 16. Measurement data of bottom diameter of ADC product freeze-dried powder cakes with different freeze-drying protectants. Note: "*" indicates mild contraction, "**" indicates moderate contraction, and "***" indicates severe contraction.

[0206] Table 17 Purity data of ADC product samples with different freeze-drying protectants before and after freeze-drying. Note: In Table 17, % represents w / v, g / 100ml.

[0207] Formulation Example 4: Surfactant Screening

[0208] 1. Surfactant addition amount screening test plan

[0209] The ADC product was ultrafiltered into a pH 5.3 10mM histidine-histidine hydrochloride 3% sucrose 3% mannitol buffer solution to dilute the ADC product concentration to approximately 15 mg / ml, obtaining the formulation solution. 0.005%, 0.02%, and 0.04% polysorbate 80 (PS80) were added to this solution, with a control group without added surfactant. After filtration through a 0.22 μm microporous membrane, four groups of ADC product samples with different surfactant contents were prepared. Sub-visible particle data were measured using Flowcam after 0, 3, and 6 freeze-thaw cycles (-40℃ to room temperature). In this example, % represents w / v, g / 100ml.

[0210] 2. Results and Analysis of Surfactant Dosage Screening

[0211] Surfactants can inhibit the formation of protein aggregates, and non-spherical particles (NSPs) in subvisible microparticles are usually protein aggregates. By analyzing the changes in the number of non-spherical particles (NSPs) in ADC product samples with different polysorbate 80 contents after freeze-thaw tests, the optimal surfactant dosage was screened.

[0212] Table 19 shows the changes in non-spherical particles (NSPs) in ADC products with different amounts of surfactant after freeze-thaw and shaking tests. After 3 and 6 freeze-thaw cycles, the number of NSPs in the unsurfed group increased significantly, while no significant increase was observed in the three groups with surfactant. Different amounts of polysorbate 80 significantly inhibited the formation of subvisible particles. Considering the stability of polysorbate 80, 0.02% polysorbate 80 was selected.

[0213] Table 19. Variation data of non-spherical subvisible particles (NSP) in freeze-thaw and shaking tests. Note: The particle size in the table refers to the equivalent sphere diameter (ESD).

[0214] Summary of formulation screening

[0215] Three buffer systems with different pH values—histidine-histidine hydrochloride, citrate-sodium citrate, and succinic acid-sodium hydroxide—were used for high-temperature and light-induced tests. The appearance changes of the ADC product in different buffer systems were observed, and purity was determined. Histidine-histidine hydrochloride at pH 5.3 was selected as the preferred buffer solution. Using a central composite design in DOE, histidine and ADC product concentrations were screened, and 10 mM histidine-histidine hydrochloride at pH 5.3 was selected as the target buffer solution with an ADC product concentration of 15 mg / ml. Different amounts of sucrose, trehalose, and mannitol were selected as stabilizers (lyophilization protectants) for the ADC product. The appearance of lyophilized powders with different lyophilization protectants was compared using a platform lyophilization process. The purity changes of the ADC product before and after lyophilization were compared, and high-temperature and light-induced stability tests were conducted after reconstitution. 3% sucrose and 3% mannitol were selected as lyophilization protectants for the ADC product. The changes in non-spherical subvisible particles (NSPs) in ADC product samples of different concentrations of polysorbate 80 after repeated freeze-thaw cycles were determined using FlowCam. Adding 0.02% polysorbate 80 was screened as a surfactant for the ADC product formulation. The optimal formulation composition of the ADC product was finally confirmed as follows: ADC product concentration 15 mg / ml, pH 5.3, 10 mM histidine-histidine hydrochloride, 0.02% (w / v) polysorbate 80, 3.0% (w / v) sucrose, and 3.0% (w / v) mannitol.

[0216] Formulation Example 5: Development of Lyophilized Formulations

[0217] Using 15 mg / ml antibody-drug conjugate (Hab33B10E8-045), 0.24 g / L histidine, 1.77 g / L histidine hydrochloride, 0.2 g / L polysorbate 80, 30 g / L sucrose, and 30 g / L mannitol as the base formulation, the buffer and stabilizer were weighed and diluted with water for injection to the target preparation volume. The mixture was stirred and stirred until homogeneous to obtain the ultrafiltration replacement buffer. The prepared antibody-drug conjugate was then exchanged into the prepared buffer.

[0218] The glass transition temperature and disintegration temperature of the sample were determined. Low-temperature DSC analysis showed that the glass transition temperature of the sample was -35.22℃, and lyophilization microscopy showed that the disintegration temperature was -30.3℃.

[0219] Pre-freezing: Based on the glass transition temperature (-35.22℃) of the ADC product stock solution, the proposed pre-freezing temperature for the small-scale test was -40℃. Before reaching -40℃, the plate was first brought to 4℃ over 30 minutes and maintained at 4℃ for another 30 minutes to ensure that all samples could be cooled from 4℃. Pre-freezing was performed at two cooling rates: 2℃ / min and 0.5℃ / min.

[0220] Single-stage drying: After pre-freezing, the sample enters the single-stage drying process, where over 90% of the water in the product, primarily free water, is removed through sublimation. The development of the single-stage drying process mainly focuses on the plate temperature, the vacuum level of the drying oven, and the duration of the single-stage drying. During the single-stage drying process, the product temperature decreases due to the endothermic effect of sublimation. The temperature of the unsublimated sample is lower than the plate temperature. Once sublimation is complete and most of the water has been removed, the product temperature rises above the plate temperature, and the single-stage drying process is finished.

[0221] To increase the sublimation rate of the product, while ensuring that the temperature of the unsublimated portion does not exceed its disintegration temperature, the plate temperature can be set above the product's allowable disintegration temperature. Pre-freezing was performed at a cooling rate of 2℃ / min, followed by desorption drying at 35℃, 0.20 mbar for 600 min. Three batches of freeze-drying tests were conducted with different vacuum settings.

[0222] After the first drying cycle, approximately 10% moisture remains adsorbed on the capillary walls and polar groups of the dried material. When this bound water reaches a certain content, it provides conditions for the growth and reproduction of microorganisms and certain chemical reactions. This portion of moisture is bound to the drug through weak molecular forces such as van der Waals forces and hydrogen bonds. To remove this moisture, a higher plate temperature is needed to transfer energy to overcome intermolecular forces and achieve the effect of removing the bound water.

[0223] During the desorption and drying stage, the plate temperature should be set according to the thermal stability of the product. The tonset temperature of the ADC product stock solution, determined by differential scanning calorimetry, is approximately 49℃. Therefore, the desorption and drying plate temperature setting needs to be below 49℃. Pre-freezing uses a cooling rate of 2℃ / min, and the first drying stage uses -5℃, a vacuum of 0.25 mbar, and a duration of 1400 min. Desorption and drying is then performed at 35℃ and 0.20 mbar for 600 min. Table 20 shows the process parameters for this freeze-drying. Table 21 shows the purity comparison data between the freeze-dried powder reconstituted solution and the stock solution. After freeze-drying, no significant changes were observed in the purity and physicochemical properties of the ADC product. The freeze-drying formulation in Table 20 can be used for pilot-scale amplification experiments.

[0224] Table 20 Optimized freeze-dried formulation for ADC product in small-scale trials

[0225] After developing a small-scale freeze-drying process for ADC products, a suitable freeze-drying process was determined. The freeze-drying process parameters are shown in Table 19. The freeze-dried ADC product obtained by this process has a good appearance and a moisture content of less than 1%. There is no significant difference in the purity and physicochemical properties of the formulation solution and the reconstituted freeze-dried powder before and after freeze-drying.

[0226] Table 21 Comparison of mass between reconstituted and original solutions of lyophilized powder

[0227] Formulation Example 6: Long-term stability of the formulation

[0228] Based on the determined optimal formulation, two batches of samples were prepared under GMP conditions according to the lyophilization process of Example 5. Stability studies were conducted on both batches. All detection methods for the research indicators were validated and proven accurate and reliable. Stability studies were performed at 5±3℃ on the un-lyophilized stock solution and the lyophilized finished product. The lyophilized powder was dissolved in 6.8 ml of water for injection before testing. Tables 22 and 23 show the stability data for the two batches of stock solution and lyophilized powder. The results show that the product quality is more stable after lyophilization than in the liquid state. Charge variants in the liquid state are more prone to producing basic variants, and the lyophilized dosage form is more conducive to the long-term storage of this product.

[0229] Table 22 Stability Results of Key Quality Indicators for GMP Batch 1

[0230] Table 23 Stability Results of Key Quality Indicators for GMP Batch 2

Claims

1. A pharmaceutical composition comprising an antibody-drug conjugate of Formula I: Where A is an antibody targeting DLL3 or its antigen-binding fragment, and R is selected from... in, R a Selected from C1-C3 alkylene groups, such as methylene, ethylene, n-propylene, and isopropylene; R b Selected from C1-C3 alkyl groups, such as methyl, ethyl, n-propyl, isopropyl; r, t, and u are each independently selected from integers from 1 to 50, preferably integers from 4 to 30; m is an integer or decimal from 1 to 12, preferably an integer or decimal from 4 to 8; The pharmaceutical composition is a liquid formulation, a lyophilized formulation, or a powder for injection, preferably a liquid formulation or a lyophilized formulation, preferably an injection, preferably an injection solution or a lyophilized injection, and more preferably a lyophilized injection. Preferably, the antibody-drug conjugate shown in Formula I has the following structure: More preferably, the antibody-drug conjugate shown in Formula I has the following structure:

2. The pharmaceutical composition of claim 1, wherein... A is an antibody or antigen-binding fragment targeting DLL3, wherein the antibody or antigen-binding fragment comprises a heavy chain and / or a light chain, the heavy chain comprising three complementarity-determining regions (CDRs), wherein the amino acid sequence of heavy chain complementarity-determining region 1 (HCDR1) is shown in SEQ ID NO:1, the amino acid sequence of heavy chain complementarity-determining region 2 (HCDR2) is shown in SEQ ID NO:2, and the amino acid sequence of heavy chain complementarity-determining region 3 (HCDR3) is shown in SEQ ID NO:3; the light chain comprises three complementarity-determining regions (CDRs), wherein the amino acid sequence of light chain complementarity-determining region 1 (LCDR1) is shown in SEQ ID NO:4, the amino acid sequence of light chain complementarity-determining region 2 (LCDR2) is shown in SEQ ID NO:5, and the amino acid sequence of light chain complementarity-determining region 3 (LCDR3) is shown in SEQ ID NO:6, wherein the CDRs are determined according to the Kabat numbering rules; More preferably, A is an antibody or antigen-binding fragment targeting DLL3, the antibody or antigen-binding fragment comprising a heavy chain and / or a light chain, the variable region (HV) sequence of the heavy chain being as shown in SEQ ID NO:9, and the variable region (LV) sequence of the light chain being as shown in SEQ ID NO:

10.

3. The pharmaceutical composition according to any one of claims 1-2, wherein the antibody-drug conjugate of formula I has the following structure: Where m is selected from 2, 4, 6, and 8; or in, y is the average number of linker-drug molecules conjugated to each antibody unit, which is an integer or decimal of 1-12, preferably an integer or decimal of 4-8; Wherein, A is Hab33B10E8, which includes two identical heavy chains and light chains. The amino acid sequence of the heavy chain is shown in SEQ ID NO:11, and the amino acid sequence of the light chain is shown in SEQ ID NO:

12.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the pharmaceutical composition is a lyophilized injection, optionally containing residual moisture.

5. The pharmaceutical composition of claim 4, wherein, The antibody-drug conjugate shown in Formula I has a mass percentage of 1-30%, preferably 5-25%, 10-25%, about 19.4%, based on 100 wt% of the drug composition described herein.

6. The pharmaceutical composition according to any one of claims 4-5, further comprising a buffer; Preferably, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycyl glycine, tromethamine (Tris), and morpholine ethanesulfonic acid (MES), with histidine buffer being preferred, and histidine-histidine hydrochloride buffer being more preferred. More preferably, the content of the buffer is 1-10%, more preferably 1-5%, and more preferably 2.6%.

7. The pharmaceutical composition according to any one of claims 4-6, further comprising a stabilizer; Preferably, the stabilizer is selected from one or more of sugars (such as sucrose and trehalose), polyols (such as mannitol and sorbitol), and amino acids (L-serine, monosodium glutamate, alanine, glycine, sarcosine, etc.), preferably sugars, and more preferably sucrose and / or mannitol. More preferably, the pharmaceutical composition comprises two stabilizers, each of which is present in a mass percentage of 1-50%, preferably 20%-40%, about 38.9%, based on 100 wt% of the pharmaceutical composition.

8. The pharmaceutical composition according to any one of claims 4-7, further comprising a surfactant; Preferably, the surfactant is selected from polysorbates, and more preferably polysorbate 80 and / or polysorbate 20; More preferably, the surfactant has a mass percentage content of 0.01%-1%, preferably 0.05%-0.4%, more preferably 0.2%, based on 100 wt% of the pharmaceutical composition.

9. The pharmaceutical composition according to any one of claims 4-8, comprising 10-30%, preferably 10-25%, more preferably 19.4% of the antibody-drug conjugate of Formula I; 1-10%, preferably 1-5%, more preferably 2.6% of a buffer, preferably histidine-histidine hydrochloride; two stabilizers, sucrose and trehalose, each stabilizer comprising 10-50%, preferably 20-40%, more preferably 38.9%; and the surfactant polysorbate 80 comprising 0.05-0.4%, preferably about 0.2%, optionally containing residual water, preferably the antibody-drug conjugate of Formula I being Hab33B10E8-045.

10. The pharmaceutical composition according to any one of claims 4-9, comprising, by weight percentage, about 19.4% of the antibody-drug conjugate of Formula I, about 2.6% of a buffer, said buffer being histidine-histidine hydrochloride, wherein the histidine content is about 0.3%; the histidine hydrochloride content is about 2.3% by weight; the stabilizer content is about 77.8% by weight; and the surfactant content is about 0.2%, optionally containing residual water; preferably, the antibody-drug conjugate of Formula I is Hab33B10E8-045. Preferably, by mass percentage, the formulation comprises about 19.4% of the antibody-drug conjugate of Formula I, about 2.6% of a buffer, wherein the buffer is histidine-histidine hydrochloride, wherein histidine is about 0.3%; the histidine hydrochloride is about 2.3% by mass; mannitol is about 38.9% by mass; sucrose is about 38.9% by mass; polysorbate 80 is about 0.2% by mass, optionally containing residual water, and preferably, the antibody-drug conjugate of Formula I is Hab33B10E8-045.

11. The pharmaceutical composition according to any one of claims 4-10, wherein the lyophilized injection can be used by dissolving it in a solvent (preferably water, more preferably water for injection) to obtain a solution, wherein the lyophilized injection is obtained by lyophilizing a stock solution formed by lyophilizing a solvent (preferably ethanol, methanol, or water) containing a predetermined amount of the pharmaceutical component.

12. The pharmaceutical composition according to any one of claims 1-3, wherein the pharmaceutical composition is a liquid formulation, preferably an injection, wherein the liquid formulation is a stock solution formed by a solvent (preferably ethanol, methanol, or water) containing a predetermined amount of the pharmaceutical component, or a solution obtained by dissolving a lyophilized injection in a solvent (preferably water, more preferably water for injection).

13. The pharmaceutical composition of claim 12, wherein the content of the antibody-drug conjugate of formula I in the liquid formulation is 1-30 mg / ml, preferably 5-25 mg / ml, more preferably 10-20 mg / ml, about 15 mg / ml.

14. The pharmaceutical composition according to any one of claims 12-13, wherein the liquid formulation further comprises a buffer; Preferably, the buffer is selected from one or more of acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycyl glycine, tromethamine (Tris), and morpholine ethanesulfonic acid (MES), with histidine buffer being preferred, and histidine-histidine hydrochloride buffer being more preferred. More preferably, the content of the buffer is 1-10 mg / ml, preferably 1-5 mg / ml, about 2.01 mg / ml; or, based on histidine, it is 5-25 mM, preferably 5-20 mM, about 10 mM; When the buffer is a histidine-histidine hydrochloride buffer, the histidine content is 0.1-1 mg / ml, preferably 0.1-0.5 mg / ml, 0.2-0.5 mg / ml, or about 0.24 mg / ml; the histidine hydrochloride content is 1-10 mg / ml, preferably 1-6 mg / ml, or about 1-4 mg / ml, or about 1.77 mg / ml.

15. The pharmaceutical composition according to any one of claims 12-14, wherein the solution, stock solution and / or liquid formulation further comprises a stabilizer; Preferably, the stabilizer is selected from one or more of sugars (such as sucrose and trehalose), polyols (such as mannitol and sorbitol), and amino acids (L-serine, monosodium glutamate, alanine, glycine, sarcosine, etc.), preferably sugars, and more preferably sucrose and / or mannitol. More preferably, the pharmaceutical composition comprises two stabilizers, each of which is present in a concentration of 10-50 mg / ml, preferably 10-40 mg / ml, 20-40 mg / ml, or about 30 mg / ml.

16. The pharmaceutical composition according to any one of claims 12-15, wherein the liquid formulation further comprises a surfactant; Preferably, the surfactant (such as polysorbate 80) has a content of 0.005-1 mg / ml, more preferably 0.05-0.4 mg / ml, about 0.2 mg / ml.

17. The pharmaceutical composition according to any one of claims 12-16, wherein the liquid formulation has a pH of 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, more preferably 4.5-5.5, more preferably 5.0-5.5, and most preferably about 5.

3.

18. The pharmaceutical composition according to any one of claims 12-17, wherein the liquid formulation comprises an antibody-drug conjugate of formula I in an amount of 1-30 mg / ml (preferably 5-25 mg / ml, 10-20 mg / ml, about 15 mg / ml) and a buffer histidine-histidine hydrochloride in an amount of 1-10 mg / ml (preferably 1-5 mg / ml, about 2.01 mg / ml), wherein, The histidine content is 0.1-1 mg / ml (preferably 0.1-0.5 mg / ml, 0.2-0.5 mg / ml, about 0.24 mg / ml), and the histidine hydrochloride content is 1-10 mg / ml (preferably 1-6 mg / ml, about 1-4 mg / ml, about 1.77 mg / ml); the two stabilizers, sucrose and mannitol, are each present in a content of 10-50 mg / ml, preferably 10-40 mg / ml, 20-40 mg / ml, about 30 mg / ml; the surfactant content is 0.005-1 mg / ml, preferably 0.05-0.4 mg / ml, about 0.2 mg / ml; or The solution, stock solution, and / or liquid formulation comprise about 15 mg / ml of the antibody-drug conjugate of Formula I, about 0.24 mg / ml of histidine, about 1.77 mg / ml of histidine hydrochloride, 0.2 mg / ml of polysorbate 80, 30 mg / ml of sucrose, and 30 mg / ml of mannitol, and the pH of the injection solution is about 5.3; or The predetermined amount of the pharmaceutical component comprises: 1-30 (preferably 5-25, 10-20, about 15) parts by weight of the antibody-drug conjugate of Formula I; 1-10 (preferably 1-5, about 2.0) parts by weight of a buffer, wherein when the buffer is histidine-histidine hydrochloride, histidine is 0.1-1 (preferably 0.1-0.5, 0.2-0.5, about 0.24) parts by weight and histidine hydrochloride is 1-10 (preferably 1-6, about 1-4, about 1.77) parts by weight of two stabilizers, sucrose and mannitol, each stabilizer being 10-50 (preferably 10-40, 20-40, about 30) parts by weight; and 0.005-1 (preferably 0.05-0.4, about 0.2) parts by weight of a surfactant (such as polysorbate 80). Preferably, the antibody-drug conjugate shown in Formula I is Hab33B10E8-045.

19. A method for preparing the pharmaceutical composition according to any one of claims 1-18, comprising the following steps: S1) Prepare the antibody-drug conjugate shown in Formula I; S2) Prepare ultrafiltration replacement buffer; S3) Replace the antibody-drug conjugate prepared in S1 with the buffer solution prepared in S2).

20. The use of the pharmaceutical composition according to any one of claims 1-18 in the preparation of a medicament for treating or preventing individual tumors; preferably, the tumor is a tumor in which tumor cells express DLL3 (DLL3+); more preferably, the tumor is a solid tumor or hematologic malignancy associated with high DLL3 expression; even more preferably, the tumor is lung cancer; more preferably, the tumor is small cell lung cancer.

21. A method for treating or preventing an individual tumor, the method comprising administering to a patient in need of a therapeutically effective dose of a pharmaceutical composition of any one of claims 1-18; said tumor being a tumor on which tumor cells express DLL3 (DLL3+); more preferably, said tumor being a solid tumor or hematologic malignancy associated with high DLL3 expression; even more preferably, said tumor being lung cancer; more preferably, said tumor being small cell lung cancer.

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