Pharmaceutical compositions containing antibody-drug conjugates and their use

Abstract

A pharmaceutical composition comprising an antibody-drug conjugate and uses thereof is provided. Specifically, a pharmaceutical composition comprising an anti-claudin antibody-drug conjugate is provided.

Description

[Technical Field]

[0001] This application claims priority to the Chinese patent application (202011061863.1) filed on September 30, 2020, and the Chinese patent application (CN202111069020.0) filed on September 13, 2021.

[0002] This disclosure relates to the field of pharmaceutical formulations, and more specifically to pharmaceutical compositions containing antibody-drug conjugates, and their use as anticancer agents. [Background technology]

[0003] The information contained herein does not necessarily constitute prior art and is provided solely as background information relevant to this disclosure.

[0004] Claudin-18 (CLDN18) is a protein encoded by the Claudin18 gene in humans. It belongs to the family of cellular tight junction proteins and can regulate molecular flow between laminar cells. The Claudin protein structure includes four transmembrane domains and two extracellular loops (its N-terminus and C-terminus are in the cytoplasm).

[0005] Claudin-18 has two splice variants, Claudin18.1 and Claudin18.2, with the two sequences differing by only eight amino acids in the first extracellular loop. Claudin18.1 and Claudin18.2 have different expression distributions; Claudin18.1 is selectively expressed in normal lung cells, while Claudin18.2, although severely restricted in normal cells, is frequently allosterically activated and overexpressed in several types of tumors (including gastric, lung, and pancreatic cancer). Claudin18.2 is considered a potential therapeutic target for gastric cancer and other types of cancer, and the discovery of this target also offers new treatment options for gastric cancer.

[0006] Antibody-drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to bioactive cytotoxins via a stable chemical linker compound. They leverage the specificity of antibody binding to surface antigens on normal and tumor cells, as well as the high efficacy of cytotoxic substances, while avoiding the drawbacks of the former (relatively low therapeutic efficacy) and the latter (excessive toxicity and side effects). This means that, compared to conventional chemotherapy drugs, antibody-drug conjugates can bind more precisely to tumor cells while reducing their impact on normal cells.

[0007] Currently, patents for antibodies and ADC agents targeting Claudin 18.2, such as WO2016166122 and WO2016165762, have been reported.

[0008] Because ADCs have more complex heterostructures than antibodies, they present even greater challenges for therapeutic ADC formulations. [Overview of the project]

[0009] This disclosure relates to a drug formulation containing an anti-Claudin 18.2 antibody-drug conjugate and its use. The formulation has advantages such as good stability and suitability in lyophilized form.

[0010] This disclosure provides a pharmaceutical composition comprising an anti-Claudin18.2 antibody-drug conjugate and a buffer, wherein the anti-Claudin18.2 antibody in the anti-Claudin18.2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, i) The heavy chain variable region has HCDR1, HCDR2, and HCDR3 having the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 6, or ii) The heavy chain variable region has HCDR1, HCDR2, and HCDR3 having the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 3, and the light chain variable region has LCDR1, LCDR2, and LCDR3 having the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 4. The buffering agent mentioned above is a histidine salt buffering agent.

[0011] In some embodiments, the pharmaceutical composition is as described in any one of the above items, wherein the buffer is a histidine acetate buffer.

[0012] In some embodiments, the pharmaceutical composition according to any one of the above claims, wherein the anti-Claudin 18.2 antibody comprises a heavy chain variable region and a light chain variable region, iii) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NOs. 15, 16, and 17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NOs. 18, 19, and 20, or iv) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NOs: 9, 10, and 11, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NOs: 12, 13, and 14.

[0013] In some embodiments, the pharmaceutical composition is as described in any one of the above clauses, wherein the anti-Claudin18.2 antibody is a mouse antibody, a chimeric antibody, or a humanized antibody.

[0014] In some embodiments, a pharmaceutical composition according to any one of the above claims, wherein the anti-Claudin 18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region, (1) The heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 3, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 4, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (2) The heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 24, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 21, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, (3) The heavy chain variable region has an amino acid sequence shown in SEQ ID NO: 5, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and the light chain variable region has an amino acid sequence shown in SEQ ID NO: 6, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, or (4) The heavy chain variable region has an amino acid sequence represented by SEQ ID NO: 31, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto, and the light chain variable region has an amino acid sequence represented by SEQ ID NO: 28, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical thereto.

[0015] In some embodiments, the pharmaceutical composition is as described in any one of the above claims, wherein the anti-Claudin18.2 antibody is a humanized antibody, and the humanized antibody comprises a framework region derived from a human antibody or a framework region variant thereof, and the framework region variant has at least 10 amino acid return mutations in the light chain framework region and / or heavy chain framework region of the human antibody.

[0016] In some embodiments, the pharmaceutical composition according to any one of the above claims, wherein the framework region variant includes a mutation selected from (a) or (b) below, i.e., (a) The light chain variable region comprises one or more amino acid restorative mutations optionally selected from 22S, 85I, and 87H, and / or the heavy chain variable region comprises one or more amino acid restorative mutations optionally selected from 48I, 82T, and 69M, or (b) The light chain variable region comprises one or more amino acid restorative mutations optionally selected from 4L or 22S, and / or the heavy chain variable region comprises one or more amino acid restorative mutations optionally selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L and 73K.

[0017] In some embodiments, the pharmaceutical composition according to any one of the above claims, wherein the framework region variant includes a mutation selected from the following, namely, (a-1) The light chain variable region includes amino acid restorative mutations of 22S, 85I and 87H, and the heavy chain variable region includes amino acid restorative mutations of 48I and 82T, or (b-1) The above light chain variable region includes a 4L amino acid restorative mutation, Of these, the 82 in the heavy chain variable region 82T corresponds to the 82A position in the Kabat rule.

[0018] In some embodiments, a pharmaceutical composition according to any one of the above claims, wherein the anti-Claudin 18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any one of the following claims, i.e. (vii) The heavy chain variable region is indicated by Sequence ID 3, and the light chain variable region is indicated by Sequence ID 4. (viii) The heavy chain variable region is indicated by sequence numbers 24, 25, 26, or 27, and the light chain variable region is indicated by sequence numbers 21, 22, or 23. (ix) The heavy chain variable region is indicated by Sequence ID 5, and the light chain variable region is indicated by Sequence ID 6, or (x) The heavy chain variable region is indicated by sequence numbers 31, 32, 33, or 34, and the light chain variable region is indicated by sequence numbers 28, 29, or 30. In some embodiments, a pharmaceutical composition according to any one of the above claims, wherein the anti-Claudin 18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any one of the following claims, i.e. (xi) The heavy chain variable region is indicated by Sequence ID 31, and the light chain variable region is indicated by Sequence ID 29, or (xii) The heavy chain variable region is shown in Sequence ID 26, and the light chain variable region is shown in Sequence ID 23.

[0019] In some embodiments, the pharmaceutical composition described in any one of the above claims, wherein the anti-Claudin18.2 antibody comprises an antibody heavy chain constant region and a light chain constant region.

[0020] In some embodiments, the heavy chain constant region is selected from the human IgG1, IgG2, IgG3, and IgG4 constant regions and their normal variants, and the light chain constant region is selected from the human antibody κ and λ chain constant regions and their normal variants. In some embodiments, the antibody includes the heavy chain constant region shown in SEQ ID NO: 7 and the light chain constant region shown in SEQ ID NO: 8.

[0021] In some embodiments, the antibody has a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the heavy chain having the amino acid sequence shown in SEQ ID NO: 35 or 42, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the light chain having the amino acid sequence shown in SEQ ID NO: 36 or 39. A light chain comprising a heavy chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a heavy chain having the amino acid sequence shown in SEQ ID NO: 37 or 49, and a light chain having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with a light chain having the amino acid sequence shown in SEQ ID NO: 38 or 46.

[0022] In some embodiments, the pharmaceutical composition described in any one of the above clauses, wherein the anti-Claudin 18.2 antibody comprises the heavy chain and light chain shown in any one of the following clauses, i.e. (c) A heavy chain whose sequence is shown in sequence number 35, and a light chain whose sequence is shown in sequence number 36, (d) A heavy chain whose sequence is shown in sequence number 42, 43, 44 or 45, and a light chain whose sequence is shown in sequence number 39, 40 or 41, (e) A heavy chain whose sequence is shown in sequence number 37, and a light chain whose sequence is shown in sequence number 38, or (f) A heavy chain whose sequence is shown in sequence number 49, 50, 51 or 52, and a light chain whose sequence is shown in sequence number 46, 47 or 48, Includes.

[0023] In some embodiments, the pharmaceutical composition described in any one of the above clauses, wherein the anti-Claudin 18.2 antibody comprises the heavy chain and light chain shown in any one of the following clauses, i.e. The heavy chain indicated by Sequence ID No. 44, and the light chain indicated by Sequence ID No. 41, or The heavy chain indicated by Sequence ID No. 49, and the light chain indicated by Sequence ID No. 47, Includes.

[0024] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-L-Y-D), that is,

Chemical formula

[0025] In some embodiments, the pharmaceutical composition described in any one of the above items, wherein the anti-Claudin 18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-LYD), Eventually, Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-, R a and R b They are homologous or homologous, and each is independently a hydrogen atom, a deuterium atom, a halogen, or C 1-6 Selected from alkyl groups, R 1 is halogenated C 1-6 Alkyl alkyl group or C 3-6 It is a cycloalkyl group, R 2 This is a hydrogen atom, halogen C 1-6 Alkyl alkyl group or C3-6 Selected from cycloalkyl groups, Alternatively, R 1 and R 2 C 3-6 Forms a cycloalkyl group, m is either 0 or 1.

[0026] In some embodiments, the pharmaceutical composition described in any one of the above claims, wherein the anti-Claudin 18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-LYD), and Y is [ka] Selected from, Of these, the O end of Y is connected to the linker unit L.

[0027] In some embodiments, the pharmaceutical composition according to any one of the above claims, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -and, L 1 is -(succinimido-3-yl-N)-WC(O)-,-CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, where W is C 1-8 Alkyl alkyl group, C 1-8 Selected from alkyl-cycloalkyl groups or linear heteroalkyl groups having 1 to 8 chain atoms, the heteroalkyl group contains 1 to 3 heteroatoms selected from N, O, or S, of which C 1-8 Alkyl alkyl group, C 1-8 Alkyl-cycloalkyl groups or linear heteroalkyl groups having 1 to 8 chain atoms can each be independently and optionally combined with a halogen, hydroxyl group, cyano group, amino group, or C. 1-6 alkyl groups, chloro 1-6 Alkyl groups, deuterated C 1-6 Alkyl alkyl group, C 1-6 Alkoxy groups and C 3-6further substituted with one or more substituents selected from cycloalkyl groups, L 2 is selected from -NR 4 (CH2CH2O)p 1 CH2CH2C(O)-, -NR 4 (CH2CH2O)p 1 CH2C(O)-, -S(CH2)p 1 C(O)- or a chemical bond, wherein p 1 is an integer from 1 to 20, L 3 is a peptide residue composed of 2 to 7 amino acids, wherein the amino acid residues are amino acid residues formed from amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and optionally further substituted with one or more substituents selected from halogen, hydroxy group, cyano group, amino group, C 1-6 alkyl group, chloro C 1-6 alkyl group, deuterated C 1-6 alkyl group, C 1-6 alkoxy group and C 3-6 cycloalkyl group, L 4 is selected from -NR 5 (CR 6 R 7 ) t -, -C(O)NR 5 -, -C(O)NR 5 (CH2) t - or a chemical bond, wherein t is an integer from 1 to 6, R 3 R 4 and R 5 are the same or different and each independently is a hydrogen atom, C 1-6 alkyl group, halogenated C 1-6 alkyl group, deuterated C 1-6 alkyl group and C 1-6 hydroxyalkyl group, R 6 and R 7 are the same or different and each independently is a hydrogen atom, halogen, C 1-6 alkyl group, halogenated C1-6 An alkyl group, deuterated C 1-6 An alkyl group and C 1-6 Selected from a hydroxyalkyl group.

[0028] In some embodiments, the pharmaceutical composition according to any one of the above, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -, L 1 is

Chemical formula

[0029] In some embodiments, the pharmaceutical composition according to any one of the above, wherein -L- is

Chemical formula

[0030] In some embodiments, the pharmaceutical composition according to any one of the above, wherein -L-Y- is optionally

Chemical formula

[0031] In some embodiments, the pharmaceutical composition described in any one of the above clauses, wherein the anti-Claudin 18.2 antibody-drug conjugate has the structure shown in any one of the following clauses, namely, [ka] Of these, Pc and n are defined as shown in the general formula (Pc-LYD).

[0032] In some embodiments, the pharmaceutical composition described in any one of the above claims, wherein the anti-Claudin 18.2 antibody-drug conjugate has a structure represented by the following formula, namely, [ka] Eventually, n is between 2 and 8, and n can be a decimal or an integer. Pc is an anti-Claudin 18.2 antibody.

[0033] In some embodiments, the pharmaceutical composition is as described in any one of the above paragraphs, wherein the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is polysorbate (e.g., polysorbate 20, polysorbate 80), poloxamer, triton, sodium dodecylsulfonate, sodium dodecyl sulfate, sodium octylglucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleum sulfobetaine, stearin sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleum sarcosine, stearin sarcosine, linoleum betaine, myristyl betaine, cetyl betaine. These are selected from lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine, isostearamidopropyl dimethylamine, methyl cocoyl sodium, methyl oleyl taurate sodium, polyethylene glycol, polypropylene glycol, copolymers of ethylene and propylene glycol, etc.

[0034] In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 80 or polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0035] In some embodiments, the pharmaceutical composition is as described in any one of the above items, wherein the concentration of the surfactant is 0.05 mg / mL to 0.5 mg / mL or 0.1 mg / mL to 0.2 mg / mL. In some embodiments, the concentration of the surfactant is 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.3 mg / mL, 0.4 mg / mL, or 0.5 mg / mL. In some embodiments, the concentration of the surfactant is 0.2 mg / mL.

[0036] In some embodiments, the pharmaceutical composition is as described in any one of the above, wherein the composition further comprises a sugar. In some embodiments, the sugar is a conventional composition (CH2O) n The sugars are selected from and their derivatives, and include monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc. The sugars may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, melibiose, melegitose, raffinose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltose alcohol, lactose alcohol, isomaltulose, etc. In some embodiments, the sugar is selected from sucrose, mannitol and trehalose. In some embodiments, the sugar is sucrose.

[0037] In some embodiments, the pharmaceutical composition is as described in any one of the above items, wherein the concentration of the sugar is 20 mg / mL to 100 mg / mL or 40 mg / mL to 80 mg / mL. In some embodiments, the concentration of the sugar is 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL. In some embodiments, the concentration of the sugar is 40 mg / mL.

[0038] In some embodiments, the pharmaceutical composition is as described in any one of the above items, wherein the concentration of the antibody-drug conjugate is 1 mg / mL to 100 mg / mL in terms of protein (i.e., antibody) concentration or 10 mg / mL to 30 mg / mL in terms of protein concentration. In some embodiments, the concentration of the antibody-drug conjugate is 5 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL in terms of protein concentration. In some embodiments, the concentration of the drug conjugate is 20 mg / mL in terms of protein concentration.

[0039] In some embodiments, the pharmaceutical composition is as described in any one of the above items, wherein the concentration of the buffer is 5 mM to 50 mM or 10 mM to 30 mM. In some embodiments, the concentration of the buffer is 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In some embodiments, the concentration of the buffer is 30 mM.

[0040] In some embodiments, the pharmaceutical composition is as described in any one of the above items, wherein the pH of the pharmaceutical composition is 5.0 to 6.5 or 5.0 to 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.4. In some embodiments, the pH of the pharmaceutical composition is 5.0 to 5.3.

[0041] In some embodiments, a pharmaceutical composition according to any one of the above claims, comprising the following components, namely, The pharmaceutical composition comprises (a) the above anti-Claudin 18.2 antibody drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) 0.1 mg / mL to 0.2 mg / mL of polysorbate, (c) 40 mg / mL to 80 mg / mL of sugar, and (d) 10 mM to 30 mM of histidine salt buffer, with a pH of approximately 5.0 to 5.5.

[0042] In some embodiments, a pharmaceutical composition according to any one of the above claims, comprising the following components, namely, The pharmaceutical composition comprises (a) the above anti-Claudin 18.2 antibody drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) polysorbate 80 at 0.1 mg / mL to 0.2 mg / mL, (c) sucrose at 40 mg / mL to 80 mg / mL, and (d) a histidine salt buffer at 10 mM to 30 mM, with a pH of approximately 5.0 to 5.5.

[0043] In some embodiments, a pharmaceutical composition according to any one of the above claims, comprising the following components, namely, The pharmaceutical composition comprises (a) the above anti-Claudin 18.2 antibody drug conjugate at a protein concentration of 20 mg / mL, (b) 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL of sucrose, and (d) 30 mM of histidine acetate buffer, and the pH of the above pharmaceutical composition is 5.0 to 5.3.

[0044] In some embodiments, the pharmaceutical composition according to any one of the above claims, wherein the anti-Claudin 18.2 antibody-drug conjugate has a structure represented by the following formula, i.e., [ka] Eventually, n is between 2 and 8, and n can be a decimal or an integer. Pc is an anti-Claudin18.2 antibody comprising a heavy chain indicated by SEQ ID NO: 49 and a light chain indicated by SEQ ID NO: 47, and the above pharmaceutical composition contains the above antibody-drug conjugate at a protein concentration of 20 mg / mL. The above pharmaceutical composition contains the following components, namely, The pharmaceutical composition contains 0.2 mg / mL of polysorbate 80, 40 mg / mL of sucrose, and 30 mM of histidine acetate buffer, and the pH of the above pharmaceutical composition is 5.0 to 5.3.

[0045] In some embodiments, the pharmaceutical composition is as described in any one of the above paragraphs, wherein the pharmaceutical composition is a liquid formulation. In some embodiments, the solvent of the liquid formulation is water.

[0046] This disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, characterized in that it can form the pharmaceutical composition described in any one of the above items after being redissolved.

[0047] This disclosure further provides lyophilized formulations which are lyophilized forms of the pharmaceutical compositions described in any one of the above paragraphs.

[0048] This disclosure further provides a method for preparing a lyophilized formulation containing an antibody-drug conjugate, the method comprising the step of lyophilizing a pharmaceutical composition described in any one of the above items.

[0049] This disclosure further provides a lyophilized formulation comprising an antibody-drug conjugate, obtained by lyophilizing a pharmaceutical composition described in any one of the above items.

[0050] In some embodiments, the freeze-drying described in any one of the above paragraphs includes, in order, the steps of pre-freezing, primary drying, and secondary drying.

[0051] In some embodiments, the freeze-drying procedure is as follows: pre-freezing at a temperature of 5°C, pre-freezing at a temperature of -45°C, primary drying at a temperature of -20°C and a vacuum of 20 Pa, and secondary drying at a temperature of 25°C and a vacuum of 1 Pa. In some embodiments, the freeze-drying procedure is as follows: pre-freezing at a temperature of 5°C for 10 min, pre-freezing at a temperature of -45°C for 50 min, primary drying at a temperature of -20°C and a vacuum of 20 Pa for 120 min, and secondary drying at a temperature of 25°C and a vacuum of 1 Pa for 60 min.

[0052] In some embodiments, the lyophilized formulation is stable at 2°C to 8°C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 28 days.

[0053] This disclosure further provides a lyophilized preparation that is a redissolved form of the lyophilized preparation described in any one of the above paragraphs.

[0054] This disclosure further provides a redissolution solution containing an antibody-drug conjugate, characterized in that it is obtained by redissolving a lyophilized formulation as described in any one of the above items.

[0055] In some embodiments, the above redissolving solution comprises the following components, namely, The redissolution solution comprises (a) the above anti-Claudin 18.2 antibody drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) 0.1 mg / mL to 0.2 mg / mL of polysorbate, (c) 40 mg / mL to 80 mg / mL of sugar, and (d) 10 mM to 30 mM of histidine salt buffer, with a pH of 5.0 to 5.5.

[0056] In some embodiments, the above redissolving solution comprises the following components, namely, The redissolution solution comprises (a) the above anti-Claudin 18.2 antibody drug conjugate at a protein concentration of 10 mg / mL to 30 mg / mL, (b) 0.1 mg / mL to 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL to 80 mg / mL of sucrose, and (d) 10 mM to 30 mM of histidine salt buffer, with a pH of 5.0 to 5.5.

[0057] In some embodiments, the above redissolving solution comprises the following components, namely, The redissolution solution comprises (a) the above anti-Claudin 18.2 antibody drug conjugate at a protein concentration of 20 mg / mL, (b) 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL of sucrose, and (d) 30 mM of histidine acetate buffer, with a pH of 5.0 to 5.3.

[0058] This disclosure further provides a product comprising a container containing the pharmaceutical composition described in any one of the above paragraphs, the lyophilized preparation described in any one of the above paragraphs, or the redissolving solution described in any one of the above paragraphs.

[0059] This disclosure further provides a method for treating a tumor or cancer, comprising administering to a subject an effective amount of the pharmaceutical composition, the lyophilized preparation, the redissolving solution, or the product described in any one of the above paragraphs.

[0060] In some embodiments, the Disclosure further provides the use of the pharmaceutical composition, lyophilized formulation, redissolvable solution, or product described in any one of the above clauses in the preparation of agents for treating tumors or cancer.

[0061] In some embodiments, the Disclosure further provides a pharmaceutical composition, a lyophilized preparation, a re-dissolving solution, or a product as described in any one of the above, which is used as a pharmaceutical agent.

[0062] In some embodiments, the tumor or cancer is preferably squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kruckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma.

[0063] In some embodiments, the lymphoma is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma.

[0064] In some embodiments, the lung cancer is selected from non-small cell lung cancer and small cell lung cancer.

[0065] In some embodiments, the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid leukemia. [Brief explanation of the drawing]

[0066] [Figure 1] This is the result of FACS detection of the binding of humanized antibodies to human Claudin 18.2 at the cellular level. [Figure 2] This is an experiment involving the endocytosis of NUGC4 cells with humanized antibodies. [Figure 3A-3C]This study detects the ADCC effect of antibodies in NUGC4 cells with different Claudin18.2 expression levels. Figure 3A shows the detection of the ADCC effect of antibodies in wild-type NUGC4 cells (low Claudin18.2 expression), Figure 3B shows the detection of the ADCC effect of antibodies in NUGC4 cells with moderate Claudin18.2 expression, and Figure 3C shows the detection of the ADCC effect of antibodies in NUGC4 cells with high Claudin18.2 expression. [Figure 4] These are the results of tumor inhibition experiments with ADC-1 as disclosed herein. [Figure 5] These are the results of tumor inhibition experiments with ADC-2 as disclosed herein. [Modes for carrying out the invention]

[0067] term To make this disclosure more easily understood, several technical and scientific terms are defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings that are ordinarily understood by those skilled in the art.

[0068] Antibody-drug conjugates (ADCs) link an antibody or antibody fragment to a bioactive cytotoxin or a small molecule drug with cytotoxic activity via a stable chemical linker compound. They leverage the antibody's specificity towards tumor cells or its binding specificity to highly expressed antigens, as well as the high efficacy of the cytotoxin, while avoiding toxicity and side effects on normal cells. Compared to conventional chemotherapy drugs, antibody-drug conjugates can accurately bind to tumor cells while reducing their impact on normal cells.

[0069] A "buffering agent" refers to a buffering agent that can withstand changes in pH due to the action of its acid-base conjugated components. Examples of buffering agents that control pH within an appropriate range include acetates, succinates, glucons, histidine salts, oxalates, lactates, phosphates, goji phosphates, tartrates, fumarates, glycylglycine, and other organic acid buffering agents.

[0070] A "histidine salt buffer" is a buffer containing histidine ions. Examples of histidine salt buffers include histidine hydrochloride, histidine acetate, histidine phosphate, and histidine sulfate, with histidine acetate buffers being preferred. Histidine acetate buffers are prepared from histidine and acetic acid, while histidine hydrochloride buffers are prepared from histidine and hydrochloric acid.

[0071] A "glutinate buffer" is a buffer containing glutinous acid ions. Examples of glutinous acid buffers include glutinous acid-sodium glutinous acid, glutinous acid-potassium glutinous acid, glutinous acid-calcium glutinous acid, and glutinous acid-magnesium glutinous acid. The preferred glutinous acid buffer is glutinous acid-sodium glutinous acid.

[0072] A "succinate buffer" is a buffer containing succinate ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, and succinate-calcium succinate salts. A preferred succinate buffer is succinate-sodium succinate. Exemplarily, the above succinate-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0073] A "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-sodium dihydrogen acid. The preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0074] "Acetate buffers" are buffers containing acetate ions. Examples of acetate buffers include sodium acetate, histidine acetate, potassium acetate, calcium acetate, and magnesium acetate. A preferred acetate buffer is sodium acetate.

[0075] "Pharmaceutical composition" refers to a mixture comprising one or more antibody-drug conjugates described herein or their physiologically / pharmaceutically acceptable salts or prodrugs, and other chemical components such as physiologically / pharmaceutically acceptable vectors or excipients. The pharmaceutical composition is intended to maintain the stability of the active ingredient, facilitate administration to the body, and contribute to the absorption of the active ingredient in order to exert further biological activity.

[0076] In this disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0077] Unless otherwise specified, the solvent in the solutions of the pharmaceutical compositions described herein is water.

[0078] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form, or a formulation or pharmaceutical composition obtained after a vacuum lyophilization process has been performed on a liquid or solution formulation.

[0079] While this disclosure provides content ranges or content values, it will be understood by those skilled in the art that such content ranges or content values ​​cover the permissible error range of the specific values ​​measured.

[0080] The pharmaceutical compositions described herein can achieve stable effects, meaning that the antibody-drug conjugates therein essentially retain their physical and / or chemical stability and / or biological activity after storage, and preferably, the pharmaceutical compositions essentially retain their physical and chemical stability and biological activity after storage. The storage period is generally selected according to the predetermined shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability, and it is possible to measure stability after storage at a selected temperature for a selected period.

[0081] A stable formulation is one that shows no significant changes when stored at refrigerated temperatures (2°C to 8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably 2 years. A stable liquid formulation also includes liquid formulations that exhibit desired characteristics after storage at temperatures including 25°C for periods including 1 month, 3 months, and 6 months. A typical example of stability is that SEC-HPLC measures that the amount of antibody monomers that have undergone aggregation or degradation does not exceed approximately 10%, preferably approximately 5%. Visual analysis reveals that the formulation is a pale yellow, nearly colorless, transparent liquid, or colorless, clear, or slightly milky white. The concentration, pH, and molar osmotic pressure of the above formulation do not change by more than ±10%. Typically, a decrease of approximately 10%, preferably approximately 5%, is observed. Typically, aggregation of approximately 10%, preferably approximately 5%, is formed.

[0082] If the antibody-drug conjugate does not show significant aggregation, precipitation, and / or denaturation when detected visually by color and / or clarity, or when measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS), then the antibody-drug conjugate is considered to "maintain its physical stability" in the drug formulation. Changes in protein conformation may be evaluated by fluorescence spectroscopy (to determine the tertiary structure of the protein) and FTIR spectroscopy (to determine the secondary structure of the protein).

[0083] If the antibody-drug conjugate does not show significant chemical changes, the antibody is considered to "maintain its chemical stability" in the drug formulation. Chemical stability can be evaluated by detecting and quantifying proteins whose chemical morphology has been altered. Degradation processes that constantly change the chemical structure of proteins include hydrolysis or cleavage (e.g., evaluated by methods such as size exclusion chromatography or CE-SDS), oxidation (e.g., evaluated by methods such as mass spectrometry or peptide mapping coupled with MALDI / TOF / MS), deamidation (e.g., evaluated by methods such as ion exchange chromatography, electrophoresis including capillary electrophoresis, peptide mapping, and measurement of isoaspartic acid), and isomerization (e.g., evaluated by measurement of isoaspartic acid content, peptide mapping, etc.).

[0084] If the biological activity of an antibody-drug conjugate at a predetermined time falls within a predetermined range of the biological activity shown at the time of preparation of the drug formulation, then the antibody-drug conjugate is said to "retain its biological activity" in the drug formulation.

[0085] The three-letter and one-letter amino acid codes used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0086] The term “antibody” as used herein is used in its broadest sense, as long as it exhibits the desired antigen-binding activity, and covers a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, or their antigen-binding fragments (also called “antigen-binding moieties”). A full-length antibody is an immunoglobulin (Ig) containing at least two heavy chains and two light chains linked to each other by disulfide bonds. Immunoglobulins differ in their antigenicity due to differences in the amino acid composition and sequence order of the heavy chain constant region. Thus, immunoglobulins can be classified into five types, or immunoglobulin isotypes, IgM, IgD, IgG, IgA, and IgE, with their corresponding heavy chains being the μ, δ, γ, α, and ε chains, respectively. Ig of the same type can be further divided into different subclasses based on differences in the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds. For example, IgG may be divided into IgG1, IgG2, IgG3, and IgG4. The light chain can be divided into a κ chain or a λ chain depending on the constant region. Each of the five types of Ig may have either a κ chain or a λ chain.

[0087] In full-length antibodies, the heavy and light chains have a variable region (abbreviated as Fv region) with significant changes in the amino acid sequence of approximately 110 amino acids near the N-terminus, while the remaining amino acid sequence near the C-terminus is relatively stable and forms the constant region. Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region contains three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL). The heavy and light chain variable regions contain a hypervariable region (also called the complementarity-determining region, abbreviated as CDR or HVR) and a relatively conserved skeletal region (also called the framework region, abbreviated as FR). Each VL and VH consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3, while the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.

[0088] The "normal variants" of the human antibody heavy chain constant region and human antibody light chain constant region described in this disclosure refer to variants of the human-derived heavy chain constant region or light chain constant region that do not alter the structure and function of the antibody variable region disclosed in the prior art. Exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that have undergone site-directed modification and amino acid substitution of the heavy chain constant region. Specific substitutions include, for example, the known YTE mutations in the prior art, L234A and / or L235A mutations, S228P mutations, 265A (e.g., D265A) and / or 297A (e.g., N297A), and / or mutations that result in a knob-into-hole structure (conferring a combination of knob-Fc and hole-Fc to the antibody heavy chain). These mutations have been confirmed to confer new properties to the antibody without altering the function of the antibody variable region.

[0089] The terms “antigen-binding fragment,” “functional fragment,” or “antigen-binding moiety” refer to one or more fragments of a complete antibody that retain the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can perform the antigen-binding function of an antibody. Exemplary examples of binding fragments included in the term “antigen-binding fragment” include (i) a monovalent fragment, the Fab fragment, consisting of VL, VH, CL, and CH1 domains; (ii) a bivalent fragment, the F(ab')2 fragment, containing two Fab fragments linked by disulfide crosslinks in the hinge region; (iii) an Fd fragment, consisting of VH and CH1 domains; (iv) an Fv fragment, consisting of the VH and VL domains of a single arm of the antibody; (v) a stable antigen-binding fragment, the dsFv, formed by interchain disulfide bonds between VH and VL; and (vi) biantibodies, bispecific antibodies, and multispecific antibodies containing fragments such as scFv, dsFv, and Fab. The two domains of the Fv fragment, VL and VH, are encoded by separate genes, but through recombination, these two domains can be linked by an artificial peptide linker that makes it possible to combine them into a single protein chain. Of these, the VL and VH regions pair up to form a monovalent molecule called single-chain Fv (scFv) (see, for example, Bird et al. (1988) Science 242:423~426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879~5883). Such single-chain antibodies are also included in the term "antigen-binding fragment" of antibodies. Such antibody fragments can be obtained by conventional techniques known to those skilled in the art, and like complete antibodies, the fragments are screened for functionality. The antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of complete immunoglobulins.

[0090] The terms "amino acid difference" or "amino acid mutation" refer to changes or mutations in amino acids in a mutant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one, two, three, or more amino acids based on the original protein or polypeptide.

[0091] The terms "antibody framework" or "FR region" refer to a portion of a variable domain (VL) or (VH) used as a stent for the antigen-binding ring (CDR) of that variable domain. In effect, it is a variable domain without a CDR.

[0092] The terms "complementarity-determining region," "CDR," or "hypervariable region" refer to one of the six main hypervariable regions in the variable domain of an antibody that promote antigen binding. Typically, each heavy chain variable region has three CDRs (HCDR1, HCDR2, HCDR3), and each light chain variable region has three CDRs (LCDR1, LCDR2, LCDR3). The amino acid sequence boundaries of CDRs can be determined by any one of several known methods, including the "Kabat" numbering system (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering system (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering system (see Lefranc MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003)). For example, in a typical format, according to the Kabat rule, the CDR amino acid residue numbers in the heavy chain variable region (VH) are 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and the CDR amino acid residue numbers in the light chain variable region (VL) are 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to the Chothia rule, the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3), and the amino acid residue numbers in VL are 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3).According to the combined Kabat and Chothia definition of CDR, the CDR consists of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. According to IMGT rules, the CDR amino acid residue numbers in VH are approximately 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3). According to IMGT rules, the CDR region of an antibody can be determined by the program IMGT / DomainGap Align. According to the AbM rules, the CDR amino acid numbers in VH are 26-32 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3), and the amino acid residue numbers in VL are 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). The heavy chain variable region and light chain variable region and their CDRs of the antibodies disclosed herein conform to the Kabat numbering rules.

[0093] "Sequence identity" of amino acids refers to the percentage of identical amino acid residues in the first and second sequences during amino acid sequence alignment (with gaps introduced as needed to maximize the percentage of sequence identity), where conservative substitutions are not considered part of sequence identity. To measure the percentage of amino acid sequence identity, alignment can be performed using various methods within the technical scope of this art, such as publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters applied to the measurement alignment, including any algorithm necessary to achieve maximum alignment over the entire length of the sequences being compared.

[0094] The engineered antibodies or antigen-binding fragments relating to this disclosure can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into a GS expression vector. The recombined immunoglobulin expression vector can stably transfect CHO cells. In a more preferred prior art, mammalian expression systems induce antibody glycosylation, particularly at the highly conserved N-terminal region of the Fc area. By expressing antibodies that bind to the antigen, stable clones can be obtained. Positive clones produce antibodies by expanding the culture in serum-free medium in a bioreactor. The culture medium from which the antibodies have been secreted can be purified by conventional methods. For example, purification is performed using an A or G Sepharose FF column containing a prepared buffer. Nonspecifically bound components are washed away. Furthermore, bound antibodies are eluted by a pH gradient method, and antibody fragments are detected and collected by SDS-PAGE. The antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers may be removed by conventional methods such as molecular sieving or ion exchange. The resulting product must be immediately frozen, for example, at -70°C, or freeze-dried.

[0095] "Conservative modification" or "conservative substitution" means substituting an amino acid in a protein with another amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, back chain conformation and rigidity) to allow the protein to be frequently modified without altering its biological activity. As is known to those skilled in the art, generally, a single amino acid substitution in a non-essential region of a polypeptide does not fundamentally alter its biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p. 224, (4th edition)). Furthermore, substitutions of amino acids with similar structure or function are less likely to disrupt biological activity. Exemplary conservative substitutions are as follows:

[0096] [Table 1]

[0097] The term "naked antibody" refers to an antibody that is not conjugated with a heterogeneous module (e.g., a cytotoxic module) or a radioactive marker. In this disclosure, the content of an antibody-drug conjugate is measured by protein concentration, i.e., the weight / volume of protein (antibody portion) in the conjugate.

[0098] The terms “linker unit” or “linker” refer to a chemical structural fragment or linkage in which one end is linked to an antibody or its antigen-binding fragment and the other end is linked to a drug, which may be linked to another linker before being linked to the drug. Preferred forms of the present disclosure are L and L 1 ~L 4 It was shown that, among them, L 1 The end is linked to the antibody, L 4 The terminus is linked to structural unit Y and then to a compound or toxin. The linker comprises an elongator, a spacer, and an amino acid unit, and can be synthesized by known methods in the art, such as the method described in US2005-0238649A1. The linker may be a “cleavable linker” that facilitates drug release in cells. For example, an acid-unstable linker (e.g., hydrazone), a protease-sensitive (e.g., peptidase-sensitive) linker, a photo-unstable linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Research 52:127-131 (1992), U.S. Patent No. 5,208,020) may be used.

[0099] The term "alkyl group" refers to a saturated aliphatic hydrocarbon group that is a linear or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 10 carbon atoms, and most preferably an alkyl group containing 1 to 6 carbon atoms. The non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, n-heptyl group, 2-methylhexyl group, 3-methylhexyl group, 4-methylhexyl group, 5-methylhexyl Syl group, 2,3-dimethylpentyl group, 2,4-dimethylpentyl group, 2,2-dimethylpentyl group, 3,3-dimethylpentyl group, 2-ethylpentyl group, 3-ethylpentyl group, n-octyl group, 2,3-dimethylhexyl group, 2,4-dimethylhexyl group, 2,5-dimethylhexyl group, 2,2-dimethylhexyl group, 3,3-dimethylhexyl group, 4,4-dimethylhexyl group, 2 This includes -ethylhexyl group, 3-ethylhexyl group, 4-ethylhexyl group, 2-methyl-2-ethylpentyl group, 2-methyl-3-ethylpentyl group, n-nonyl group, 2-methyl-2-ethylhexyl group, 2-methyl-3-ethylhexyl group, 2,2-diethylpentyl group, n-decyl group, 3,3-diethylhexyl group, 2,2-diethylhexyl group, and various branched isomers thereof.More preferably, the lower alkyl group contains 1 to 6 carbon atoms, and non-limiting examples include methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, n-pentyl group, 1,1-dimethylpropyl group, 1,2-dimethylpropyl group, 2,2-dimethylpropyl group, 1-ethylpropyl group, 2-methylbutyl group, 3-methylbutyl group, n-hexyl group, 1-ethyl-2-methylpropyl group, 1,1,2-trimethylpropyl group, 1,1-dimethylbutyl group, 1,2-dimethylbutyl group, 2,2-dimethylbutyl group, 1,3-dimethylbutyl group, 2-ethylbutyl group, 2-methylpentyl group, 3-methylpentyl group, 4-methylpentyl group, 2,3-dimethylbutyl group, and the like. The alkyl group may be substituted or not, and if substituted, the substituent may be substituted at any available linking site, and preferably the substituent is independently one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0100] The term "heteroalkyl group" refers to an alkyl group containing one or more heteroatoms selected from N, O, or S, where the alkyl group is as defined above.

[0101] The term "alkylene group" refers to a saturated linear or branched aliphatic hydrocarbon group having two residues derived by removing two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. An alkylene group is a linear or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 12 carbon atoms, and more preferably containing 1 to 6 carbon atoms. Non-restrictive examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylidene (-CH(CH3)-), 1,2-ethylidene (-CH2CH2)-, 1,1-propyridene (-CH(CH2CH3)-), 1,2-propyridene (-CH2CH(CH3)-), 1,3-propyridene (-CH2CH2CH2-), 1,4-butylidene (-CH2CH2CH2CH2-), and 1,5-butylidene (-CH2CH2CH2CH2CH2-). Alkylene groups may or may not be substituted. When substitution occurs, the substituent may be substituted at any available linking point, and it is preferable that the substituent is independently and optionally substituted with one or more groups selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocyclyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0102] The term "alkoxy group" refers to -O-(alkyl group) and -O-(unsubstituted cycloalkyl group), with the definitions of alkyl group and cycloalkyl group being as described above. Non-restrictive examples of alkoxy groups include methoxy group, ethoxy group, propoxy group, butoxy group, cyclopropoxy group, cyclobutoxy group, cyclopentyloxy group, and cyclohexyloxy group. Alkoxy groups may be optionally substituted or unsubstituted, and if substituted, the substituent is preferably one or more groups independently selected from alkyl group, alkenyl group, alkynyl group, alkoxy group, alkylthio group, alkylamino group, halogen, mercapto group, hydroxyl group, nitro group, cyano group, cycloalkyl group, heterocycloalkyl group, aryl group, heteroaryl group, cycloalkoxy group, heterocycloalkoxy group, cycloalkylthio group, and heterocycloalkylthio group.

[0103] The term "cycloalkyl group" refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituents. A cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 7 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, and cyclooctyl groups, while polycyclic cycloalkyl groups include cycloalkyl groups of spiro rings, fused rings, and crosslinked rings.

[0104] The term "heterocyclyl group" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, of which one or more ring atoms are nitrogen, oxygen, or S(O). mThe heteroatoms are selected from (where m is an integer from 0 to 2), but do not contain the -OO-, -OS-, or -SS- ring portion, and the remaining ring atoms are carbon. A heterocyclyl group preferably contains 3 to 12 ring atoms, of which 1 to 4 are heteroatoms, and a more preferred cycloalkyl ring contains 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, and homopiperazinyl groups. Polycyclic heterocyclyl groups include spiro-ring, fused ring, and bridging ring heterocyclyl groups.

[0105] The term "spiroheterocyclyl group" refers to a polycyclic heterocyclyl group with 5 to 20 members in which two monocyclic rings share one atom (called a spiro atom), and one or more of these ring atoms are nitrogen, oxygen, or S(O). m The heteroatom is selected from (where m is an integer from 0 to 2), and the remaining ring atom is carbon. It may contain one or more double bonds, but there are no rings with a fully conjugated π-electron system. Preferably it is 6- to 14-membered, and more preferably 7- to 10-membered. The spiroheterocyclyl group is divided into a monospiroheterocyclyl group, a bisspiroheterocyclyl group, or a polyspiroheterocyclyl group depending on the number of spiroatoms shared between the rings, preferably a monospiroheterocyclyl group and a bisspiroheterocyclyl group. More preferably it is a 4-membered / 4-membered, 4-membered / 5-membered, 4-membered / 6-membered, 5-membered / 5-membered, or 5-membered / 6-membered monospiroheterocyclyl group. Non-limiting examples of spiroheterocyclyl groups are: [ka]

[0106] The term "condensed heterocyclyl group" refers to a 5- to 20-membered polycyclic heterocyclyl system in which each ring shares pairs of adjacent atoms with other rings in the system. In a condensed heterocyclyl group, one or more rings may contain one or more double bonds, but none of the rings have a fully conjugated π-electron system, and one or more of the ring atoms are nitrogen, oxygen, or S(O). m The heteroatom is selected from (where m is an integer from 0 to 2), and the remaining ring atom is carbon. The condensed heterocyclyl group is preferably 6- to 14-membered, and more preferably 7- to 10-membered. Depending on the number of rings that make up the group, the condensed heterocyclyl group can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic or tricyclic, and more preferably a 5-membered / 5-membered or 5-membered / 6-membered bicyclic condensed heterocyclyl group. Non-limiting examples of condensed heterocyclyl groups are: [ka] Includes.

[0107] The term "crosslinked cycloalkyl group" refers to a 5- to 14-membered polycyclic heterocyclyl group in which any two rings share two atoms that are not directly linked. A crosslinked cycloalkyl group may contain one or more double bonds, but none of the rings have a fully conjugated π-electron system, and one or more of the ring atoms are nitrogen, oxygen, or S(O). m The heteroatom is selected from (where m is an integer from 0 to 2), and the remaining ring atom is carbon. The crosslinked cycloalkyl group is preferably 6-14 membered, more preferably 7-10 membered. Depending on the number of rings it comprises, the crosslinked cycloalkyl group can be classified as bicyclic, tricyclic, tetracyclic, or polycyclic, preferably bicyclic, tricyclic, or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of crosslinked heterocyclyl groups are: [ka]

[0108] The above heterocyclyl ring may be condensed with an aryl group, a heteroaryl group, or a cycloalkyl ring, of which the ring linked to the parent structure is a heterocyclyl group, and non-limiting examples include: [ka] This includes, among others.

[0109] The heterocyclyl group may be optionally substituted or left unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, mercapto groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, heterocycloalkylthio groups, and oxo groups.

[0110] The term "aryl group" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic group (i.e., a ring sharing adjacent carbon atom pairs) having a conjugated π-electron system. The aryl group is preferably 6- to 10-membered, for example, a phenyl group and a naphthyl group, preferably a phenyl group. The above aryl ring may be fused to a heteroaryl group, a heterocyclyl group, or a cycloalkyl group, where the ring linked to the parent structure is an aryl ring, and non-limiting examples include: [ka] Includes.

[0111] The aryl group may or may not be substituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, sulfhydryl groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0112] The term "heteroaryl group" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, of which the heteroatoms are selected from oxygen, sulfur, and nitrogen. Heteroaryl groups are preferably 5 to 10 membered, more preferably 5 or 6 membered, and examples include furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, and tetrazolyl groups. The above heteroaryl ring may be condensed with an aryl group, a heterocyclyl group, or a cycloalkyl ring, and the ring linked to the parent structure is a heteroaryl ring, and non-limiting examples include: [ka] Includes.

[0113] The heteroaryl group may be optionally substituted or left unsubstituted. If substituted, the substituent is preferably one or more groups independently selected from alkyl groups, alkenyl groups, alkynyl groups, alkoxy groups, alkylthio groups, alkylamino groups, halogens, sulfhydryl groups, hydroxyl groups, nitro groups, cyano groups, cycloalkyl groups, heterocycloalkyl groups, aryl groups, heteroaryl groups, cycloalkoxy groups, heterocycloalkoxy groups, cycloalkylthio groups, and heterocycloalkylthio groups.

[0114] The term "amino protecting group" refers to a group that protects an amino group with an easily removable group so that the amino group is not altered when other parts of the molecule react. Non-limiting examples include the 9-fluorenylmethyloxycarbonyl group, the tert-butoxycarbonyl group, the acetyl group, the benzyl group, the allyl group, and the p-methoxybenzyl group. These groups can be optionally substituted with one to three substituents selected from halogens, alkoxy groups, or nitro groups. The 9-fluorenylmethyloxycarbonyl group is preferred as the amino protecting group.

[0115] The term "cycloalkylalkyl group" refers to a group in which one or more hydrogen atoms on an alkyl group are substituted with a cycloalkyl group, preferably one cycloalkyl group, where the alkyl group is as defined above, and the cycloalkyl group is as defined above.

[0116] The term "haloalkyl group" refers to an alkyl group in which one or more hydrogen atoms are substituted with halogens, where the alkyl group is as defined above.

[0117] The term "deuterated alkyl group" refers to an alkyl group in which one or more hydrogen atoms are substituted with deuterium atoms, where the alkyl group is as defined above.

[0118] The term "hydroxyl group" refers to the -OH group.

[0119] The term "halogen" refers to fluorine, chlorine, bromine, or iodine.

[0120] The term "amino group" refers to -NH2.

[0121] The term "nitro group" refers to -NO2.

[0122] "Optionally" or "optionally" means that the event or situation described thereafter may occur, but is not necessarily required, and this description includes both cases in which the event or situation occurs and cases in which it does not. For example, "optionally containing 1 to 3 antibody heavy chain variable regions" means that antibody heavy chain variable regions of a specific sequence may be present, but are not necessarily required.

[0123] "Substituting" means that one or more hydrogen atoms in a group, preferably five or fewer, more preferably one to three hydrogen atoms, are substituted by a number of substituents that correspond to each other independently. Of course, substituents can only be located at their chemically possible sites, and those skilled in the art can determine possible or impossible substitutions with little effort (by experiment or theory). For example, an amino group or hydroxyl group with free hydrogen can become unstable when bonded to a carbon atom with an unsaturated (e.g., olefin) bond.

[0124] The term "drug load" refers to the average amount of cytotoxic drug loaded onto each antibody or its antigen-binding fragment in an ADC molecule, and may be expressed as a ratio of drug amount to antibody amount, with each antibody or its antigen-binding fragment (Pc) being linked to 0 to 12, preferably 1 to 10, more preferably 2 to 8, and most preferably 3.5 to 4.5 cytotoxic drugs (D). In embodiments of this disclosure, the drug load is denoted by n, where an exemplary n may be one or more calculated average values ​​of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drug on each ADC molecule after the coupling reaction can be identified by conventional methods such as UV / Vis spectroscopy, mass spectrometry, ELISA, and HPLC.

[0125] In one embodiment of this disclosure, a cytotoxic drug is coupled by a linker unit to the N-terminal amino group of an antibody or its antigen-binding fragment, the ε-amino group of a lysine residue, and / or a mercapto group. Generally, in a coupling reaction, the number of drug molecules that can be coupled to the antibody is less than the theoretical maximum.

[0126] The loading dose of cytotoxic drugs is (1) Controlling the molar ratio of the linking reagent to the monoclonal antibody, (2) Controlling reaction time and temperature, (3) Selecting different reaction reagents, It can be controlled in a non-restrictive manner, including [mention specific methods].

[0127] The preparation of ordinary medicinal compositions is described in the Chinese Pharmacopoeia.

[0128] The term "vector," as used in the pharmaceutical compositions of this disclosure, refers to a system capable of altering how a drug enters and is distributed within the human body, controlling the rate of drug release, and transporting the drug to a target organ. The release and targeting systems of drug vectors can reduce drug degradation and loss, decrease side effects, and improve bioavailability. For example, polymeric surfactants as vectors, due to their unique amphiphilic structure, can self-assemble to form various forms of aggregates. Preferred examples include micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates have the ability to encapsulate drug molecules and exhibit good permeability to membranes, making them good drug vectors.

[0129] When applied to animals, humans, experimental subjects, cells, tissues, organs, or biofluids, “administration,” “giving,” and “processing” mean the contact of exogenous drugs, therapeutic agents, diagnostic agents, or compositions with animals, humans, subjects, cells, tissues, organs, or biofluids. “Administration,” “giving,” and “processing” may also mean, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods. Processing of cells includes contact between reagents and cells, and contact between reagents and fluids, where the fluid comes into contact with the cells. “Administration,” “giving,” and “processing” also mean the treatment of, for example, cells in vitro and in vitro with reagents, diagnostics, binding compositions, or through other types of cells. When applied to humans, veterinary medicine, or research subjects, “processing” means therapeutic treatment, preventive or precautionary measures, research, and diagnostic applications.

[0130] "Treatment" means, for example, administering an oral or topical therapeutic agent to a patient, the patient having one or more disease symptoms, and the known therapeutic agents having a therapeutic effect on these symptoms. Typically, the patient or group receiving treatment is given the therapeutic agent in a dose that effectively relieves one or more disease symptoms, thereby inducing the resolution of these symptoms or preventing them from progressing to any clinically measurable degree. The amount of therapeutic agent that effectively relieves any specific disease symptom (also called the "therapeutic dose") can vary depending on various factors, including the patient's disease state, age and weight, and the drug's ability to produce the desired therapeutic effect in the patient. Whether the disease symptoms have been reduced can be assessed by any clinical detection method commonly used by physicians or other professional healthcare providers to assess the severity and progression of the symptoms. Embodiments of the present disclosure (e.g., treatment methods or products) may be ineffective in alleviating each target disease symptom, but any statistical test known in the art, such as the Student t-test, chi-squared test, Mann and Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, should reduce the target disease symptoms in a statistically significant number of patients.

[0131] An "effective dose" includes an amount sufficient to improve or prevent the symptoms or condition of a medical disease. An effective dose also refers to an amount sufficient to allow or promote a diagnosis. The effective dose used for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's overall health, the method, route and dosage of administration, and the severity of side effects. The effective dose may also be the maximum dose or administration plan that avoids significant side effects or toxic effects.

[0132] "Substitution" refers to the substitution of the solvent system in which the antibody or ADC is dissolved. For example, a high-salt or high-osmotic-pressure solvent system containing the antibody or ADC is substituted in a buffer system that stabilizes the formulation by a physical manipulation method so that the antibody protein is present in a stable formulation. The above physical manipulation methods include, but are not limited to, ultrafiltration, dialysis, or redissolution after centrifugation.

[0133] Examples The present disclosure will be further explained below in conjunction with examples, but these examples are not intended to limit the scope of the present disclosure. In the examples of the present disclosure, experimental methods for which specific conditions are not specified generally follow the usual conditions, such as those described in the "Antibody Technology Experiment Manual" and the "Molecular Clone Manual" published by the Reizei Minato Laboratory, or the conditions recommended by the raw material or product manufacturer. Reagents for which the specific source is not specified are commercially available, ordinary reagents.

[0134] 1. Antibody-drug conjugates Preparation of anti-Claudin18.2 antibody Example 1-1: Construction of a cell line with high expression of Claudin18.2 Using Lipofectamine 3000 transfection reagent, pCDH-hClaudin18.2 lentiviral expression vector plasmid and pVSV-G or pCMV-dR8.91 lentiviral packaging vector were transfected into 293T lentiviral packaging cells. The supernatant containing the virus was collected, filtered, and ultrafast centrifugation was performed. The concentrated virus was used to infect human gastric signet ring cell carcinoma cell line NUGC4, which was then sorted with puromycin for 2-3 weeks before FACS single-cell sorting was performed.

[0135] Based on the tumor IHC (Intravascular Health) score, Claudin18.2 expression levels were differentiated. Cells corresponding to a tumor IHC score of 3 were classified as high-expression cells, while cells corresponding to a tumor IHC score of 2 were classified as moderate-expression cells.

[0136] By detecting Claudin18.2 expression on the surface of lentivirus-infected NUGC4 cells using FACS, we selected NUGC4 / hClaudin18.2 monoclonal cell lines with high Claudin18.2 expression levels. Simultaneously, by detecting Claudin18.2 expression on the surface of wild-type NUGC4 cells using FACS, we selected NUGC4 clonal cell lines with moderate Claudin18.2 expression levels. Wild-type NUGC4 cells were found to have low Claudin18.2 expression levels.

[0137] The selected monoclonal cell lines were cultured on a large scale and frozen in a refrigerator in preparation for subsequent experiments.

[0138] Claudin18.2 Array Genbank:NP_001002026(Sequence ID 1): MAVTACQGLGFVVSLIGIAGIIAATCMDQWSTQDLYNNPVTAVFNYQGLWRSCVRESSGFTECRGYFTLLGLPAMLQAVRALMIVGIVLGAIGLLVSIFALKCIRIGSMEDSAKANMTLTSGIMFIVSGLC AIAGVSVFANMLVTNFWMSTANMYTGMGGMVQTVQTRYTFGAALFVGWVAGGLTLIGGVMMCIACRGLAPEETNYKAVSYHASGHSVAYKPGGFKASTGFGSNTKNKKIYDGGARTEDEVQSYPSKHDYV.

[0139] Claudin18.2 DNA sequence (SEQ ID NO: 2): AGAATTGCGC TGTCCACTTG TCGTGTGGCT CTGTGTCGAC ACTGTGCGCC ACCATGGCCG TGACTGCCTG TCAGGGCTTG GGGTTCGTGG TTTCACTGAT TGGGATTGCG GGCATCATTG CTGCCACCTG CATGGACCAG TGGAGCACCC AAGACTTGTA CAACAACCCC GTAACAGCTG TTTTCAACTA CCAGGGGCTG TGGCGCTCCT GTGTCCGAGA GAGCTCTGGC TTCACCGAGT GCCGGGGCTA CTTCACCCTG CTGGGGCTGC CAGCCATGCT GCAGGCAGTG CGAGCCCTGA TGATCGTAGG CATCGTCCTG GGTGCCATTG GCCTCCTGGT ATCCATCTTT GCCCTGAAAT GCATCCGCATTGGCAGCATG GAGGACTCTG CCAAAGCCAA CATGACACTG ACCTCCGGGA TCATGTTCAT TGTCTCAGGT CTTTGTGCAA TTGCTGGAGT GTCTGTGTTT GCCAACATGC TGGTGACTAA CTTCTGGATG TCCACAGCTA ACATGTACAC CGGCATGGGT GGGATGGTGC AGACTGTTCA GACCAGGTAC ACATTTGGTG CGGCTCTGTT CGTGGGCTGG GTCGCTGGAG GCCTCACACT AATTGGGGGT GTGATGATGT GCATCGCCTG CCGGGGCCTG GCACCAGAAG AAACCAACTA CAAAGCCGTT TCTTATCATG CCTCAGGCCA CAGTGTTGCC TACAAGCCTG GAGGCTTCAAGGCCAGCACT GGCTTTGGGT CCAACACCAA AAACAAGAAG ATATACGATG GAGGTGCCCG CACAGAGGAC GAGGTACAAT CTTATCCTTC CAAGCACGAC TATGTGTAAT GCTCTAAGACCTCTCAGCACGGGCGGAAGA AACTCCCGGA GAGCTCACCC AAAAAACAAG GAGATCCCAT CTAGATTTCT TCTTGCTTTT GACTCACAGC TGGAAGTTAG AAAAGCCTCG ATTTCATCTT TGGAGAGGCC AAATGGTCTT AGCCTCAGTC TCTGTCTCTA AATATTCCAC CATAAAACAG CTGAGTTATT TATGAATTAG AGGCTATAGC TCACATTTTC AATCCTCTAT TTCTTTTTTT AAATATAACT TTCTACTCTG ATGAGAGAAT GTGGTTTTAA TCTCTCTCTC ACATTTTGAT GATTTAGACA GACTCCCCCT CTTCCTCCTA GTCAATAAAC CCATTGATGA TCTATTTCCC AGCTTATCCC CAAGAAAACT TTTGAAAGGA AAGAGTAGAC CCAAAGATGT TATTTTCTGC TGTTTGAATT TTGTCTCCCC ACCCCCAACT TGGCTAGTAA TAAACACTTA CTGAAGAAGA AGCAATAAGA GAAAGATATT TGTAATCTCT CCAGCCCATG ATCTCGGTTT TCTTACACTG TGATCTTAAA AGTTACCAAA CCAAAGTCAT TTTCAGTTTG AGGCAACCAA ACCTTTCTAC TGCTGTTGAC ATCTTCTTAT TACAGCAACA CCATTCTAGG AGTTTCCTGA GCTCTCCACT GGAGTCCTCT TTCTGTCGCG GGTCAGAAAT TGTCCCTAGA TGAATGAGAA AATTATTTT TTTAATTTAA GTCCTAAATA TAGTTAAAAT AAATAATGTT TTAGTAAAAT GATACACTAT CTCTGTGAAA TAGCCTCACC CCTACATGTG GATAGAAGGA AATGAAAAAA TAATTGCTTT GACATTGTCT ATATGGTACT TTGTAAAGTC ATGCTTAAGT ACAAATTCCA TGAAAAGCTC ACTGATCCTA ATTCTTTCCC TTTGAGGTCT CTATGGCTCT GATTGTACAT GATAGTAAGT GTAAGCCATG TAAAAAGTAA ATAATGTCTG GGCACAGTGG CTCACGCCTG TAATCCTAGCACTTTGGGAG GCTGAGGAGG AAGGATCACT TGAGCCCAGA AGTTCGAGAC TAGCCTGGGCAACATGGAGAAGCCCTGTCT CTACAAAATA CAGAGAGAAA AAATCAGCCA GTCATGGTGG CCTACACCTG TAGTCCCAGC ATTCCGGGAG GCTGAGGTGG GAGGATCACT TGAGCCCAGGGAGGTTGGGG CTGCAGTGAG CCATGATCAC ACCACTGCAC TCCAGCCAGG TGACATAGCGAGATCCTGTC TAAAAAAATA AAAAATAAAT AATGGAACAC AGCAAGTCCT AGGAAGTAGGTTAAAACTAA TTCTTTAAAA AAAAAAAAAAA GTTGAGCCTG AATTAAATGT AATGTTTCCA AGTGACAGGT ATCCACATTT GCATGGTTAC AAGCCACTGC CAGTTAGCAG TAGCACTTTC CTGGCACTGT GGTCGGTTTT GTTTTGTTTT GCTTTGTTTA GAGACGGGGT CTCACTTTCC AGGCTGGCCT CAAACTCCTG CACTCAAGCA ATTCTTCTAC CCTGGCCTCC CAAGTAGCTG GAATTACAGG TGTGCGCCAT CACAACTAGC TGGTGGTCAG TTTTGTTACT CTGAGAGCTG TTCACTTCTC TGAATTCACC TAGAGTGGTT GGACCATCAG ATGTTTGGGC AAAACTGAAA GCTCTTTGCA ACCACACAC TTCCCTGAGC TTACATCACT GCCCTTTTGA GCAGAAAGTC TAAATTCCTT CCAAGACAGT AGAATTCCAT CCCAGTACCA AAGCCAGATA GGCCCCCTAGGAAACTGAGG TAAGAGCAGT CTCTAAAAAC TACCCACAGC AGCATTGGTG CAGGGGAACT TGGCCATTAG GTTATTATT GAGAGGAAG TCCTCACATC AATAGTACAT ATGAAAGTGACCTCCAAGGG GATTGGTGAA TACTCATAAG GATCTTCAGG CTGAACAGAC TATGTCTGGG GAAAGAACGG ATTATGCCCC ATTAAATAAC AAGTTGTGTT CAAGAGTCAG AGCAGTGAGCTCAGAGGCCC TTCTCACTGA GACAGCAACA TTTAAACCAA ACCAGAGGA GTATTTGTGG AACTCACTGC CTCAGTTTGG GTAAAGGATG AGCAGACAAG TCAACTAAAG AAAAAAGAAAAGCAAGGGAGGGGTTGAGC AATCTAGAGC ATGGAGTTTG TTAAGTGCTC TCTGGATTTG AGTTGAAGAG CATCCATTTG AGTTGAAGGC CACAGGGCAC AATGAGCTCT CCCTTCTACC ACCAGAAAGT CCCTGGTCAG GTCTCAGGTA GTGCGGTGTG GCTCAGCTGG GTTTTTAATT AGCGCATTCT CTATCCAACA TTTAATTGTT TGAAAGCCTC CATATAGTTA GATTGTGCTT TGTAATTTTG TTGTTGTTGC TCTATCTTAT TGTATATGCA TTGAGTATTA ACCTGAATGT TTTGTTACTT AAATATTAAA AACACTGTTA TCCTACAGTT。

[0140] Example 1-2: Production of Anti-Human Claudin18.2 Monoclonal Antibody 1 Immunization Anti-human Claudin18.2 monoclonal antibody was produced using immunized mice.

[0141] Experimental SJL white mice were female, 6 to 8 weeks old (Beijing Vital River Laboratory Animal Technology Co., Ltd., Charles River Laboratory Animal Technology Co., Ltd., Beijing, Animal Production License Number: SCXK(Beijing) 2012-0001). Breeding environment: SPF grade. After purchasing the mice, they were adjusted to a 12 / 12 hour light / dark cycle and bred in a laboratory environment with a temperature of 20°C to 25°C and a humidity of 40% to 60% for one week. The mice adapted to the environment were immunized according to the following protocol. The immunizing antigen was huClaudin18.2-HEK293 cells (HEK-293 stable transfected cell line transfected with the human Claudin18.2 plasmid).

[0142] Immunization protocol: Before the first immunization of the cells, TiterMax (R) Gold Adjuvant (Sigma Cat No. T2684) was injected intraperitoneally (IP) into the mice at 0.1 mL / mouse. After 30 minutes, 1×10 of each mouse was intraperitoneally (IP) injected with 0.1 mL of physiological saline 8Cell solution diluted to a concentration of / mL was injected. After uniformly dispersing the cells, inoculation was performed on days 0, 14, 28, 42, and 56. Blood was collected on days 21, 35, 49, and 63, and antibody titers in mouse serum were determined by ELISA. After the 4th and 5th immunizations, mice with high and stable antibody titers in their serum were selected for spleen cell fusion. An additional immunization was performed 3 days before spleen cell fusion, and 1 × 10⁶ cells were injected intraperitoneally (IP). 7 They injected the cells.

[0143] 2. Fusion of spleen cells PEG-mediated fusion step allows splenic lymphocytes and myeloma cells (Sp2 / 0 cells) (ATCC) to fuse. (R) CRL-8287 TM ) were fused to obtain hybridoma cells. The hybridoma cells were 0.5 × 10 6 / mL ~ 1 × 10 6 The cells were resuspended in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI) at a density of / mL, seeded at 100 μL / well in a 96-well plate, incubated at 37°C and 5% CO2 for 3-4 days, then supplemented with 100 μL / well of HAT complete medium and cultured for 3-4 days until clones formed. The supernatant was removed, 200 μL / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI) was added, incubated at 37°C and 5% CO2 for 3 days, and then detected by ELISA.

[0144] 3. Selection of hybridoma cells The culture supernatant was detected using a binding ELISA method based on the growth density of hybridoma cells. Cells that showed strong binding ability to huClaudin18.2-HEK293 cells but no binding to HEK293 cells were selected, amplified and cryopreserved in a timely manner, and subcloned 2 to 3 times until single-cell clones were obtained.

[0145] Each time cells were subcloned, cell binding experiments had to be performed. After selection based on these experiments, hybridomaclons were obtained, and antibodies were further prepared using serum-free cell culture. The antibodies were then purified according to the purification example and prepared for use in the detection example.

[0146] Examples 1-3: Humanization of mouse antibodies We selected the monoclonal hybridoma cell lines mAb1901 and mAb1902, which exhibit high in vitro activity, cloned the monoclonal antibody sequences from these cells, and then performed humanization, recombinant expression, and activity evaluation.

[0147] The process for cloning sequences from hybridomas is as follows: Collect hybridoma cells in the logarithmic growth phase, extract RNA with Trizol (Invitrogen, 15596-018) (following the steps in the reagent kit instructions), and reverse transcribe (PrimeScript). TM Reverse Transcriptase (Takara, cat # 2680A). The cDNA obtained by reverse transcription was amplified by PCR using a mouse Ig-Primer Set (Novagen, TB326 Rev.B 0503) and sent to a sequencing company for sequencing. The amino acid sequences corresponding to the obtained DNA sequences are shown in SEQ ID NOs: 3-6.

[0148] mAb1901 mouse heavy chain variable region (SEQ ID NO: 3) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQGTSVTVSS; mAb1901 mouse light chain variable region (SEQ ID NO: 4) DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKLELK; mAb1902 mouse heavy chain variable region (SEQ ID NO: 5) EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSS; mAb1902 mouse light chain variable region (SEQ ID NO: 6) DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIK; The mouse heavy chain variable region and light chain variable region described above were linked to the heavy chain constant region and human κ light chain constant region of the human IgG1 antibody, respectively, to form the chimeric antibodies ch1901 and ch1902.

[0149] The constant region of each antibody is selected from the following sequences. Heavy chain constant region of human IgG1 antibody: (SEQ ID NO: 7) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; Human κ-light chain constant region: (SEQ ID NO: 8) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0150] Like the methods disclosed in many documents in this field, the humanization of mouse monoclonal antibodies was performed. That is, instead of the parental (mouse antibody) constant domain, a human constant domain was used, and based on the homology between the mouse antibody and the human antibody, a human germline sequence was selected and CDR transplantation was performed. The present invention selects candidate molecules with good activity for humanization, and the results are as follows.

[0151] 1. CDR regions of mouse antibodies In Table 1, the amino acid residues of VH / VL CDR are determined and annotated by the Kabat numbering system.

[0152] The CDR sequences of the mouse antibodies are as shown in Table 1.

[0153] [Table 2]

[0154] 2. Selection of human germline FR region sequences Based on the typical structures of the obtained mouse antibody VH / VL CDRs, the heavy and light chain variable region sequences were compared with the antibody Germline database, and a highly homologous human germline template was obtained. Among them, the human germline light chain framework region was derived from the human κ light chain gene.

[0155] 2.1 Design of humanized modification and revertant mutation of mAb1901 Appropriate human antibody germline was selected, and the mAb1901 mouse antibody was humanized. Instead of the humanized variable region, the CDR region of the mouse antibody mAb1901 was transplanted into the selected humanized template and then recombined with the IgG constant region to form a complete antibody. Simultaneously, the FR region in the V region of the humanized antibody was reversed. Exemplary reverse mutation forms and combinations are as follows.

[0156] [Table 3]

[0157] [Table 4] In the table above, the heavy chain variable region is linked to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, while the light chain variable region is linked to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody. In other embodiments, the heavy chain variable region and the light chain variable region may be linked to other heavy chain constant regions and light chain constant regions, respectively, to form the full-length antibody.

[0158] 2.2 Design of humanized modifications and reverse mutations of mAb1902 Appropriate human antibody germline was selected, and the mAb1902 mouse antibody was humanized. Instead of the humanized variable region, the CDR region of the mouse antibody mAb1902 was transplanted into the selected humanized template and further recombined with the IgG constant region to form a complete antibody. Simultaneously, the FR region in the V region of the humanized antibody was reversed. Exemplary reverse mutation forms and combinations are as follows.

[0159] [Table 5]

[0160] [Table 6]

[0161] In the table above, the heavy chain variable region is linked to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, while the light chain variable region is linked to the human κ light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody.

[0162] Chimeric antibody ch1901 ch1901 heavy chain: (SEQ ID NO: 35) EVQLMESGGGLVKPGGSLKLSCAASGFTFSDYGIHWVRQAPEMGLEWIAYISRGSSTIYYADTVKGRFTMSRDNAKNTLFLQMTSLRSEDTAMYYCARGGYDTRNAMDYWGQ GTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; ch1901 light chain: (Sequence number 36) DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLNSGNQKNYLAWYQQKPGQPPKLLIYGASTRASGVPDRFTGSGSGTDFTLTISSVQAEDLAIYHCQNDLYYPLTFGAGTKL ELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC; Chimeric antibody ch1902 ch1902 heavy chain: (SEQ ID NO: 37) EVQLQESGAELVKPGASVKLSCKASGYIFTSYWMHWVKQRPGQGLEWIGMIHPNSGSTNYNEKFKGKATLTLDKSSSTAYMQLSSLPSEDSAVYYCARLKTGNSFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; ch1902 light chain: (SEQ ID NO: 38) DIVLTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNAYTYPFTFGSGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0163]

Table 7

[0164] The light and heavy chain sequences of the full-length antibody are as follows.

[0165]

Table 8-1

Table 8-2

[0166] [Table 9]

[0167] The light-heavy chain sequences of the full-length antibody are as follows:

[0168] [Table 10] [Table 11]

[0169] The positive control antibody for this disclosure is IMAB-362 (according to WO2016166122). Heavy chain (SEQ ID NO: 53) QVQLQQPGAE LVRPGASVKL SCKASGYTFT SYWINWVKQR PGQGLEWIGN IYPSDSYTNY NQKFKDKATL TVDKSSSTAY MQLSSPTSED SAVYYCTRSW RGNSFDYWGQ GTTLTVSSAS TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLLSPGK; Light chain (Sequence ID 54) DIVMTQSPSS LTVTAGEKVT MSCKSSQSLL NSGNQKNYLT WYQQKPGQPP KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCQNDYSY PFTFGSGTKL EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL QSGNSQESVT EQDSKDSTYS LSSTLTLSKA DYEKHKVYAC EVTHQGLSSP VTKSFNRGEC. The antibodies described above were cloned, expressed, and purified using conventional gene cloning and recombinant expression methods.

[0170] Preparation of anti-Claudin18.2 ADC complex drugs The drug portion of the anti-Claudin 18.2 ADC conjugate of this disclosure may be any preferred drug. Particularly preferred drugs are described, for example, in PCT Publication No. WO2020063676A1 (which is incorporated in its entirety by reference). Compound 9-A of the present disclosure is N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolidino[1,2-b]quinoline-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole-1-yl)hexaneamide, which has the following structure.

[0171] [ka] Analysis of drug loading dose of ADC stock solution 1. UV-HPLC method After placing cuvettes containing sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell, respectively, and subtracting the solvent blank, the cuvettes containing the sample solution awaiting testing were placed in the sample measurement absorption cell, and the absorbances at 280 nm and 370 nm were measured.

[0172] Calculation of results: The ADC stock solution load was measured using ultraviolet spectrophotometry (instrument used: Thermo nanodrop 2000 ultraviolet spectrophotometer). The principle is that the total absorbance of the ADC stock solution at a certain wavelength is equal to the sum of the absorbances of the drug and the monoclonal antibody at that wavelength, i.e., (1)A 280nm =ε mab-280 bC mab +ε Drug-280 bC Drug ε Drug-280 The average molar extinction coefficient of the drug at 280 nm is 5100. C Drug : Drug concentration, ε mab-280 The average molar extinction coefficient at 280 nm of the monoclonal antibody stock solution is 214600. C mab :Concentration of monoclonal antibody stock solution, b: The optical path length is 1 cm.

[0173] Similarly, the total absorbance equation for the sample at 370 nm can be obtained: (2)A 370nm =ε mab-370 bC mab +ε Drug-370 bC Drug ε Drug-370 The average molar extinction coefficient of the drug at 370 nm is 19000. C Drug : Drug concentration, ε mab-370 The extinction coefficient of monoclonal antibodies at 370 nm is 0. C mab :Concentration of monoclonal antibody stock solution, b: The optical path length is 1 cm. By using the two equations (1) and (2), the drug loading can be calculated by combining the extinction coefficient and concentration data of the monoclonal antibody and the drug at two detection wavelengths. Drug loading = C Drug / C mab .

[0174] 2.RP-HPLC method Naked antibody and awaiting ADC sample (concentration 1 mg / mL) were reduced with 4 μL of DDT (sigma), bathed in water at 37°C for 1 hour, then removed and placed in an inner tube. Detection was performed using an Agilent 1200 high-performance liquid chromatograph, with an Agilent PLRP-S 1000A 8 μm 4.6 mm × 250 mm column selected. The column temperature was 80°C, the DAD detector wavelength was 280 nm, the flow rate was 1 mL / min, and the injection volume was 40 μL. Subsequently, the positions of light and heavy chains were distinguished by spectral comparison of the sample and the naked antibody, and the DAR value was calculated by integrating the spectrum of the detected sample.

[0175] Solution preparation: 1) 0.25 M DTT solution: Preparation example: 5.78 mg of DTT was taken, dissolved thoroughly in 150 μL of purified water, and a 0.25 M DTT solution was obtained and stored at -20°C.

[0176] 2) Mobile phase A (0.1% TFA aqueous solution): Preparation example: 1000 mL of purified water was measured using a graduated cylinder, 1 mL of TFA (sigma) was added, and the mixture was thoroughly and uniformly mixed before use. The solution was then stored at 2°C to 8°C for 14 days.

[0177] 3) Mobile phase B (0.1% TFA acetonitrile solution): Preparation example: 1000 mL of acetonitrile was measured out using a graduated cylinder, 1 mL of TFA was added, and the mixture was thoroughly and uniformly mixed before use. The solution was then stored at 2°C to 8°C for 14 days. Data analysis: By comparing the spectra of the sample and the bare antibody, the positions of the light and heavy chains were distinguished, and the DAR value was calculated by integrating the spectrum of the detected sample. The calculation formula is as follows:

[0178] [Table 12]

[0179] Sum of LC peak areas = LC peak area + LC + 1 peak area, Sum of HC peak areas = HC peak area + HC + 1 peak area + HC + 2 peak area + HC + 3 peak area LC DAR = Σ(number of linked drugs × percentage of peak area) / sum of LC peak areas. HC DAR = Σ(number of linked drugs × percentage of peak area) / sum of HC peak areas. DAR = LC DAR + HC DAR.

[0180] Examples 1-4: ADC-1 / ADC-2 Under conditions of 37°C, an aqueous solution of tri(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.03 mL, 110.3 μmol) was added to PBS buffer containing antibody h1902-5 (0.05 M PBS buffer at pH=6.5, 10.0 mg / mL, 320.0 mL, 21.62 μmol). The mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours until the reaction was stopped.

[0181] The reaction mixture was cooled to 25°C in a water bath. Compound 9-A (350 mg, 303 mol) was dissolved in 13.2 mL of acetonitrile and 6.6 mL of DMSO, then added to the reaction mixture. The mixture was placed in a water bath shaker and allowed to react at 25°C for 3 hours while shaking, until the reaction was stopped.

[0182] The reaction mixture was purified by passing it through an ultrafiltration membrane to remove small molecules. For purification, 5 L of 50 mM pH=6.5 PBS buffer (4% acetonitrile, 2% DMSO) and 5 L of 10 mM pH=5.3 succinate buffer were used in sequence. Then, sucrose was added to the purified solution up to 60 mg / mL and Tween-20 up to 0.2 mg / mL to prepare ADC-1 (10 mM pH=5.3 succinate buffer, 10 mg / mL, 2.626 g) with a yield of 81.81%. Subsequently, it was prepared as a lyophilized powder at 20 mg / flask.

[0183] The average value was calculated using UV-HPLC: n=6.8. Using the method described above, ADC-2 was prepared by using antibody h1901-11 instead of antibody h1902-5 in combination with compound 9-A, resulting in n=7.1.

[0184] Examples 1-5: ADC-3 Under conditions of 37°C, an aqueous solution of PBS buffer containing antibody h1901-11 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1 mL, 67.5 nmol) was added to an aqueous solution of the prepared TCEP (10 mM, 10.1 μL, 101 nmol). The mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0185] Compound 9-A (0.58 mg, 540 nmol) was dissolved in 34 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted with shaking for 3 hours at 25°C until the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.5 and containing 0.001 M EDTA) to obtain ADC-3 in PBS buffer (0.72 mg / mL, 11.2 mL), which was stored at 4°C. The mean value was calculated by RP-HPLC: n=2.51.

[0186] Examples 1-6: ADC-4 Under conditions of 37°C, an aqueous solution of PBS buffer containing antibody h1901-11 (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1 mL, 67.5 nmol) was added to an aqueous solution of the prepared TCEP (10 mM, 16.9 μL, 169 nmol). The mixture was placed in a water bath shaker and reacted with shaking at 37°C for 3 hours, after which the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0187] Compound 9-A (0.73 mg, 680 nmol) was dissolved in 43 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted with shaking for 3 hours at 25°C until the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.5 and containing 0.001 M EDTA) to obtain ADC-4 in PBS buffer (0.62 mg / mL, 12.5 mL), which was stored at 4°C. The mean value was calculated by RP-HPLC: n=4.06.

[0188] Examples 1-7: ADC-5 Under conditions of 37°C, an aqueous solution of antibody h1901-11 buffered in PBS (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1 mL, 67.5 nmol) was added to an aqueous solution of the prepared TCEP (10 mM, 35.8 μL, 358 nmol). The mixture was placed in a water bath shaker and reacted at 37°C for 3 hours while shaking, until the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0189] Compound 9-A (1.09 mg, 1015 nmol) was dissolved in 64 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted with shaking for 3 hours at 25°C until the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.5 and containing 0.001 M EDTA) to obtain ADC-5 in PBS buffer (0.54 mg / mL, 12.5 mL), which was stored at 4°C. The mean value was calculated by RP-HPLC: n=6.8.

[0190] Examples 1-8: ADC-6 Under conditions of 37°C, an aqueous solution of antibody h1902-5 buffered in PBS (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added to an aqueous solution of the prepared TCEP (10 mM, 10.9 μL, 109 nmol). The mixture was placed in a water bath shaker and reacted at 37°C for 3 hours while shaking, until the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0191] Compound 9-A (0.63 mg, 587 nmol) was dissolved in 40 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted with shaking for 3 hours at 25°C until the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.5 and containing 0.001 M EDTA) to obtain ADC-6 in PBS buffer (0.7 mg / mL, 13.0 mL), which was stored at 4°C. The mean value was calculated by RP-HPLC: n=2.69.

[0192] Examples 1-9: ADC-7 Under conditions of 37°C, an aqueous solution of antibody h1902-5 buffered in PBS (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added to an aqueous solution of the prepared TCEP (10 mM, 18.3 μL, 183 nmol). The mixture was placed in a water bath shaker and reacted at 37°C for 3 hours while shaking, until the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0193] Compound 9-A (0.79 mg, 736 nmol) was dissolved in 50 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted with shaking for 3 hours at 25°C until the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.5 and containing 0.001 M EDTA) to obtain ADC-7 in PBS buffer (0.6 mg / mL, 14.0 mL), which was stored at 4°C. The mean value was calculated by RP-HPLC: n=4.25.

[0194] Examples 1-10: ADC-8 Under conditions of 37°C, an aqueous solution of antibody h1902-5 buffered in PBS (0.05 M PBS buffer solution with pH=6.5, 10.0 mg / mL, 1.08 mL, 72.9 nmol) was added to an aqueous solution of the prepared TCEP (10 mM, 38.7 μL, 387 nmol). The mixture was placed in a water bath shaker and reacted at 37°C for 3 hours while shaking, until the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0195] Compound 9-A (1.18 mg, 1099 nmol) was dissolved in 70 μL of DMSO and added to the reaction mixture. The mixture was placed in a water bath shaker and reacted with shaking for 3 hours at 25°C until the reaction was stopped. The reaction mixture was desalted and purified using a Sephadex G25 gel column (eluent phase: 0.05 M PBS buffer aqueous solution with pH 6.5 and containing 0.001 M EDTA) to obtain ADC-8 in PBS buffer (0.56 mg / mL, 14.2 mL), which was stored at 4°C. The mean value was calculated by RP-HPLC: n=7.01.

[0196] Example 1-11: ADC-9 Under conditions of 12°C, the prepared TCEP histidine buffer (10 mM histidine-acetic acid-Tris and 2.5 mM EDTA buffer, pH 7.2, 20.6 g / L, 6.49 L, 0.91 mmol) containing antibody h1902-5 was added to a histidine-acetic acid-Tris / EDTA buffer (pH 7.2, 10 mM histidine buffer, 1.717 mM, 1.16 L, 1.99 mmol). The mixture was placed in a constant temperature water bath and reacted with stirring at 12°C for 2 hours until the reaction was stopped, yielding intermediate solution I.

[0197] Compound 9-A (4.72 g, 4.39 mmol) was dissolved in 0.38 L of DMSO to prepare a DMSO solution of compound 9-A. 0.38 L of DMSO was added to the above intermediate solution I, and then the DMSO solution of compound 9-A was added. The mixture was placed in a constant temperature water bath and reacted with stirring at 12°C for 1 hour until the reaction was stopped.

[0198] The above reaction mixture was purified using a Capto S Impact cation chromatography column. Each of the nine column volumes was washed with 0.05 M acetic acid buffer (pH=5.0) containing 10% (v / v) DMSO, and each of the six column volumes was washed with 0.05 M acetic acid buffer (pH=5.0). The mixture was then eluted with 0.05 M acetic acid and 0.30 M sodium chloride buffer (pH=5.5) to remove free toxins and residual solvent from the reaction mixture. Ultrafiltration was performed at 22°C using seven times the volume of the cation eluent and the same volume (a 30 KD polycellulose membrane package was used as the ultrafiltration membrane package) to obtain the product ADC-9. The average value was calculated by RP-HPLC: n=4.1.

[0199] Biological evaluation Test Example 1: Cell-level ELISA coupling experiment Cell-based ELISA experiments were used to detect the binding properties of the Claudin18.2 antibody. NUGC4 cells expressing Claudin18.2 were stably transfected and cultured in 96-well cell plates (Corning, 3599). When the cells grew to 90% density, 4% paraformaldehyde was added and the cells were fixed for 1 hour. The plates were then washed three times with PBST buffer (PBS at pH 7.4 containing 0.05% Tween-20), and 200 μL / well of 5% skim milk (Kōmyō skim milk powder) blocking solution diluted in PBS was added. The cells were incubated at 37°C for 2.5 hours or blocked overnight at 4°C (16-18 hours). After blocking was complete, the blocking solution was discarded, the plate was washed three times with PBST buffer, and then 50 μL / well of different concentrations of test-ready antibodies diluted with sample diluent (PBS with 1% skim milk at pH 7.4) were added and incubated at 37°C for 2 hours. After incubation was complete, the plate was washed five times with PBST, and 100 μL / well of HRP-labeled sheep anti-human secondary antibody (Jackson Immuno Research, 109-035-003) diluted with sample diluent was added and incubated at 37°C for 1 hour. After washing the plate six times with PBST, 50 μL / well of TMB chromogenic substrate (KPL, 52-00-03) was added, incubated at room temperature for 10-15 minutes, and the reaction was stopped by adding 50 μL / well of 1 M H2SO4. The absorption was read at 450 nm using an MD Versa Max Tm microplate reader, and the binding EC50 value of the Claudin18.2 antibody to Claudin18.2 was calculated.

[0200] [Table 13]

[0201] [Table 14]

[0202] [Table 15]

[0203] Test Example 2: Antibody-Cell Binding Experiment NUGC4 cells expressing Claudin18.2, obtained by stable transfection, were incubated in FACS buffer (2% fetal bovine serum (Gibco, 10099141), pH 7.4 PBS (Sigma, P4417-100TAB)) at a rate of 1 × 10⁶ times. 6 Cell suspensions were prepared at 100 μL / mL and added to 96-well round-bottom plates (Corning, 3795) at 100 μL / well. After removing the supernatant by centrifugation, 50 μL / well of different concentrations of test-ready Claudin18.2 antibody diluted with FACS buffer was added, and the cells were incubated in the dark at 4°C for 1 hour. After washing by centrifugation three times with 300 g of FACS buffer, anti-human IgG(H+L) (invitrogen, A-11013) coated with working concentration Alexa Fluor 488 was added, and the cells were incubated in the dark at 4°C for 40 minutes. After washing by centrifugation three times with 300 g of FACS buffer, geometric mean fluorescence intensity was detected using a BD FACS Canto II flow cytometer, and the binding EC50 values ​​of Claudin18.2 antibody to NUGC4 cells expressing Claudin18.2 after stable transfection were calculated. The results are shown in Figure 1.

[0204] Test Example 3: Antibody Endocytosis Experiment Claudin 18.2 antibody, pre-labeled with DyLight 488 NHS Ester (thermofisher, 46403) and awaiting testing, was administered at a final concentration of 5 μg / mL in 1 × 10⁶ units. 6NUGC4 cells expressing Claudin18.2 were stably transfected with 1 mL of Claudin18.2 and incubated on ice in the dark for 1 hour. The cells were then washed by centrifugation three times with pre-cooled FACS buffer (PBS at pH 7.4, 2% fetal bovine serum), the supernatant was removed, and the cells were added to preheated complete medium and placed in a 5% CO2 cell incubator at 37°C. Cells were removed at 0, 0.5, 1, 2, and 4 hours, respectively, and stored on ice in the dark. After collecting all the samples, 300 g was centrifuged at low temperature to remove the supernatant, and elution buffer (0.05 M glycine and 0.1 M sodium chloride at pH 1.7) was added. The samples were incubated at room temperature for 7 minutes, washed once by centrifugation with 300 g of FACS buffer, and the geometric mean fluorescence intensity was detected using a BD FACS Canto II flow cytometer to calculate the endocytosis efficiency of the Claudin18.2 antibody into NUGC4 cells expressing Claudin18.2 after stable transfection. The results (see Figure 2) show that the humanized antibody has good cell endocytosis efficiency.

[0205] Test Example 4: Measurement of antibody affinity based on flow cytometry On the day of the experiment, HEK293 / hClaudin18.2 cells were collected in a U-bottom 96-well plate, with 1 × 10⁶ cells per well. 5 pieces~2×10 5 The sample consisted of [number] cells. Claudin18.2 antibody at an initial concentration of 5 μg / mL, 2× gradient dilution (12 concentration points), was added and incubated at 4°C for 1 hour. A positive control (IMAB362) was set up, along with a negative control (without antibody). After removing the antibody by centrifugation, FITC anti-human IgG Fc antibody (200×) was added at 100 μL / well and incubated in the dark at 4°C for 30 minutes. After washing twice with PBS + 2% FBS, flow cytometry was performed. The BD FACS CantoII was started, preheating was completed, the BD FACSDiva software was opened, a new experiment was established, the HEK293 / hClaudin18.2 negative control sample was detected, and the FSC and SSC voltages were adjusted to appropriate values ​​and saved. Quantum TMFollowing the instructions for the FITC-5 MESF Kit, blank sample B and calibration curve 1 were detected, and the FITC voltage was adjusted to an appropriate value and saved. The saved voltage was used to detect samples in a U-bottom 96-well plate, and the data was recorded. The experimental data was analyzed using Flowjo software to obtain Geo Mean values, and the Quantum... TM The MESF-Geo Mean calibration curve was fitted according to the FITC-5 MESF Kit instructions. The molar concentration and free antibody concentration of Claudin18.2 antibody bound to HEK293 / hClaudin18.2 cells were calculated based on the concentration fluorescence values ​​of the FITC anti-human IgG Fc antibody. The antibody's Bmax and dissociation constant KD were then calculated using the Scatchard plot method. The results are shown in Table 14.

[0206] [Table 16]

[0207] Test Example 5: Evaluation of the ADCC effect of antibodies Various NUGC4 cells (high / moderate / low expression of Claudin 18.2) were digested, centrifuged at 1000 rpm, resuspended, and counted. The cells were counted in 3 × 10⁶ units. 5 Cells were resuspended at a density of cells / mL in phenol red-free RPMI 1640 (Gibco, 11835-030) supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum, Gibco, 1921005PJ). 25 μL of cells per well (7500 cells / well) were added to a 96-well plate (Corning, 3903). Antibodies were diluted in the above phenol red-free medium to prepare 3× antibody diluents, and 25 μL / well of antibody was added to the cell plate. Incubation was performed at 37°C in a 5% CO2 incubator for 0.5 hours.

[0208] Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, resuspended, and counted. The cells were counted in 3 × 10⁶ cells. 6The cells were resuspended at a density of cells / mL in phenol red-free RPMI 1640 with 10% FBS (New Zealand very low IgG fetal bovine serum) added, and 25 μL of cells (7.5 × 10⁶) were placed per well in an experimental plate. 4 (1 cell / well) was added. Incubated for 6 hours in a 37°C, 5% CO2 incubator.

[0209] 75 μL / well of Bright-Glo (Promega, E2610) was added to each well of the experimental plate, and chemiluminescence was detected using a microplate reader (PerkinElmer, VITOR3).

[0210] As a result (see Table 15 and Figures 3A-3C), antibodies h1901-11 and h1902-5 both showed very strong ADCC activity in NUGC4 cells expressing Claudin 18.2 at different levels: low (Figure 3A), moderate (Figure 3B), and high (Figure 3C).

[0211] [Table 17]

[0212] Experiment Example 6: Cellular Activity Experiment of ADC Molecules This experiment detected the in vitro damaging effects of ADC molecules on human gastric cancer cell lines using the CellTiter-Glo Luminescence Cell Viability Assay. On day 1, cells with low, moderate, and high expression of NUGC4-claudin18.2 were collected and their density was set to 2.5 × 10⁶. 4The sample was adjusted to 1 / mL and added to a 96-well white transparent plate at 90 μL / well, with approximately 2500 cells per well. The cells were incubated overnight at 37°C in a 5% CO2 incubator. On day 2, the sample was diluted in a U-bottom 96-well plate, with an initial concentration of 5 μM, 4× gradient dilution, and 9 concentration points. 10 μL / well of the diluted sample was added to the cell plate. The cells were incubated at 37°C in 5% CO2 for 6 days. On day 8, the cell culture plate was removed, 50 μL / well of Cell Titer-Glo Reagent was added, and the plate was left at room temperature for 2-3 minutes. Luminescence values ​​were read using a PHERAstar FS microplate reader. Data analysis was performed using GraphPad Prism software. The results are shown in Table 16.

[0213] [Table 18]

[0214] Test Example 7: Evaluation of the efficacy of ADC molecules in the body Human gastric cancer cells NUGC4 (moderate expression of Claudin 18.2) cells (5 × 10) 6 Balb / c nudes were subcutaneously inoculated with 50% of the Matrigel (containing Matrigel / animal) in the right rib region. On day 0, they were divided into groups of 8 animals / group, for a total of 8 groups. The average tumor volume was approximately 84.41 mm². 3 That was the case.

[0215] ADC was administered intraperitoneally in a total of three doses, with each animal receiving 10 g / 0.1 mL based on its body weight, on days 0, 4, and 11, respectively.

[0216] ADC was injected intraperitoneally on the day of group division, for a total of four doses, administered every five days, with each animal receiving 10 g / 0.1 mL based on its body weight.

[0217] The tumor's volume and weight were measured twice a week, and the data was recorded.

[0218] Using Excel 2003 statistical software, the mean was calculated using the average (avg), the standard deviation (SD) was calculated using STDEV, the SEM value was calculated using STDEV / SQRT, and the group difference (P-value) was calculated using TTEST.

[0219] Tumor volume (V) calculation formula: V = 1 / 2 × L 長 ×L 短 2 Relative volume (RTV) = VT / V0 Tumor inhibition rate (%) = (CRTV - TRTV) / CRTV (%) Of these, V0 and VT represent the tumor volume at the start of the experiment (day 0 being the day of the first dose) and at the end of the experiment, respectively. CRTV and TRTV represent the relative tumor volume of the blank control group (Vehicle) and the experimental group at the end of the experiment, respectively. The results are shown in Table 17 and Figures 4 and 5.

[0220] [Table 19]

[0221] 2. Formulations The equipment used during the preparation and detection of the formulations, and the methods for calculating the results, are as follows: SEC molecular exclusion chromatography: This analytical method separates solutes based on the correlation between the pore size of the gel pores and the coil dimensions of the polymer sample molecules.

[0222] SEC% (percentage of SEC monomer content) = A monomer / A total × 100% (A monomer is the peak area of ​​the main peak monomer in the sample, and A total is the sum of all peak areas).

[0223] SEC measurement equipment: Agilent 1260, Column: Waters, XBrige BEH 200Å SEC (300 mm × 7.8 mm, 3.5 μm).

[0224] CE capillary gel electrophoresis: Electrophoresis is a method of separating samples based on their molecular weight using a constant voltage, by moving a gel onto a capillary tube as a support medium.

[0225] The purity percentage of reduced CE = A main peak / A total × 100% (A main peak is the peak area of ​​the light chain main peak + heavy chain main peak in the sample, and A total is the sum of all peak areas).

[0226] CE measuring instrument: Beckman model number: plus800.

[0227] Measurement of osmotic pressure: The osmotic pressure was measured using the freezing point method, and based on the fact that the freezing point depression is directly proportional to the molar concentration of the solution, a high-sensitivity thermometer was used to measure the freezing point of the solution, and the result was converted to osmotic pressure using electrical energy. Equipment manufacturer: Loser, model number: OM815.

[0228] Measurement of protein concentration: Since the drug in the antibody-drug conjugate is absorbed at 280 nm, the protein concentration is corrected using the following formula. A280 = Cd * ε280d + Cmab * ε280mab; A370 = Cd * ε370d; Cd represents the drug concentration, Cmab represents the protein concentration, ε280d represents the extinction coefficient of the drug at 280 nm, ε280mab represents the extinction coefficient of the protein at 280 nm, and ε370d represents the extinction coefficient of the drug at 370 nm. ε280mab = 1.49 mg-1 cm-1 mL, ε280d = 5000 (molar extinction coefficient of the drug at 280 nm) / 1074.13 (molecular weight of the drug) = 4.65 mg-1 cm-1 mL, ε370d = 19000 (molar extinction coefficient of the drug at 370 nm) / 1074.13 (molecular weight of the drug) = 17.69 mg-1 cm-1 mL. The above extinction coefficients are mass extinction coefficients.

[0229] Protein concentration measuring instrument: UV-Vis spectrophotometer, model number: Nano Drop oneC, optical path 1 mm.

[0230] Example 2-1: Selection of formulation buffer systems and pH values Formulations were prepared containing 20 mg / mL (protein concentration) of ADC-9, the following various buffer systems, and 0.1 mg / mL of polysorbate 80 (PS80).

[0231] 1) 10 mM citrate - sodium citrate (CA), pH 5.5 2) 10 mM succinate - sodium succinate (SA), pH 5.0 3) 10 mM succinate - sodium succinate, pH 5.5 4) 10 mM histidine hydrochloride (His-HCl), pH 5.5 5) 10 mM histidine hydrochloride, pH 6.0 6) 10 mM histidine hydrochloride, pH 6.5 7) 10 mM histidine acetate (His-AA), pH 5.0 8) 10 mM histidine acetate, pH 5.5 9) 10 mM phosphate (PB), pH 6.5.

[0232] Each formulation was filtered, filled into containers, plugged, and sealed. Samples were then taken and subjected to high-temperature stability (40°C) and shaking (25°C, 300 rpm) studies, and their appearance, SEC, and reduced CE were examined. The results are shown in Table 18.

[0233] After shaking for 11 days, only samples 2), 3), 7), and 9) became transparent in appearance, and after being left at 40°C for 15 days, samples 1), 2), 3), 7), and 8) also became transparent in appearance. In other words, judging from appearance, the formulations of samples 2), 3), and 7) are relatively superior.

[0234] After leaving the samples at 40°C for 15 days, detection by SEC revealed that the monomers in samples 4), 5), 6), 7), and 8) showed a reduction of approximately 4%, while the monomers in the other formulations showed a reduction of 7% to 10%.

[0235] After leaving the samples at 40°C for 15 days, detection by reducing CE showed that samples 4), 5), 7), and 8) exhibited a reduction in the main peak of approximately 1% to 2%, which was superior to the other samples.

[0236] Based on the data above, sample 7), a 10 mM His-AA, pH 5.0 formulation, was selected as the final buffer because it performed better than the other formulations in terms of appearance and various chemical detection parameters.

[0237] [Table 20]

[0238] Example 2-2: Selection of surfactant type and concentration Formulations containing polysorbates of various types and concentrations, along with 10 mM His-AA, pH 5.0 buffer, 80 mg / mL sucrose, and ADC-9 with a protein concentration of 20 mg / mL, were prepared. Each formulation was filtered, filled into containers, plugged, and sealed. High-temperature stability studies (40°C) and freeze-thaw studies were performed on the samples. In the freeze-thaw study, five freeze-thaw cycles (FT5C, 35°C to 2°C to 8°C) were performed, followed by 3 days at room temperature (25°C, D3). Appearance, SEC, and reduced CE were examined, and the specific formulation designs are shown in Table 19.

[0239] The results are shown in Table 20. According to the experimental results, the formulation using 0.2 mg / mL PS80 showed the best appearance and chemical detection parameters under each condition.

[0240] Based on these results, the type and concentration of the surfactant were determined to be PS80 at 0.2 mg / mL.

[0241] [Table 21]

[0242] [Table 22]

[0243] Examples 2-3: Selection of sugar types Preparations were made containing sucrose, trehalose, and mannitol, respectively, and further containing 10 mM His-AA (pH 5.0) buffer, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. Each preparation was filtered, filled into containers, plugged, and sealed. High-temperature stability studies (40°C) were performed on the samples, followed by freeze-thaw cycles at -35°C / 4°C, and then left at room temperature for 3 days. Appearance, SEC, and reduced CE were examined.

[0244] The results are shown in Table 21. Under various freeze-thaw conditions for sugar preparations, the sucrose-based sample had a superior appearance compared to the trehalose-based or mannitol-based sample. According to SEC detection results, the sucrose-based or trehalose-based sample was superior to the mannitol-based sample. After being left at 40°C for one month, the sucrose-based sample had a superior appearance compared to the trehalose-based sample. SEC and reduced CE detection results also showed that the sucrose-based sample was slightly superior to the trehalose-based sample.

[0245] [Table 23]

[0246] Preparations were made containing sucrose, trehalose, and mannitol, respectively, and further containing 10 mM His-AA (pH 5.0) buffer, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. Each preparation was filtered, filled into containers, partially plugged, lyophilized, fully plugged, capped, and left to stand for high-temperature stability (40°C) testing. Appearance, SEC, and reduced CE were examined. The lyophilization process is described in Table 22, lyophilization process parameters 1.

[0247] [Table 24]

[0248] After being left at 40°C for one month, the SEC detection results (see Table 23) showed that the sucrose-based sample was slightly better than the trehalose-based sample and even better than the mannitol-based sample. The reduced CE detection results (see Table 23) showed that the sucrose-based sample was comparable to the trehalose-based sample and also superior to the mannitol-based sample.

[0249] [Table 25]

[0250] Example 2-4: Experiment to optimize the appearance of a sample after freeze-drying. A stock solution was prepared using 10 mM His-AA, pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. The stock solution was filtered and filled into containers, then freeze-dried according to freeze-drying process parameter 1 (see Table 22). The appearance of the freeze-dried sample was examined. The freeze-dried sample was a white solid powder with a flat surface, but the bottom edge of the solid powder was slightly shrunk.

[0251] The sugar concentration of the sample was further reduced to 60 mg / mL, and a stock solution was prepared using 10 mM His-AA, pH 5.0, 60 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. After filtering and filling into containers, the mixture was freeze-dried according to freeze-drying process parameter 1 in Table 22. After freeze-drying, the solid powder had a smooth, wrinkle-free surface, and the bottom edge of the solid powder was slightly shrunk.

[0252] The sugar concentration was reduced to 40 mg / mL, but in this case, the osmotic pressure of the finished product was too low, and there was a risk of osmotic pressure decreasing during clinical administration. To ensure the osmotic pressure of the finished product, the ionic strength of the buffer was increased to 30 mM. A stock solution was prepared with 30 mM His-AA, pH 5.0, 40 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. After filtration and filling into containers, it was freeze-dried according to freeze-drying process parameter 1. After freeze-drying, the surface of the sample solid powder was flat without depressions, and the bottom edge of the solid powder was intact.

[0253] The results for the three formulations are shown in Table 24.

[0254] [Table 26]

[0255] Examples 2-5: Stability of samples after freeze-drying The stock solution was prepared according to the formulations in Table 25, filtered, and filled into containers. After lyophilization according to lyophilization process parameter 1 (see Table 22), the lyophilized sample was left to stand at 40°C under M1 conditions, then redissolved and its stability change was detected.

[0256] The stability results are shown in Table 25. Under conditions of 40°C and M1, the lyophilized sample of formulation 3 showed a decrease in reduced CE of approximately 3.7%, while the remaining two formulations showed no significant changes in any of the chemical detection parameters, clearly demonstrating superior stability compared to formulation 3. The pH of the stock solution of formulation 2 before lyophilization was 5.04, and the pH of the redissolved solution after lyophilization was 5.27.

[0257] [Table 27]

[0258] Examples 2-6: Stability of lyophilized solution formulations The formulations were prepared according to the formulations in Table 26, filtered, filled into containers, plugged, and sealed. After undergoing a freeze-thaw cycle at -35°C / 4°C, the samples were left at room temperature for 3 days for study, followed by 11 days of shaking studies and high-temperature stability studies (40°C). Changes in the appearance, SEC, and reduced CE of the samples under the corresponding conditions were examined.

[0259] The stability results are shown in Table 26. After reducing the sucrose concentration and improving the ionic strength of the buffer solution, formulation #2 showed no significant difference from formulation #1 in terms of changes in appearance and purity, and under high-temperature conditions, the reduction in reduced CE was slightly better than that of formulation #1.

[0260] [Table 28] Furthermore, the present invention includes the following embodiments. [Aspect 1] A pharmaceutical composition comprising an anti-Claudin18.2 antibody-drug conjugate and a buffer, wherein the anti-Claudin18.2 antibody in the anti-Claudin18.2 antibody-drug conjugate includes a heavy chain variable region and a light chain variable region, of which, i) The heavy chain variable region has HCDR1, HCDR2, and HCDR3 having the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 5, and the light chain variable region includes LCDR1, LCDR2, and LCDR3 having the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 6, or ii) The heavy chain variable region has HCDR1, HCDR2, and HCDR3 having the same amino acid sequence as the heavy chain variable region shown in SEQ ID NO: 3, and the light chain variable region has LCDR1, LCDR2, and LCDR3 having the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 4. The buffer is a histidine salt buffer, preferably a histidine acetate buffer. Pharmaceutical composition. [Aspect 2] The aforementioned anti-Claudin18.2 antibody includes a heavy chain variable region and a light chain variable region, of which, iii) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NOs. 15, 16, and 17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NOs. 18, 19, and 20, or iv) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NOs. 9, 10, and 11, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NOs. 12, 13, and 14. Preferably, the anti-Claudin18.2 antibody includes a heavy chain variable region and a light chain variable region, of which, v) The heavy chain variable region is indicated by Sequence ID 31, and the light chain variable region is indicated by Sequence ID 29, or vi) The heavy chain variable region is indicated by Sequence ID 26, and the light chain variable region is indicated by Sequence ID 23. More preferably, the anti-Claudin18.2 antibody is vii) The heavy chain shown in Sequence ID No. 49, and the light chain shown in Sequence ID No. 47, or viii) The heavy chain shown in Sequence ID No. 44, and the light chain shown in Sequence ID No. 41, A pharmaceutical composition according to embodiment 1, comprising: [Aspect 3] The aforementioned anti-Claudin 18.2 antibody-drug conjugate has a structure represented by the general formula (Pc-LYD), namely, [ka] Eventually, Y is -O-(CRa R b ) m -CR 1 R 2 -C(O)-, -O-CR 1 R 2 -(CR a R b ) m -, -O-CR 1 R 2 -, -NH-(CR a R b ) m -CR 1 R 2 -C(O)- and -S-(CR) a R b ) m -CR 1 R 2 Selected from -C(O)-, R a and R b These are homologous or homologous, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, hydroxyl groups, amino groups, cyano groups, nitro groups, hydroxyalkyl groups, cycloalkyl groups, and heterocyclyl groups. Or, R a and R b These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocyclyl group. R 1 This is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups. R 2 This is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups. Or, R 1 and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocyclyl group. Or, R a and R 2 These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocyclyl group. m is an integer between 0 and 4. n is between 1 and 10, and n can be a decimal or an integer. L is the linker unit, Pc is an anti-Claudin18.2 antibody. Preferably, the anti-Claudin18.2 antibody-drug conjugate has a structure represented by the following formula, i.e.,

change

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Claims

1. A pharmaceutical composition comprising an anti-Claudin 18.2 antibody-drug conjugate and a histidine salt buffer, wherein the anti-Claudin 18.2 antibody in the anti-Claudin 18.2 antibody-drug conjugate comprises a heavy chain variable region and a light chain variable region, where, iii) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, or iv) The heavy chain variable region includes HCDR1, HCDR2, and HCDR3, respectively, as indicated by SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and the light chain variable region includes LCDR1, LCDR2, and LCDR3, respectively, as indicated by SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO:

14. Pharmaceutical composition.

2. v) The heavy chain variable region is indicated by Sequence ID 31, and the light chain variable region is indicated by Sequence ID 29, or vi) The heavy chain variable region is shown in sequence number 26, and the light chain variable region is shown in sequence number 23. The pharmaceutical composition according to claim 1.

3. The anti-Claudin 18.2 antibody is vii) The heavy chain shown in Sequence ID No. 49, and the light chain shown in Sequence ID No. 47, or viiii) The heavy chain indicated by Sequence ID No. 44, and the light chain indicated by Sequence ID No. 41, A pharmaceutical composition according to claim 1, comprising:

4. The anti-Claudin 18.2 antibody-drug conjugate has the general formula (Pc-L-Y-D): 【Chemistry 1】 [In the formula, Y is selected from -O-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)-, -O-CR １ R ２ -(CR ａ R ｂ ), ｍ -O-CR １ R ２ -, -NH-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)- and -S-(CR ａ R ｂ ), ｍ -CR １ R ２ -C(O)-; R ａ and R ｂ These are homologous or different, and each is independently selected from hydrogen atoms, deuterium atoms, halogens, alkyl groups, haloalkyl groups, deuterated alkyl groups, alkoxy groups, hydroxyl groups, amino groups, cyano groups, nitro groups, hydroxyalkyl groups, cycloalkyl groups, and heterocyclyl groups. Or, R ａ and R ｂ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocyclyl group. R １ This is selected from halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups. R ２ This is selected from hydrogen atoms, halogens, haloalkyl groups, deuterated alkyl groups, cycloalkyl groups, cycloalkylalkyl groups, alkoxyalkyl groups, heterocyclyl groups, aryl groups, and heteroaryl groups. Or, R １ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocyclyl group. Or, R ａ and R ２ These, together with the carbon atoms linked to them, form a cycloalkyl group or a heterocyclyl group. m is an integer between 0 and 4. n is between 1 and 10, and n can be a decimal or an integer. L is the linker unit, Pc is the anti-Claudin 18.2 antibody as defined in claim 1. Having the structure shown, The pharmaceutical composition according to claim 1.

5. Y is -O-(CR a R b) m -CR 1 R 2 -C(O)-, Ra and Rb are homologous or homologous, and each is independently selected from a hydrogen atom, a deuterium atom, a halogen, and a C1-6 alkyl group. R1 is a C1-6 alkyl group or a C3-6 cycloalkyl group. R2 is selected from a hydrogen atom, a C1-6 alkyl halogen, and a C3-6 cycloalkyl group. Alternatively, R1 and R2, together with the carbon atoms linked to them, form a C3-6 cycloalkyl group. m is 0 or 1, The linker unit -L- is -L1 -L2 -L3 -L4-, L1 is 【Chemistry 2】 And s1 is an integer from 2 to 8, L2 is a chemical bond, L3 is a tetrapeptide residue, L4 is -NR5(CR6R7)t-, where R5, R6 and R7 are homologous or different and each is independently a hydrogen atom or a C1-6 alkyl group, and t is 1 or 2. Here, the L1 end is connected to Pc, and the L4 end is connected to Y. The pharmaceutical composition according to claim 4.

6. The anti-Claudin 18.2 antibody-drug conjugate has the following formula: 【Transformation 3】 [In the formula, n is between 2 and 8, and n can be a decimal or an integer. Pc is the anti-Claudin 18.2 antibody as defined in claim 1. Having the structure shown, The pharmaceutical composition according to claim 4.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a surfactant.

8. The pharmaceutical composition according to claim 7, wherein the surfactant is polysorbate 80 or polysorbate 20.

9. The pharmaceutical composition according to claim 7, wherein the surfactant is polysorbate 80.

10. The pharmaceutical composition according to claim 7, wherein the concentration of the surfactant is 0.05 mg / mL to 0.5 mg / mL.

11. The pharmaceutical composition according to claim 7, wherein the concentration of the surfactant is 0.1 mg / mL to 0.2 mg / mL.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the composition further comprises sugar.

13. The pharmaceutical composition according to claim 12, wherein the sugar is selected from sucrose, mannitol, and trehalose.

14. The pharmaceutical composition according to claim 12, wherein the sugar is sucrose.

15. The pharmaceutical composition according to claim 12, wherein the concentration of the sugar is 20 mg / mL to 100 mg / mL.

16. The pharmaceutical composition according to claim 12, wherein the concentration of the sugar is 40 mg / mL to 80 mg / mL.

17. The pharmaceutical composition according to any one of claims 1 to 16, wherein the concentration of the anti-Claudin 18.2 antibody-drug conjugate is 1 mg / mL to 100 mg / mL in terms of protein concentration.

18. The pharmaceutical composition according to any one of claims 1 to 16, wherein the concentration of the anti-Claudin 18.2 antibody-drug conjugate is 10 mg / mL to 30 mg / mL in terms of protein concentration.

19. The pharmaceutical composition according to claim 1, wherein the histidine salt buffer is a histidine acetate buffer.

20. The pharmaceutical composition according to claim 1, wherein the concentration of the buffering agent is 5 mM to 50 mM.

21. The pharmaceutical composition according to claim 1, wherein the concentration of the buffering agent is 10 mM to 30 mM.

22. A pharmaceutical composition according to any one of claims 1 to 21, wherein the pH is 5.0 to 6.

5.

23. The pharmaceutical composition according to any one of claims 1 to 21, wherein the pH is 5.0 to 5.

5.

24. The pharmaceutical composition according to any one of claims 1 to 21, wherein the pH is 5.0 to 5.

3.

25. The following ingredients, namely, A pharmaceutical composition according to any one of claims 1 to 24, comprising (a) the anti-Claudin 18.2 antibody drug conjugate in a protein concentration of 10 mg / mL to 30 mg / mL, (b) 0.1 mg / mL to 0.2 mg / mL of polysorbate, (c) 40 mg / mL to 80 mg / mL of sugar, and (d) 10 mM to 30 mM of histidine salt buffer, wherein the pH is 5.0 to 5.

5.

26. A drug conjugate containing an anti-Claudin 18.2 antibody at a protein concentration of 20 mg / mL, 0.2 mg / mL of polysorbate 80, 40 mg / mL sucrose and It contains a 30 mM histidine acetate buffer, The pH is 5.0 to 5.

3. The anti-Claudin 18.2 antibody-drug conjugate has the following formula: 【Chemistry 4】 [In the formula, n is between 2 and 8, and n can be a decimal or an integer. Pc is an anti-Claudin 18.2 antibody containing the heavy chain shown in SEQ ID NO: 49 and the light chain shown in SEQ ID NO:

47. Having the structure shown, Pharmaceutical composition.

27. A lyophilized preparation comprising an antibody-drug conjugate, which is a lyophilized form of the pharmaceutical composition according to any one of claims 1 to 26.

28. A pharmaceutical composition according to any one of claims 1 to 26, or a lyophilized preparation according to claim 27, for the treatment of a tumor or cancer, Herein, the tumor or cancer is selected from squamous cell carcinoma of the head and neck, head and neck cancer, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, central nervous system cancer, neuroendocrine tumor, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatocellular carcinoma, hepatobiliary cancer, pancreatic cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, colon cancer, colorectal cancer, kidney cancer, clear cell renal cell carcinoma, ovarian cancer, endometrial cancer, cervical cancer, bladder cancer, prostate cancer, testicular cancer, skin cancer, melanoma, leukemia, lymphoma, bone cancer, chondrosarcoma, myeloma, multiple myeloma, myelodysplastic syndrome, Kruckenberg tumor, myeloproliferative neoplasm, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis, and Merkel cell carcinoma, and is a pharmaceutical composition or lyophilized preparation.

29. The pharmaceutical composition or lyophilized preparation according to claim 28, wherein the lymphoma is selected from Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma and lymphoplasmacytic lymphoma; the lung cancer is selected from non-small cell lung cancer and small cell lung cancer; and the leukemia is selected from chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid leukemia.

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