Pharmaceutical composition comprising antibody-drug conjugate, and use of pharmaceutical composition

Abstract

Provided are a pharmaceutical composition comprising an antibody-drug conjugate, and use of the pharmaceutical composition. Specifically provided is a pharmaceutical composition, comprising an anti-claudin antibody-drug conjugate.

Description

A pharmaceutical composition containing an antibody-drug conjugate and its use

[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. Technical Field

[0002] The present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising an antibody-drug conjugate, and its use as an anti-cancer drug. Background Art

[0003] The statements herein merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0004] Claudin-18 (CLDN18) is a protein encoded by the Claudin18 gene in humans. It belongs to the family of tight junction proteins and controls the flow of molecules between intercellular layers. The Claudin protein structure includes four transmembrane regions and two extracellular loops (with its N-terminus and C-terminus located in the cytoplasm).

[0005] Claudin-18 has two splice variants, Claudin 18.1 and Claudin 18.2, whose sequences differ only by eight amino acids in the first extracellular loop. The expression distributions of Claudin 18.1 and Claudin 18.2 differ: Claudin 18.1 is selectively expressed in normal lung cells, while Claudin 18.2 has highly restricted expression in normal cells but is frequently ectopically activated and overexpressed in various tumors (gastric, lung, and pancreatic cancers, among others). Claudin 18.2 is considered a potential therapeutic target for gastric cancer and other cancer types, and its discovery provides a new option for the treatment of gastric cancer.

[0006] Antibody drug conjugates (ADCs) link monoclonal antibodies or antibody fragments to biologically active cytotoxins via stable chemical linker compounds. These ADCs leverage the specificity of antibodies for binding to surface antigens on both normal and tumor cells and the high efficiency of cytotoxic substances, while avoiding the drawbacks of the former, such as low efficacy and the latter's excessive toxic side effects. This means that compared to traditional chemotherapy drugs, ADCs can more precisely bind to tumor cells while minimizing their effects on normal cells.

[0007] There are currently patent reports on antibodies and ADC drugs targeting Claudin18.2, such as WO2016166122 and WO2016165762.

[0008] Since ADCs have a more complex and heterogeneous structure than antibodies, they pose greater challenges to ADC formulation for therapeutic purposes.

[0009] Summary of the Invention

[0010] The present disclosure relates to pharmaceutical preparations containing anti-Claudin18.2 antibody-drug conjugates and their uses. The preparations have advantages such as good stability and a good lyophilized form.

[0011] The present 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, wherein:

[0012] i) the heavy chain variable region comprises 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 comprises LCDR1, LCDR2, and LCDR3 having the same amino acid sequence as the light chain variable region shown in SEQ ID NO: 6; or

[0013] ii) the heavy chain variable region has HCDR1, HCDR2, and HCDR3 having the same amino acid sequence as the heavy chain variable region set forth 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 set forth in SEQ ID NO: 4;

[0014] The buffer is a histidine buffer.

[0015] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the buffer is a histidine-acetate buffer.

[0016] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0017] iii) the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20, respectively; or

[0018] iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0019] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody is a murine antibody, a chimeric antibody, or a humanized antibody.

[0020] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein: comprises a heavy chain variable region and a light chain variable region, wherein:

[0021] (1) the heavy chain variable region amino acid sequence is as 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 amino acid sequence is as shown in SEQ ID NO:4, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto;

[0022] (2) the heavy chain variable region amino acid sequence is as 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 amino acid sequence is as shown in SEQ ID NO:21, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto;

[0023] (3) the heavy chain variable region amino acid sequence is as 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 amino acid sequence is as 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

[0024] (4) the heavy chain variable region amino acid sequence is as shown in 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 amino acid sequence is as shown in SEQ ID NO:28, or is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical thereto.

[0025] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody is a humanized antibody, which comprises a framework region derived from a human antibody or a framework region variant thereof, wherein the framework region variant has a back mutation of up to 10 amino acids in the light chain framework region and / or the heavy chain framework region of the human antibody, respectively.

[0026] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the framework region variant comprises a mutation selected from the following (a) or (b):

[0027] (a) the light chain variable region comprises one or more amino acid back mutations selected from 22S, 85I and 87H, and / or the heavy chain variable region comprises one or more amino acid back mutations selected from 48I, 82T and 69M; or

[0028] (b) the light chain variable region comprises one or more amino acid back mutations selected from 4L or 22S, and / or the heavy chain variable region comprises one or more amino acid back mutations selected from 38K, 40R, 48I, 66K, 67A, 69L, 71L and 73K.

[0029] In some embodiments, the pharmaceutical composition of any of the above items, wherein the framework region variant comprises a mutation selected from the group consisting of:

[0030] (a-1) amino acid reversion mutations comprising 22S, 85I, and 87H in the light chain variable region, and amino acid reversion mutations comprising 48I and 82T in the heavy chain variable region; or

[0031] (b-1) the light chain variable region comprises a 4L amino acid back mutation;

[0032] Wherein, the 82 in the 82T of the heavy chain variable region is the 82A position according to the Kabat rule.

[0033] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any of the following:

[0034] (vii) the heavy chain variable region is shown in SEQ ID NO: 3 and the light chain variable region is shown in SEQ ID NO: 4;

[0035] (viii) the heavy chain variable region is represented by SEQ ID NO: 24, 25, 26 or 27 and the light chain variable region is represented by SEQ ID NO: 21, 22 or 23;

[0036] (ix) the heavy chain variable region is shown in SEQ ID NO: 5 and the light chain variable region is shown in SEQ ID NO: 6; or

[0037] (x) the heavy chain variable region is set forth in SEQ ID NO: 31, 32, 33, or 34, and the light chain variable region is set forth in SEQ ID NO: 28, 29, or 30;

[0038] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region as shown in any of the following:

[0039] (xi) the heavy chain variable region is set forth in SEQ ID NO: 31 and the light chain variable region is set forth in SEQ ID NO: 29; or

[0040] (xii) the heavy chain variable region is shown in SEQ ID NO: 26 and the light chain variable region is shown in SEQ ID NO: 23.

[0041] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises an antibody heavy chain constant region and a light chain constant region.

[0042] In some embodiments, the heavy chain constant region is selected from human IgG1, IgG2, IgG3, and IgG4 constant regions and conventional variants thereof, and the light chain constant region is selected from human antibody kappa and lambda chain constant regions and conventional variants thereof. In some embodiments, the antibody comprises a heavy chain constant region as set forth in SEQ ID NO: 7 and a light chain constant region as set forth in SEQ ID NO: 8.

[0043] In some embodiments, the antibody comprises: a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a heavy chain having a sequence of amino acids set forth in SEQ ID NO: 35 or 42, and a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a light chain having a sequence of amino acids set forth in SEQ ID NO: 36 or 39; or a heavy chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a heavy chain having a sequence of amino acids set forth in SEQ ID NO: 37 or 49, and a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a light chain having a sequence of amino acids set forth in SEQ ID NO: The light chain of the amino acid sequence shown in NO:38 or 46 has a light chain that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical.

[0044] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises a heavy chain and a light chain as shown in any of the following:

[0045] (c) a heavy chain with a sequence as shown in SEQ ID NO: 35 and a light chain with a sequence as shown in SEQ ID NO: 36;

[0046] (d) a heavy chain having a sequence as shown in SEQ ID NO: 42, 43, 44 or 45 and a light chain having a sequence as shown in SEQ ID NO: 39, 40 or 41;

[0047] (e) a heavy chain having a sequence as shown in SEQ ID NO: 37 and a light chain having a sequence as shown in SEQ ID NO: 38; or

[0048] (f) a heavy chain having a sequence as shown in SEQ ID NO: 49, 50, 51 or 52 and a light chain having a sequence as shown in SEQ ID NO: 46, 47 or 48.

[0049] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody comprises a heavy chain and a light chain as shown in any of the following:

[0050] The heavy chain represented by SEQ ID NO:44, and the light chain represented by SEQ ID NO:41; or

[0051] The heavy chain is represented by SEQ ID NO:49, and the light chain is represented by SEQ ID NO:47.

[0052] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-LYD):

[0053]

[0054] in:

[0055] 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)-;

[0056] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group, and a heterocyclic group;

[0057] Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0058] R 1 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl;

[0059] R 2 is selected from the group consisting of a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group;

[0060] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0061] Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl group or a heterocyclyl group;

[0062] m is an integer from 0 to 4;

[0063] n is 1 to 10, and n is a decimal or an integer;

[0064] L is the joint unit;

[0065] Pc is anti-Claudin18.2 antibody;

[0066] In some embodiments, the pharmaceutical composition of any of the above items, wherein n is a decimal or an integer, can be 2 to 8, 3 to 7, 3.5 to 4.5, 2, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9.

[0067] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-LYD),

[0068] in:

[0069] Y is -O-(CR a R b ) m -CR 1 R 2 -C(O)-;

[0070] R a and R b are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen or a C 1-6 alkyl;

[0071] R 1 Halogenated C 1-6 Alkyl or C 3-6 Cycloalkyl;

[0072] R 2 Selected from hydrogen atoms, halogenated C 1-6 Alkyl or C 3-6 Cycloalkyl;

[0073] Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms C 3-6 Cycloalkyl;

[0074] m is 0 or 1.

[0075] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-LYD), wherein Y is selected from:

[0076]

[0077] The O end of Y is connected to the linker unit L.

[0078] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0079] L 1 Selected from -(succinimide-3-yl-N)-WC(O)-, -CH2-C(O)-NR 3 -WC(O)- or -C(O)-WC(O)-, wherein W is selected from C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight-chain heteroalkyl of 1 to 8 chain atoms, said heteroalkyl containing 1 to 3 heteroatoms selected from N, O or S, wherein said C 1-8 Alkyl, C 1-8 Alkyl-cycloalkyl or straight chain heteroalkyl of 1 to 8 chain atoms are each independently optionally further selected from halogen, hydroxy, cyano, amino, C 1-6 Alkyl, chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 substituted by one or more substituents of the cycloalkyl group;

[0080] L 2 Selected from-NR 4 (CH2CH2O)p 1 CH2CH2C(O)-、-NR 4 (CH2CH2O)p 1 CH2C(O)-、-S(CH2)p 1 C(O)- or chemical bond, where p 1 is an integer from 1 to 20;

[0081] L 3 The peptide residue is composed of 2 to 7 amino acids, wherein the amino acid residue is selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid, and is optionally further selected from halogen, hydroxyl, cyano, amino, C 1-6Alkyl, chloro C 1-6 Alkyl, deuterated C 1-6 Alkyl, C 1-6 Alkoxy and C 3-6 substituted by one or more substituents in the cycloalkyl group;

[0082] L 4 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;

[0083] R 3 、R 4 and R 5 are the same or different and are each independently selected from hydrogen atom, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 hydroxyalkyl;

[0084] R 6 and R 7 are the same or different and are each independently selected from hydrogen atom, halogen, C 1-6 Alkyl, halogenated C 1-6 Alkyl, deuterated C 1-6 Alkyl and C 1-6 Hydroxyalkyl.

[0085] In some embodiments, the pharmaceutical composition as described in any one of the above items, wherein the linker unit -L- is -L 1 -L 2 -L 3 -L 4 -,

[0086] L 1 for s 1 is an integer from 2 to 8;

[0087] L 2 is a chemical bond;

[0088] L 3 is a tetrapeptide residue; preferably a tetrapeptide residue of GGFG (SEQ ID No: 55);

[0089] L 4 -NR 5 (CR 6 R 7 )t-,R 5、R 6 or R 7 The same or different, and each independently a hydrogen atom or a C 1-6 Alkyl, t is 1 or 2;

[0090] The L 1 The end is connected to PC, L 4 Connect the Y end.

[0091] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein -L- is:

[0092]

[0093] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein LY- is optionally selected from:

[0094]

[0095] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody drug conjugate is selected from any of the following structures:

[0096]

[0097]

[0098] wherein Pc and n are as defined in the general formula (Pc-LYD).

[0099] In some embodiments, the pharmaceutical composition as described in any of the above items, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the following formula:

[0100]

[0101] in:

[0102] n is 2 to 8, and n is a decimal or an integer;

[0103] Pc is an anti-Claudin18.2 antibody.

[0104] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the pharmaceutical composition further comprises a surfactant. In some embodiments, the surfactant is selected from polysorbates (such as polysorbate 20, polysorbate 80), polyhydroxyalkylene, Triton, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl- Oleyl-Betaine, Myristyl-Betaine, Cetyl-Betaine, Lauramidopropyl-Betaine, Cocamidopropyl-Betaine, Linoleamidopropyl-Betaine, Myristamidopropyl-Betaine, Palmitamidopropyl-Betaine, Isostearamidopropyl-Betaine, Myristamidopropyl-Dimethylamine, Palmitamidopropyl-Dimethylamine, Isostearamidopropyl-Dimethylamine, Sodium Methyl Cocoyl, Sodium Methyl Oleyl Taurate, Polyethylene Glycol, Polypropylene Glycol, Copolymer of Ethylene and Propylene Glycol, etc.

[0105] 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.

[0106] In some embodiments, the pharmaceutical composition of any one of the above, wherein the surfactant concentration is 0.05 mg / mL to 0.5 mg / mL or 0.1 mg / mL to 0.2 mg / mL. In some embodiments, the surfactant concentration 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, 0.5 mg / mL. In some embodiments, the surfactant concentration is 0.2 mg / mL.

[0107] In some embodiments, the pharmaceutical composition as described in any one of the above, wherein the composition further comprises a sugar. In some embodiments, the sugar is selected from the conventional composition (CH2O) nand derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, and the like. The sugar may be selected from glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerol, erythritol, glycerol, arabitol, sylitol, sorbitol, mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, iso-maltulose, and the like. In some embodiments, the sugar is selected from sucrose, mannitol, and trehalose. In some embodiments, the sugar is sucrose.

[0108] In some embodiments, the pharmaceutical composition of any of the above, wherein the sugar concentration is 20 mg / mL to 100 mg / mL or 40 mg / mL to 80 mg / mL. In some embodiments, the sugar concentration 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 sugar concentration is 40 mg / mL.

[0109] In some embodiments, the pharmaceutical composition as described in any of the above, wherein the antibody drug conjugate concentration is 1 mg / mL to 100 mg / mL based on protein (i.e., antibody) concentration or 10 mg / mL to 30 mg / mL based on protein concentration. In some embodiments, the antibody drug conjugate concentration 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 based on protein concentration. In some embodiments, the antibody drug conjugate concentration is 20 mg / mL based on protein concentration.

[0110] In some embodiments, the pharmaceutical composition of any one of the above, wherein the concentration of the buffer is 5mM to 50mM or 10mM to 30mM. In some embodiments, the concentration of the buffer is 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM or 50mM. In some embodiments, the concentration of the buffer is 30mM.

[0111] In some embodiments, the pharmaceutical composition of any of the above items, wherein the pH of the pharmaceutical composition is 5.0-6.5 or 5.0-5.5. 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 5.0-5.4. In some embodiments, the pH of the pharmaceutical composition is 5.0-5.3.

[0112] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0113] (a) 10 mg / mL to 30 mg / mL of the anti-Claudin18.2 antibody drug conjugate at a protein concentration, (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 histidine salt buffer; the pH of the pharmaceutical composition is about 5.0-5.5.

[0114] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0115] (a) 10 mg / mL to 30 mg / mL of the anti-Claudin18.2 antibody drug conjugate at a protein concentration, (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 histidine buffer; the pH of the pharmaceutical composition is about 5.0-5.5.

[0116] In some embodiments, the pharmaceutical composition as described in any of the above items comprises the following components:

[0117] (a) 20 mg / mL of the anti-Claudin18.2 antibody-drug conjugate at a protein concentration, (b) 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL of sucrose, and (d) 30 mM histidine-acetate buffer, wherein the pH of the pharmaceutical composition is 5.0-5.3.

[0118] In some embodiments, in the pharmaceutical composition described in any one of the above items, the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the following formula:

[0119]

[0120] in:

[0121] n is 2 to 8, and n is a decimal or an integer;

[0122] Pc is an anti-Claudin18.2 antibody comprising a heavy chain represented by SEQ ID NO: 49 and a light chain represented by SEQ ID NO: 47; the pharmaceutical composition contains the antibody-drug conjugate at a protein concentration of 20 mg / mL;

[0123] The pharmaceutical composition further comprises the following components:

[0124] 0.2 mg / mL polysorbate 80, 40 mg / mL sucrose and 30 mM histidine-acetate buffer, the pH of the pharmaceutical composition is 5.0-5.3.

[0125] In some embodiments, the pharmaceutical composition as described in any one of the above items is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water.

[0126] The present disclosure also provides a lyophilized preparation containing an antibody drug conjugate, characterized in that the preparation can form any of the pharmaceutical compositions described above after reconstitution.

[0127] The present disclosure also provides a lyophilized preparation, which is a lyophilized form of the pharmaceutical composition as described above.

[0128] The present disclosure also provides a method for preparing a lyophilized formulation containing an antibody drug conjugate, comprising the step of freeze-drying the pharmaceutical composition as described in any one of the above items.

[0129] The present disclosure also provides a lyophilized formulation containing an antibody drug conjugate, wherein the formulation is obtained by freeze-drying the pharmaceutical composition as described in any one of the above items.

[0130] In some embodiments, the freeze-drying as described in any one of the above items comprises the steps of pre-freezing, primary drying and secondary drying in sequence.

[0131] In some embodiments, the freeze-drying procedure is as follows: pre-freezing at 5°C; pre-freezing at -45°C; primary drying at -20°C and a vacuum degree of 20 Pa; secondary drying at 25°C and a vacuum degree of 1 Pa. In some embodiments, the freeze-drying procedure is as follows: pre-freezing at 5°C and a time of 10 min; pre-freezing at -45°C and a time of 50 min; primary drying at -20°C and a vacuum degree of 20 Pa and a time of 120 min; secondary drying at 25°C and a vacuum degree of 1 Pa and a time of 60 min.

[0132] In some embodiments, the lyophilized formulation is stable at 2-8° 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.

[0133] The present disclosure also provides a lyophilized preparation, which is a reconstituted form of the lyophilized preparation described in any one of the above items.

[0134] The present disclosure also provides a reconstituted solution containing an antibody drug conjugate, characterized in that the reconstituted solution is prepared by reconstituted the lyophilized preparation as described in any one of the above items.

[0135] In some embodiments, the reconstitution solution described above comprises the following components:

[0136] (a) 10 mg / mL to 30 mg / mL of the anti-Claudin18.2 antibody drug conjugate at a protein concentration, (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 histidine buffer; the pH of the reconstituted solution is about 5.0-5.5.

[0137] In some embodiments, the reconstitution solution described above comprises the following components:

[0138] (a) 10 mg / mL to 30 mg / mL of the anti-Claudin18.2 antibody drug conjugate at a protein concentration, (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 histidine buffer; the pH of the reconstitution solution is about 5.0-5.5.

[0139] In some embodiments, the reconstitution solution described above comprises the following components:

[0140] (a) 20 mg / mL of the anti-Claudin18.2 antibody drug conjugate at a protein concentration, (b) 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL of sucrose, and (d) 30 mM histidine-acetate buffer, wherein the pH of the reconstitution solution is 5.0-5.3.

[0141] The present disclosure also provides a product, comprising a container containing the pharmaceutical composition as described in any one of the above items, the lyophilized formulation as described in any one of the above items, or the reconstituted solution as described in any one of the above items.

[0142] The present disclosure also provides a method for treating tumors or cancer, comprising administering to a subject an effective amount of the pharmaceutical composition, lyophilized formulation, reconstituted solution, or product described above.

[0143] In some embodiments, the present disclosure also provides use of the pharmaceutical composition as described in any of the above, the lyophilized formulation as described in any of the above, the reconstituted solution as described in any of the above, or the product as described in any of the above in the preparation of a drug for treating tumors or cancer.

[0144] In some embodiments, the present disclosure also provides a pharmaceutical composition as described above, a lyophilized formulation as described above, a reconstituted solution as described above, or a product as described above for use as a medicament.

[0145] In some embodiments, the tumor or cancer is preferably head and neck squamous cell carcinoma, 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, hepatoma, 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, Krukenberg tumor, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma.

[0146] In some embodiments, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary mediastinal large B-cell lymphoma, mantle cell lymphoma, small lymphocytic lymphoma, T-cell / histiocyte-rich large B-cell lymphoma, and lymphoplasmacytic lymphoma.

[0147] In some embodiments, the lung cancer is selected from the group consisting of non-small cell lung cancer and small cell lung cancer.

[0148] In some embodiments, the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloid cell leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0149] Figure 1: FACS detection results of humanized antibody binding to human Claudin18.2 at the cellular level.

[0150] Figure 2: NUGC4 cell endocytosis assay of humanized antibodies.

[0151] Figures 3A to 3C: Detection of the ADCC effect of the antibody in NUGC4 cells with varying levels of Claudin18.2 expression. Figure 3A shows the ADCC effect of the antibody in wild-type NUGC4 cells (low Claudin18.2 expression); Figure 3B shows the ADCC effect of the antibody in NUGC4 cells with moderate Claudin18.2 expression; Figure 3C shows the ADCC effect of the antibody in NUGC4 cells with high Claudin18.2 expression.

[0152] FIG4 shows the results of tumor inhibition experiments of ADC-1 disclosed herein.

[0153] FIG5 shows the results of tumor inhibition experiments of ADC-2 disclosed herein. DETAILED DESCRIPTION

[0154] the term

[0155] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise explicitly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present disclosure belongs.

[0156] Antibody-drug conjugates (ADCs) link antibodies or antibody fragments to biologically active cytotoxins or small molecule drugs with cell-killing activity via a stable chemical linker. These ADCs leverage the antibody's specificity for tumor cell-specific or highly expressed antigens and the high efficacy of the cytotoxin, while avoiding toxic side effects on normal cells. Compared to traditional chemotherapy drugs, ADCs can precisely bind to tumor cells while minimizing their effects on normal cells.

[0157] "Buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components. Examples of buffers that control pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffers.

[0158] 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; histidine acetate buffer is preferred. Histidine acetate buffer is a mixture of histidine and acetic acid, while histidine hydrochloride buffer is a mixture of histidine and hydrochloric acid.

[0159] "Citrate buffer" is a buffer comprising citrate ions. Examples of citrate buffers include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like. A preferred citrate buffer is citric acid-sodium citrate.

[0160] A "succinate buffer" is a buffer comprising succinate ions. Examples of succinate buffers include succinic acid-sodium succinate, succinic acid-potassium succinate, succinic acid-calcium succinate, and the like. A preferred succinate buffer is succinic acid-sodium succinate. For example, the succinic acid-sodium succinate can be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0161] "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, disodium hydrogen phosphate-citric acid, and the like. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0162] "Acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include acetate-sodium acetate, histidine acetate, acetate-potassium acetate, acetate-calcium acetate, acetate-magnesium acetate, and the like. A preferred acetate buffer is acetate-sodium acetate.

[0163] A "pharmaceutical composition" refers to a mixture containing one or more antibody-drug conjugates described herein, or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredient, facilitate administration to an organism, and promote absorption of the active ingredient, thereby exerting its biological activity.

[0164] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0165] Unless otherwise specified, the solvent in the solution form of the pharmaceutical composition described in the present disclosure is water.

[0166] "Lyophilized formulation" refers to a pharmaceutical composition in liquid or solution form or a formulation or pharmaceutical composition obtained after a liquid or solution formulation has been subjected to a vacuum freeze-drying step.

[0167] Although the present disclosure provides content ranges or content values, persons of ordinary skill in the art understand that the content ranges or content values ​​encompass an acceptable error range for the specific values ​​determined.

[0168] The pharmaceutical compositions disclosed herein can achieve a stable effect: the antibody drug conjugates therein substantially retain their physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical compositions substantially retain their physical and chemical stability and their biological activity after storage. The storage period is generally selected based on the intended shelf life of the pharmaceutical composition. Currently, there are various analytical techniques for measuring protein stability after storage at a selected temperature for a selected period of time.

[0169] A stable formulation is one in which no significant change is observed after storage at refrigerated temperatures (2-8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. In addition, a stable liquid formulation includes a liquid formulation that exhibits the desired characteristics after storage at a temperature of 25°C for a period of 1 month, 3 months, or 6 months. A typical example of stability is that no more than about 10%, preferably no more than about 5%, of the antibody monomers aggregate or degrade as measured by SEC-HPLC. By visual analysis, the formulation is a pale yellow, nearly colorless, clear liquid or colorless, or clear to slightly milky white. The concentration, pH, and weight-gram molecular osmotic pressure concentration of the formulation vary by no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5%, is usually observed. Aggregates of no more than about 10%, preferably no more than about 5%, are usually formed.

[0170] The antibody drug conjugate "retains its physical stability" in the pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation as measured by visual inspection of color and / or clarity, or by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).

[0171] An antibody drug conjugate "retains its chemical stability" in a pharmaceutical formulation if the antibody drug conjugate shows no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein. Degradation processes that often change the chemical structure of a protein include hydrolysis or truncation (assessed by methods such as size exclusion chromatography and CE-SDS), oxidation (assessed by methods such as peptide mapping in combination with mass spectrometry or MALDI / TOF / MS), deamidation (assessed by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartate measurement), and isomerization (assessed by measuring isoaspartate content, peptide mapping, etc.).

[0172] An antibody drug conjugate "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody drug conjugate at a given time is within a predetermined range of the biological activity exhibited when the pharmaceutical formulation is prepared.

[0173] The three letter and one letter codes for amino acids used in this disclosure are as described in J. biol. chem, 243, p3558 (1968).

[0174] The term "antibody" disclosed herein is used in the broadest sense and covers various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), full-length antibodies, or antigen-binding fragments thereof (also referred to as "antigen-binding portions"), as long as they exhibit the desired antigen-binding activity. A full-length antibody is an immunoglobulin (Ig) comprising at least two heavy chains and two light chains interconnected by disulfide bonds. The amino acid composition and arrangement order of the constant region of the immunoglobulin heavy chain are different, so their antigenicity is also different. Accordingly, immunoglobulins can be divided into five categories, or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgA, and IgE, and their corresponding heavy chains are μ chain, δ chain, γ chain, α chain, and ε chain, respectively. The same class of Ig can be divided into different subclasses based on the difference in the amino acid composition of its hinge region and the number and position of the heavy chain disulfide bonds, such as IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains are divided into κ chains or λ chains based on the difference in the constant region. Each of the five Ig classes can have either kappa or lambda chains.

[0175] The approximately 110 amino acids near the N-terminus of a full-length antibody heavy and light chain vary greatly in sequence and constitute the variable region (abbreviated as the Fv region). The remaining amino acid sequences near the C-terminus are relatively stable and constitute the constant region. Each heavy chain is composed of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH). The heavy chain constant region comprises three domains: CH1, CH2, and CH3. Each light chain is composed 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 include hypervariable regions (also called complementarity-determining regions, abbreviated as CDRs or HVRs) and relatively conserved framework regions (also called framework regions, abbreviated as FRs). Each VL and VH is composed of three CDRs and four FRs arranged from amino to carboxyl terminus in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The three CDR regions of the light chain are referred to as LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2, and HCDR3.

[0176] The "conventional variants" of the human antibody heavy chain constant region and the human antibody light chain constant region described in the present disclosure refer to variants of the heavy chain constant region or light chain constant region derived from humans that have been disclosed in the prior art and do not change the structure and function of the antibody variable region. Exemplary variants include IgG1, IgG2, IgG3 or IgG4 heavy chain constant region variants with site-directed modification and amino acid substitution of the heavy chain constant region, specific substitutions such as the YTE mutation known in the prior art, L234A and / or L235A mutation, S228P mutation, 265A (e.g., D265A) and / or 297A (e.g., N297A), and / or mutations to obtain a knob-into-hole structure (so that the antibody heavy chain has a knob-Fc and hole-Fc combination). These mutations have been shown to impart new properties to the antibody without changing the function of the antibody variable region.

[0177] The term "antigen-binding fragment" or "functional fragment" or "antigen-binding portion" refers to one or more fragments of an intact antibody that retains the ability to specifically bind to an antigen. It has been shown that fragments of a full-length antibody can be used to perform the antigen-binding function of an antibody. Exemplary binding fragments encompassed by the term "antigen-binding fragment" include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single arm of an antibody; (v) a dsFv, a stable antigen-binding fragment formed by an interchain disulfide bond between VH and VL; and (vi) diabodies, bispecific antibodies, and multispecific antibodies comprising fragments such as scFv, dsFv, and Fab. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, recombinant methods can be used to join the two domains through an artificial peptide linker that enables them to form a single protein chain, in which VL and VH pair to form a monovalent molecule called a single-chain Fv (scFv) (see, e.g., 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 an antibody. Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for functionality in the same manner as for intact antibodies. Antigen-binding portions can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0178] The term "amino acid difference" or "amino acid mutation" refers to the presence of amino acid changes or mutations in a variant protein or polypeptide compared to the original protein or polypeptide, including insertions, deletions or substitutions of 1, 2, 3 or more amino acids based on the original protein or polypeptide.

[0179] The term "antibody framework" or "FR region" refers to a portion of a variable domain VL or VH that serves as a scaffold for the antigen binding loops (CDRs) of the variable domain. Essentially, it is a variable domain without CDRs.

[0180] The term "complementarity determining region," "CDR," or "hypervariable region" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. Typically, there are three CDRs (HCDR1, HCDR2, HCDR3) in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region. The amino acid sequence boundaries of the CDRs can be determined using any of a variety of well-known schemes, including the "Kabat" numbering convention (see Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD), the "Chothia" numbering convention (see Al-Lazikani et al., (1997) JMB 273:927-948), and the ImMunoGenTics (IMGT) numbering convention (see Lefranc et al., (1998) JMB 274:927-948). MP, Immunologist, 7, 132-136 (1999); Lefranc, MP et al., Dev. Comp. Immunol., 27, 55-77 (2003) etc. For example, for the classical format, following the Kabat rule, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). Following the Chothia rule, the CDR amino acid residues in VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in VL are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3). Amino acid residues are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of Kabat and Chothia, the CDRs are composed 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. Following the IMGT rule, the CDR amino acid residues in VH are approximately numbered 26-35 (CDR1), 51-57 (CDR2), and 93-102 (CDR3), and the CDR amino acid residues in VL are approximately numbered 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR3).Following the IMGT rules, the CDR regions of the antibodies can be determined using the program IMGT / DomainGap Align. Following the AbM rules, the CDR amino acids in VH are numbered 26-32 (HCDR1), 50-58 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). The heavy chain variable region and light chain variable region of the antibodies disclosed herein and their CDRs conform to the Kabat numbering convention.

[0181] Amino acid sequence "identity" refers to the percentage of identical amino acid residues in a first sequence and a second sequence when the amino acid sequences are aligned (allowing for the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity); wherein conservative substitutions are not considered part of the sequence identity. To determine the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. One skilled in the art can determine parameters suitable for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences.

[0182] The engineered antibodies or antigen-binding fragments disclosed herein can be prepared and purified using conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. As a more preferred existing technology, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that bind to antigens. Positive clones are expanded in serum-free culture medium in a bioreactor to produce antibodies. The culture medium secreting the antibodies can be purified using conventional techniques. For example, purification can be performed using an A or G Sepharose FF column containing an adjusted buffer. Non-specifically bound components are washed away. The bound antibodies are then eluted using a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed using conventional methods, such as molecular sieves and ion exchange. The resulting product should be immediately frozen, such as at -70°C, or lyophilized.

[0183] "Conservative modification" or "conservative substitution or replacement" refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.), so that changes can be made frequently without changing the biological activity of the protein. It is known to those skilled in the art that, in general, single amino acid replacements in non-essential regions of a polypeptide do not substantially change the biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224, (4th ed.)). In addition, replacement of amino acids with similar structure or function is unlikely to destroy biological activity. Exemplary conservative replacements are as follows:

[0184] Table a. Conservative substitutions of amino acids

[0185] The original residues were conservatively substituted with Ala(A)Gly; SerArg(R)Lys; HisAsn(N)Gln; His; AspAsp(D)Glu; AsnCys(C)Ser; Ala; ValGln(Q)Asn; GluGlu(E)Asp; GlnGly(G)AlaHis(H)Asn; GlnIle(I)Leu; ValLeu(L)Ile; ValLys(K)Arg; HisMet(M)Leu; Ile; TyrPhe(F)Tyr; Met; LeuPro(P)AlaSer(S)ThrThr(T)SerTrp(W)Tyr; Phe

[0186] Tyr(Y)Trp;PheVal(V)Ile;Leu

[0187] The term "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or a radiolabel. In this disclosure, the content of the antibody drug conjugate is measured as protein concentration, i.e., the weight / volume of the protein (antibody portion) in the conjugate.

[0188] The term "linker unit" or "linker" refers to a chemical structure fragment or bond that is connected to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and can also be connected to other linkers before being connected to the drug. The preferred embodiment of the present disclosure is represented by L and L 1 To L 4 , where L 1 The end is connected to the antibody, L 4The end is connected to the structural unit Y and then connected to the compound or toxin. Linkers, including extenders, spacers and amino acid units, can be synthesized by methods known in the art, such as those described in US2005-0238649A1. The linker can be a "cleavable linker" that facilitates release of the drug in the cell. For example, an acid-labile linker (e.g., a hydrazone), a protease-sensitive (e.g., a peptidase-sensitive) linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Research 52:127-131 (1992); U.S. Patent No. 5,208,020).

[0189] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, which is a straight or branched chain 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. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-Dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl, 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl, and various branched-chain isomers thereof. More preferred are lower alkyl groups containing 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, and the like. The alkyl group may be substituted or unsubstituted. When substituted, the substituent may be substituted at any available point of attachment. The substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo.

[0190] The term "heteroalkyl" refers to an alkyl group containing one or more heteroatoms selected from N, O or S, wherein alkyl is as defined above.

[0191] The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms. An alkylene group is a straight or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene (-CH2-), 1,1-ethylene (-CH(CH3)-), 1,2-ethylene (-CH2CH2)-, 1,1-propylene (-CH(CH2CH3)-), 1,2-propylene (-CH2CH(CH3)-), 1,3-propylene (-CH2CH2CH2-), 1,4-butylene (-CH2CH2CH2CH2-), and 1,5-butylene (-CH2CH2CH2CH2CH2-). An alkylene group may be substituted or unsubstituted. When substituted, the substituents may be substituted at any available point of attachment and are preferably independently substituted with one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio and oxo.

[0192] The term "alkoxy" refers to -O-(alkyl) and -O-(unsubstituted cycloalkyl), wherein the definition of alkyl or cycloalkyl is as described above. Non-limiting examples of alkoxy include: methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy can be optionally substituted or unsubstituted, and when substituted, substituents are preferably one or more following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio.

[0193] The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent. The 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 cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, etc.; polycyclic cycloalkyls include spirocyclic, fused ring, and bridged ring cycloalkyls.

[0194] The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent containing 3 to 20 ring atoms, one or more of which is selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, but does not include -OO-, -OS- or -SS- ring moieties, the remaining ring atoms being carbon. The heterocyclic group preferably comprises 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably, the cycloalkyl ring comprises 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclic groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, etc. Polycyclic heterocyclic groups include spirocyclic, fused ring and bridged heterocyclic groups.

[0195] The term "spiroheterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclic group in which the rings share one atom (called a spiro atom), wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer 0 to 2) heteroatom, and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated π electron system. It is preferably 6 to 14 members, more preferably 7 to 10 members. According to the number of shared spiral atoms between the rings, the spiro heterocyclic group is divided into a monospiro heterocyclic group, a dispiro heterocyclic group or a polyspiro heterocyclic group, preferably a monospiro heterocyclic group and a dispiro heterocyclic group. It is more preferably 4 yuan / 4 yuan, 4 yuan / 5 yuan, 4 yuan / 6 yuan, 5 yuan / 5 yuan or 5 yuan / 6 yuan monospiro heterocyclic group. Non-limiting examples of spiro heterocyclic groups include:

[0196]

[0197] The term "fused heterocyclyl" refers to a 5- to 20-membered polycyclic heterocyclic ring system in which each ring shares a pair of adjacent atoms with the other rings in the system. In a fused heterocyclyl, one or more rings may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m (wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. The fused heterocyclic group is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, the fused heterocyclic group can be divided into bicyclic, tricyclic, tetracyclic or polycyclic rings; preferably bicyclic or tricyclic; more preferably 5-membered / 5-membered, or 5-membered / 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

[0198]

[0199] The term "bridged heterocyclic group" refers to a 5- to 14-membered polycyclic heterocyclic group in which any two rings share two atoms that are not directly connected. A bridged heterocyclic group may contain one or more double bonds, but no ring has a completely conjugated π electron system, wherein one or more ring atoms are selected from nitrogen, oxygen or S(O) m(wherein m is an integer from 0 to 2) heteroatoms, and the remaining ring atoms are carbon. The bridged heterocyclic group is preferably 6 to 14 members, more preferably 7 to 10 members. Depending on the number of constituent rings, the bridged heterocyclic group can be classified as bicyclic, tricyclic, tetracyclic or polycyclic; preferably bicyclic, tricyclic or tetracyclic; more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

[0200]

[0201] The heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring attached to the parent structure is a heterocyclyl, non-limiting examples of which include:

[0202] wait.

[0203] Heterocyclic radical can be optionally substituted or unsubstituted. When substituted, substituent is preferably one or more following groups, it is independently selected from alkyl, alkenyl, alkynyl, alkoxyl, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, oxo.

[0204] The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic or fused polycyclic (i.e., rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system. Aryl is preferably 6- to 10-membered, such as phenyl and naphthyl, with phenyl being preferred. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the aryl ring, non-limiting examples of which include:

[0205]

[0206] Aryl can be substituted or unsubstituted. When substituted, the substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0207] The term "heteroaryl" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. The heteroaryl group is preferably 5 to 10-membered, more preferably 5-membered or 6-membered, such as furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring, non-limiting examples of which include:

[0208]

[0209] Heteroaryl can be optionally substituted or unsubstituted. When substituted, substituent is preferably one or more of the following groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, sulfhydryl, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio.

[0210] The term "amino-protecting group" refers to a group that is easily removable to protect the amino group while other parts of the molecule react to maintain the amino group intact. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, tert-butyloxycarbonyl, acetyl, benzyl, allyl, and p-methoxybenzyl. These groups may optionally be substituted with 1-3 substituents selected from halogen, alkoxy, or nitro. The amino-protecting group is preferably 9-fluorenylmethyloxycarbonyl.

[0211] The term "cycloalkylalkyl" refers to a hydrogen atom on an alkyl group replaced by one or more cycloalkyl groups, preferably one cycloalkyl group, wherein alkyl is as defined above and cycloalkyl is as defined above.

[0212] The term "haloalkyl" refers to an alkyl group where a hydrogen atom is replaced by one or more halogen atoms, wherein alkyl is as defined above.

[0213] The term "deuterated alkyl" refers to an alkyl group in which hydrogen atoms are replaced by one or more deuterium atoms, wherein alkyl is as defined above.

[0214] The term "hydroxy" refers to an -OH group.

[0215] The term "halogen" refers to fluorine, chlorine, bromine or iodine.

[0216] The term "amino" refers to -NH2.

[0217] The term "nitro" refers to -NO2.

[0218] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region of the specified sequence may but need not be present.

[0219] "Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms, in a group are replaced independently of one another by a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and those skilled in the art can determine (by experiment or theory) possible or impossible substitutions without undue effort. For example, an amino or hydroxyl group with free hydrogen may be unstable when combined with a carbon atom with an unsaturated (e.g., olefinic) bond.

[0220] The term "drug loading" refers to the average amount of cytotoxic drug loaded per antibody or antigen-binding fragment thereof in an ADC molecule, and can also be expressed as the ratio of the amount of drug to the amount of antibody. The drug loading range can be 0-12, preferably 1-10, more preferably 2-8, and most preferably 3.5-4.5 cytotoxic drugs (D) attached per antibody or antigen-binding fragment thereof (Pc). In the embodiments disclosed herein, the drug loading is expressed as n, and exemplary n can be the calculated average of one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The average amount of drug per ADC molecule after the conjugation reaction can be determined using conventional methods such as UV / visible spectroscopy, mass spectrometry, ELISA assays, and HPLC characterization.

[0221] In one embodiment of the present disclosure, the cytotoxic drug is conjugated to the N-terminal amino group, the ε-amino group of a lysine residue, and / or the sulfhydryl group of an antibody or its antigen-binding fragment via a linker unit. Generally, the number of drug molecules that can be conjugated to the antibody in a conjugation reaction will be less than the theoretical maximum.

[0222] Cytotoxic drug loading can be controlled by the following non-limiting methods, including:

[0223] (1) Control the molar ratio of the linker and the monoclonal antibody,

[0224] (2) Control reaction time and temperature,

[0225] (3) Select different reaction reagents.

[0226] The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

[0227] The term "carrier" as used in the pharmaceutical compositions disclosed herein refers to a system that can change the way a drug enters the human body and its distribution in the body, control the rate of drug release, and deliver the drug to a targeted organ. Drug carrier release and targeting systems can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymer surfactants that can be used as carriers can self-assemble to form various forms of aggregates due to their unique amphiphilic structure, preferably micelles, microemulsions, gels, liquid crystals, vesicles, and the like. These aggregates have the ability to encapsulate drug molecules and have good permeability to membranes, making them excellent drug carriers.

[0228] "Administer," "give," and "treat" as applied to an animal, a human, a laboratory subject, a cell, a tissue, an organ, or a biological fluid, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, a human, a subject, a cell, a tissue, an organ, or a biological fluid. "Administer," "give," and "treat" can refer to, for example, therapeutic, pharmacokinetics, diagnostics, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administer," "give," and "treat" also mean the in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnostic, a binding composition, or by another cell. "Treatment," as applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research, and diagnostic applications.

[0229] "Treatment" means administering an internal or external therapeutic agent, such as a composition comprising any of the binding compounds of the present disclosure, to a patient who has one or more symptoms of a disease for which the therapeutic agent is known to have a therapeutic effect. Typically, the therapeutic agent is administered in an amount effective to alleviate one or more symptoms of the disease in the treated patient or population to induce regression of such symptoms or inhibit the development of such symptoms to any clinically measurable degree. The amount of a therapeutic agent effective to alleviate any specific disease symptom (also referred to as a "therapeutically effective amount") can vary according to a variety of factors, such as the patient's disease state, age, and weight, and the ability of the drug to produce the desired therapeutic effect in the patient. Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other health care professional to assess the severity or progression of the symptoms. Although embodiments of the present disclosure (e.g., methods of treatment or articles of manufacture) may not be effective in alleviating every symptom of the target disease, they should alleviate the target disease symptoms in a statistically significant number of patients as determined by any statistical test known in the art, such as Student's t-test, chi-square test, U test according to Mann and Whitney, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test.

[0230] An "effective amount" encompasses an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical condition. An effective amount also means an amount sufficient to permit or facilitate diagnosis. The effective amount 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 route and dosage of administration, and the severity of side effects. An effective amount can be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0231] "Displacement" refers to the replacement of the solvent system used to dissolve the antibody or ADC. For example, a high-salt or hypertonic solvent system containing the antibody or ADC is replaced by a physical manipulation using a buffer system that stabilizes the formulation, thereby stabilizing the antibody protein in the formulation. Such physical manipulations include, but are not limited to, ultrafiltration, dialysis, or centrifugation followed by reconstitution.

[0232] Example

[0233] The present disclosure is further described below with reference to the following examples, but these examples are not intended to limit the scope of this disclosure. Experimental methods in the examples disclosed herein that do not specify specific conditions were generally performed according to conventional conditions, such as those in the Cold Spring Harbor Laboratory's "Antibody Techniques Laboratory Manual" and "Molecular Cloning Manual," or according to the conditions recommended by the raw material or product manufacturer. Reagents whose specific sources are not specified were commercially available.

[0234] 1. Antibody-drug conjugates

[0235] Preparation of anti-Claudin18.2 antibody

[0236] Example 1-1: Construction of a cell line that highly expresses Claudin18.2

[0237] Using Lipofectamine 3000 transfection reagent, the pCDH-hClaudin18.2 lentiviral expression vector plasmid and the pVSV-G or pCMV-dR8.91 lentiviral system packaging vector were transfected into 293T viral packaging cells. The virus-containing culture supernatant was collected, filtered, and subjected to ultracentrifugation. The concentrated virus was used to infect the human gastric signet ring cell carcinoma cell line NUGC4. The cells were selected with puromycin for two to three weeks, and then single-cell sorting was performed by FACS.

[0238] The expression level of Claudin18.2 was determined based on the tumor IHC score: cells with a Claudin18.2 expression level comparable to that of a tumor with an IHC score of 3 were considered high-expressing cells, and cells with a Claudin18.2 expression level comparable to that of a tumor with an IHC score of 2 were considered moderate-expressing cells.

[0239] Based on FACS analysis of Claudin18.2 expression on the surface of lentivirus-infected NUGC4 cells, NUGC4 / hClaudin18.2 monoclonal cell lines with high Claudin18.2 expression were selected. Simultaneously, FACS analysis of Claudin18.2 expression on the surface of wild-type NUGC4 cells was used to select NUGC4 clones with moderate Claudin18.2 expression. Wild-type NUGC4 cells express low Claudin18.2.

[0240] The selected monoclonal cell lines were expanded and cultured, and cryopreserved for subsequent experiments.

[0241] Claudin18.2 sequence Genbank: NP_001002026 (SEQ ID NO: 1):

[0242]

[0243] Claudin18.2 DNA sequence (SEQ ID NO: 2):

[0244]

[0245]

[0246]

[0247] Examples 1-2: Generation of anti-human claudin18.2 monoclonal antibody

[0248] 1 Immunization

[0249] Anti-human Claudin18.2 monoclonal antibody was generated by immunizing mice.

[0250] SJL white mice were used in the experiment, female, 6-8 weeks old (Charles River Laboratory Animal Technology Co., Ltd., Beijing, VITAL RIVER LABORATORY ANIMALS TECHNOLOGY CO., LTD., Beijing; Animal production license number: SCXK (Beijing) 2012-0001). Feeding environment: SPF level. After the mice were purchased, they were raised in the laboratory environment for 1 week, with a 12 / 12 hour light / dark cycle adjustment, temperature 20-25°C; humidity 40-60%. The mice that had adapted to the environment were immunized according to the following protocol. The immunization antigen was huClaudin18.2-HEK293 cells (HEK-293 stable transfected cell line transfected with human Claudin18.2 plasmid).

[0251] Immunization protocol: Before the first immunization with cells, Gold Adjuvant (Sigma Cat No. T2684) 0.1 ml / animal was injected intraperitoneally (IP) into the mice; half an hour later, 0.1 ml of normal saline diluted to 1×10 8 / ml concentration of cell fluid. After the cells are evenly dispersed, they are inoculated on days 0, 14, 28, 42, and 56. Blood is collected on days 21, 35, 49, and 63, and the antibody titer in the mouse serum is determined by ELISA. After the 4th to 5th immunization, mice with high antibody titers in the serum and titers that tend to plateau are selected for spleen cell fusion. Three days before spleen cell fusion, boost immunization is performed, and 1×10 7 cell.

[0252] 2. Spleen cell fusion

[0253] The spleen lymphocytes were fused with myeloma cells Sp2 / 0 cells ( CRL-8287 TM ) were fused to obtain hybridoma cells. Hybridoma cells were 0.5-1×10 6 Resuspend the cells at a density of 1:1 / ml in complete medium (IMDM medium containing 20% ​​FBS, 1×HAT, and 1×OPI), seed 100 μl / well in a 96-well plate, incubate at 37°C, 5% CO2 for 3-4 days, then add 100 μl / well of HAT complete medium and continue culturing for 3-4 days until colonies form. Remove the supernatant, add 200 μl / well of HT complete medium (IMDM medium containing 20% ​​FBS, 1×HT, and 1×OPI), and incubate at 37°C, 5% CO2 for 3 days before performing ELISA analysis.

[0254] 3. Hybridoma Cell Screening

[0255] Based on the hybridoma cell growth density, the culture supernatant was tested using the binding ELISA method. Cells with strong binding ability to huClaudin18.2-HEK293 cells but no binding to HEK293 cells were selected and promptly expanded and frozen. After two to three subcloning steps, single cell clones were obtained.

[0256] Each time the cells are subcloned, a cell binding experiment is required. Through the above experimental screening, hybridoma clones are obtained, and antibodies are further prepared using serum-free cell culture methods. The antibodies are purified according to the purification example and used in the detection example.

[0257] Example 1-3: Humanization of murine antibodies

[0258] Monoclonal hybridoma cell lines mAb1901 and mAb1902 with high in vitro activity were selected; the monoclonal antibody sequences therein were cloned, and then humanized, recombinantly expressed, and activity evaluated.

[0259] The process of cloning sequences from hybridomas is as follows: Hybridoma cells in logarithmic growth phase were collected, RNA was extracted using Trizol (Invitrogen, 15596-018) (according to the kit instructions) and reverse transcribed (PrimeScript TM The cDNA obtained by reverse transcription was amplified by PCR using 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:

[0260] mAb1901 murine heavy chain variable region (SEQ ID NO: 3)

[0261]

[0262] mAb1901 murine light chain variable region (SEQ ID NO: 4)

[0263]

[0264] mAb1902 murine heavy chain variable region (SEQ ID NO: 5)

[0265]

[0266] mAb1902 murine light chain variable region (SEQ ID NO: 6)

[0267]

[0268] The above-mentioned murine heavy chain variable region and light chain variable region were respectively connected to the heavy chain constant region of the following human IgG1 antibody and the human κ light chain constant region to form chimeric antibodies ch1901 and ch1902.

[0269] The constant region of each antibody was selected from the following sequences:

[0270] Heavy chain constant region of human IgG1 antibody: (SEQ ID NO: 7)

[0271]

[0272] Human κ light chain constant region: (SEQ ID NO: 8)

[0273]

[0274] Humanization of murine monoclonal antibodies is performed using methods described in numerous publications in the field. Briefly, human constant domains are substituted for the parental (murine antibody) constant domains. Human germline antibody sequences are selected based on homology between the murine and human antibodies, and CDR grafting is performed. The present invention selected candidate molecules with good activity for humanization, with the following results.

[0275] 1. CDR region of mouse antibody

[0276] The amino acid residues of the VH / VL CDRs in Table 1 are identified and annotated using the Kabat numbering system.

[0277] The CDR sequences of the mouse antibodies are shown in Table 1:

[0278] Table 1. CDR sequences of mouse antibodies

[0279] Antibody mAb1901HCDR1DYGIH (SEQ ID NO: 9) HCDR2YISRGSSTIYYADTVKG (SEQ ID NO: 10) HCDR3GGYDTRNAMDY (SEQ ID NO: 11) LCDR1KSSQSLLNSGNQKNYLA (SEQ ID NO: 12) LCDR2GASTRAS (SEQ ID NO: 13) LCDR3QNDLYYPLT (SEQ ID NO: 11) NO: 14) Antibody mAb1902HCDR1SYWMH (SEQ ID NO: 15) HCDR2MIHPNSGSTNYNEKFKGR (SEQ ID NO: 16) HCDR3LKTGNSFDY (SEQ ID NO: 17) LCDR1KSSQSLLNSGNQKNYLT (SEQ ID NO: 18) LCDR2WASTRES (SEQ ID NO: 19) LCDR3QNAYTYPFT (SEQ ID NO:20)

[0280] 2. Select human germline FR region sequences

[0281] Based on the typical structures of the murine antibody VH / VLC DCR, the heavy and light chain variable region sequences were compared with the antibody Germline database to obtain a human germline template with high homology. The human germline light chain framework region is derived from the human kappa light chain gene.

[0282] 2.1 Humanization and backmutation design of mAb1901

[0283] An appropriate human antibody germline was selected to humanize the murine mAb1901 antibody. The CDR regions of the murine mAb1901 were transplanted onto the selected humanization template, replacing the humanized variable regions. The humanized variable regions were then recombined with the IgG constant regions to form a complete antibody. Simultaneously, the FR regions within the V region of the humanized antibody were backmutated. Exemplary backmutation methods and combinations are as follows:

[0284] Table 2. mAb1901 humanized antibody and reverse mutation *

[0285]

[0286] *All amino acid position numbers in the table are based on the Kabat numbering convention. In the heavy chain variable region, position 82 is position 82A according to the Kabat numbering convention.

[0287] Table 3. Sequences of the light chain variable region and heavy chain variable region of mAb1901 humanized antibody

[0288]

[0289] The corresponding heavy chain variable region in the table above is linked to the human IgG1 heavy chain constant region set forth in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is linked to the human kappa light chain constant region set forth in SEQ ID NO: 8 to form the light chain of the full-length antibody. In other embodiments, the heavy chain variable region and light chain variable region can also be linked to other heavy chain constant regions and light chain constant regions, respectively, to form the full-length antibody.

[0290] 2.2 Humanization and backmutation design of mAb1902

[0291] An appropriate human antibody germline was selected to humanize the murine mAb1902 antibody. The CDR regions of the murine mAb1902 were transplanted onto the selected humanization template, replacing the humanized variable regions. The humanized variable regions were then recombined with the IgG constant regions to form a complete antibody. Simultaneously, the FR regions within the V region of the humanized antibody were backmutated. Exemplary backmutation methods and combinations are as follows:

[0292] Table 4. mAb1902 humanized antibody and its back mutation design *

[0293]

[0294] *All amino acid position numbers in the table are based on the Kabat numbering convention.

[0295] Table 5. Sequences of the light chain variable region and heavy chain variable region of mAb1902 humanized antibody

[0296]

[0297] The corresponding heavy chain variable region in the above table is connected to the human IgG1 heavy chain constant region shown in SEQ ID NO: 7 to form the heavy chain of the full-length antibody, and the light chain variable region is connected to the human kappa light chain constant region shown in SEQ ID NO: 8 to form the light chain of the full-length antibody.

[0298] Chimeric antibody ch1901

[0299] ch1901 heavy chain: (SEQ ID NO: 35)

[0300]

[0301] ch1901 light chain: (SEQ ID NO: 36)

[0302]

[0303]

[0304] Chimeric antibody ch1902

[0305] ch1902 heavy chain: (SEQ ID NO: 37)

[0306]

[0307] ch1902 light chain: (SEQ ID NO: 38)

[0308]

[0309] Table 6. mAb1901 humanized antibody

[0310] Light and heavy chains H1H2H3H4L1h1901-1h1901-2h1901-3h1901-4L2h1901-5h1901-6h1901-7h1901-8L3h1901-9h1901-10h1901-11h1901-12

[0311] The full-length antibody light and heavy chain sequences are shown below:

[0312] Table 7. mAb1901 humanized antibody light chain and heavy chain sequences

[0313]

[0314]

[0315]

[0316] Table 8. mAb1902 humanized antibody

[0317] Light and heavy chains H11H12H13H14L11h1902-1h1902-2h1902-3h1902-4L12h1902-5h1902-6h1902-7h1902-8L13h1902-9h1902-10h1902-11h1902-12

[0318] The full-length antibody light and heavy chain sequences are shown below:

[0319] Table 9. mAb1901 humanized antibody light chain and heavy chain sequences

[0320]

[0321]

[0322] The positive control antibody disclosed herein is IMAB-362 (from WO2016166122).

[0323] Heavy chain (SEQ ID NO: 53)

[0324]

[0325] Light chain (SEQ ID NO: 54)

[0326]

[0327] The above antibodies were cloned, expressed and purified respectively using conventional gene cloning and recombinant expression methods.

[0328] Preparation of anti-Claudin18.2 ADC conjugates

[0329] drug

[0330] The drug moiety of the anti-Claudin18.2 ADC conjugates disclosed herein can be any suitable drug. Particularly suitable drugs are described, for example, in PCT Publication No. WO2020063676A1 (incorporated herein by reference in its entirety). Compound 9-A disclosed herein 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]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide, which has the following structure:

[0331]

[0332] ADC stock solution drug loading analysis

[0333] 1. UV-HPLC method

[0334] Place the cuvette filled with sodium succinate buffer in the reference absorption cell and the sample measurement absorption cell respectively, deduct the solvent blank, and then place the cuvette filled with the sample solution to be tested in the sample measurement absorption cell to measure the absorbance at 280nm and 370nm.

[0335] Result calculation: The ADC stock solution loading was determined by ultraviolet spectrophotometry (using the instrument: Thermo nanodrop2000 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, that is:

[0336] (1)A 280nm =ε mab-280 oeLh mab +ε Drug-280 oeLh Drug

[0337] ε Drug-280 : The average molar extinction coefficient of the drug at 280nm is 5100;

[0338] C Drug : drug concentration;

[0339] ε mab-280 : The average molar extinction coefficient of the single antigen solution at 280nm is 214600;

[0340] C mab : concentration of single antigen solution;

[0341] b: The optical path length is 1 cm.

[0342] Similarly, the total absorbance equation of the sample at 370nm can be obtained:

[0343] (2)A 370nm =ε mab-370 oeLh mab +ε Drug-370 oeLh Drug

[0344] ε Drug-370 : The average molar extinction coefficient of the drug at 370nm is 19000;

[0345] C Drug : drug concentration;

[0346] ε mab-370 : The extinction coefficient of the single antigen solution at 370nm is 0;

[0347] C mab : concentration of single antigen solution;

[0348] b: The optical path length is 1 cm.

[0349] The drug loading can be calculated by combining equations (1) and (2) with the extinction coefficients and concentration data of the monoclonal antibody and drug at two detection wavelengths.

[0350] Drug loading = C Drug / C mab .

[0351] 2. RP-HPLC method

[0352] Naked antibody and ADC sample to be tested (concentration: 1 mg / ml) were reduced with 4 μl of DDT (Sigma), incubated in a 37°C water bath for 1 hour, and then removed to an inner tube. Detection was performed using an Agilent 1200 HPLC using an Agilent PLRP-S 1000A 8μm 4.6x250mm column, 80°C column temperature, DAD detector wavelength of 280 nm, flow rate of 1 mL / min, and injection volume of 40 μL. The light and heavy chains were then identified by comparing the spectra of the sample with those of the naked antibody. The spectra of the test sample were then integrated to calculate the DAR value.

[0353] Solution preparation:

[0354] 1) 0.25M DTT solution:

[0355] Preparation example: Take 5.78 mg of DTT and add 150 μl of purified water to fully dissolve it to prepare 0.25 M DTT solution. Store at -20°C.

[0356] 2) Mobile phase A (0.1% TFA aqueous solution):

[0357] Preparation example: Measure 1000 ml of purified water into a graduated cylinder, add 1 mL of TFA (Sigma), mix thoroughly before use, and store at 2-8°C for 14 days.

[0358] 3) Mobile phase B (0.1% TFA acetonitrile solution):

[0359] Preparation example: Measure 1000 ml of acetonitrile into a graduated cylinder, add 1 mL of TFA, mix thoroughly before use, and store at 2-8°C for 14 days.

[0360] Data Analysis:

[0361] By comparing the spectra of the sample and the naked antibody, the positions of the light chain and heavy chain are distinguished, and then the spectrum of the test sample is integrated to calculate the DAR value.

[0362] The calculation formula is as follows:

[0363] Table 10. ADC light chain and heavy chain drug labeling table

[0364] Name connected drug number LC0LC+12HC0HC+12HC+24HC+36

[0365] Sum of LC peak areas = LC peak area + LC+1 peak area;

[0366] The sum of HC peak areas = HC peak area + HC+1 peak area + HC+2 peak area + HC+3 peak area;

[0367] LC DAR = Σ(number of linked drugs * peak area percentage) / total LC peak area;

[0368] HC DAR = Σ(number of connected drugs * peak area percentage) / sum of HC peak areas;

[0369] DAR=LC DAR+HC DAR.

[0370] Example 1-4: ADC-1 / ADC-2

[0371] At 37°C, an aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.03 mL, 110.3 μmol) was added to PBS buffer (pH = 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 320.0 mL, 21.62 μmol) containing antibody h1902-5. The mixture was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction.

[0372] The reaction solution was cooled to 25°C using a water bath, and compound 9-A (350 mg, 303 mol) was dissolved in 13.2 ml of acetonitrile and 6.6 ml of DMSO and added to the reaction solution. The mixture was placed in a water bath shaker and shaken at 25°C for 3 hours to stop the reaction.

[0373] The reaction solution was purified by ultrafiltration to remove small molecules. Purification was performed using 5 L of 50 mM PBS buffer (4% acetonitrile, 2% DMSO) at pH 6.5, followed by 5 L of 10 mM succinate buffer (pH 5.3). Sucrose was then added to the purified solution to a concentration of 60 mg / mL, and Tween-20 to a concentration of 0.2 mg / mL. ADC-1 (10 mM, pH 5.3 succinate buffer; 10 mg / mL, 2.626 g) was obtained with a yield of 81.81%. The product was then prepared as a lyophilized powder at a concentration of 20 mg / vial.

[0374] UV-HPLC calculated average value: n=6.8.

[0375] Using the above method, antibody h1901-11 can be used instead of h1902-5, and compound 9-A can be used to prepare ADC-2, with n=7.1.

[0376] Example 1-5: ADC-3

[0377] At 37°C, a solution of antibody h1901-11 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 1 mL, 67.5 nmol) was added with a prepared TCEP aqueous solution (10 mM, 10.1 μL, 101 nmol). The solution was shaken in a water bath at 37°C for 3 hours, and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0378] Compound 9-A (0.58 mg, 540 nmol) was dissolved in 34 μl of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-3 in PBS buffer (0.72 mg / mL, 11.2 mL) and stored at 4°C. RP-HPLC calculated average: n = 2.51.

[0379] Example 1-6: ADC-4

[0380] At 37°C, a solution of antibody h1901-11 in PBS buffer (0.05 M PBS buffer, pH 6.5; 10.0 mg / mL, 1 mL, 67.5 nmol) was added with a prepared TCEP aqueous solution (10 mM, 16.9 μL, 169 nmol). The solution was shaken in a water bath at 37°C for 3 hours, and the reaction was stopped. The reaction solution was cooled to 25°C in a water bath.

[0381] Compound 9-A (0.73 mg, 680 nmol) was dissolved in 43 μl of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, 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. RP-HPLC calculated average: n = 4.06.

[0382] Example 1-7: ADC-5

[0383] To a solution of antibody h1901-11 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1 mL, 67.5 nmol) was added a prepared TCEP aqueous solution (10 mM, 35.8 μL, 358 nmol) at 37°C. The mixture was shaken in a water bath at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath.

[0384] Compound 9-A (1.09 mg, 1015 nmol) was dissolved in 64 μl of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-5 in PBS buffer (0.54 mg / mL, 12.5 mL) and stored at 4°C. RP-HPLC calculated average: n = 6.8.

[0385] Example 1-8: ADC-6

[0386] To a solution of antibody h1902-5 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.08 mL, 72.9 nmol) at 37°C, add the prepared TCEP aqueous solution (10 mM, 10.9 μL, 109 nmol). Place in a water bath shaker and shake at 37°C for 3 hours to stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0387] Compound 9-A (0.63 mg, 587 nmol) was dissolved in 40 μl of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-6 in PBS buffer (0.7 mg / mL, 13.0 mL). The solution was stored at 4°C. RP-HPLC calculated average: n = 2.69.

[0388] Example 1-9: ADC-7

[0389] To a solution of antibody h1902-5 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.08 mL, 72.9 nmol) at 37°C, add the prepared TCEP aqueous solution (10 mM, 18.3 μL, 183 nmol). Place in a water bath shaker and shake at 37°C for 3 hours to stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0390] Compound 9-A (0.79 mg, 736 nmol) was dissolved in 50 μl of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-7 in PBS buffer (0.6 mg / mL, 14.0 mL) and stored at 4°C. RP-HPLC calculated average: n = 4.25.

[0391] Example 1-10: ADC-8

[0392] To a solution of antibody h1902-5 in PBS buffer (pH 6.5, 0.05 M PBS buffer; 10.0 mg / mL, 1.08 mL, 72.9 nmol) at 37°C, add the prepared TCEP aqueous solution (10 mM, 38.7 μL, 387 nmol). Place in a water bath shaker and shake at 37°C for 3 hours to stop the reaction. Cool the reaction solution to 25°C in a water bath.

[0393] Compound 9-A (1.18 mg, 1099 nmol) was dissolved in 70 μl of DMSO and added to the above reaction solution. The mixture was shaken in a water bath at 25°C for 3 hours, and the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution at pH 6.5, containing 0.001 M EDTA) to obtain ADC-8 in PBS buffer (0.56 mg / mL, 14.2 mL) and stored at 4°C. RP-HPLC calculated average: n = 7.01.

[0394] Example 1-11: ADC-9

[0395] At 12°C, prepared TCEP histidine buffer (10 mM histidine buffer; 1.717 mM, 1.16 L 1.99 mmol) was added to histidine-acetate-Tris / EDTA buffer (10 mM histidine-acetate-Tris and 2.5 mM EDTA buffer at pH 7.2; 20.6 g / L, 6.49 L, 0.91 mmol) containing antibody h1902-5. The mixture was placed in a thermostatic water bath and stirred at 12°C for 2 hours. The reaction was stopped to obtain an intermediate I solution.

[0396] Dissolve compound 9-A (4.72 g, 4.39 mmol) in 0.38 L DMSO to generate a DMSO solution of compound 9-A. Add 0.38 L DMSO to the intermediate I solution above, then add the DMSO solution of compound 9-A above. Place in a thermostatic water bath and stir at 12°C for 1 hour to stop the reaction.

[0397] The reaction solution was purified by Capto S Impact cationic chromatography, washing with 9 column volumes of 0.05 M acetate buffer (pH 5.0) containing 10% (v / v) DMSO and 6 column volumes of 0.05 M acetate buffer (pH 5.0), followed by elution with 0.05 M acetic acid, 0.30 M sodium chloride buffer (pH 5.5) to remove free toxins and residual solvent. The cationic eluate was then subjected to 7 equal volumes of ultrafiltration at 22°C (using a 30 kD polycellulose membrane) to obtain the product ADC-9. RP-HPLC calculated average: n = 4.1.

[0398] Biological evaluation

[0399] Test Example 1: Cell-level ELISA binding experiment

[0400] A cell-based ELISA assay was used to test the binding properties of the Claudin18.2 antibody. NUGC4 cells stably expressing Claudin18.2 were cultured in 96-well cell plates (Corning, 3599). When cells reached 90% confluency, 4% paraformaldehyde was added to fix the cells for 1 hour. The plates were washed three times with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20). Then, 200 μl / well of blocking solution (5% skim milk (Bright skim milk powder) diluted in PBS) was added. The plates were incubated at 37°C for 2.5 hours or at 4°C overnight (16-18 hours) for blocking. After blocking, the blocking solution was discarded, and the plates were washed three times with PBST buffer. Then, 50 μl / well of the test antibody diluted in sample diluent (pH 7.4 PBS containing 1% skim milk) was added and incubated at 37°C for 2 hours. After incubation, the plate was washed five times with PBST and 100 μl / well of HRP-labeled goat anti-human secondary antibody (Jackson Immuno Research, 109-035-003) diluted in 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 and incubated at room temperature for 10-15 minutes. The reaction was terminated by adding 50 μl / well of 1 M H2SO4. The absorbance was read at 450 nm using an MD Versa Max™ microplate reader, and the EC50 value of claudin18.2 antibody binding to claudin18.2 was calculated.

[0401] Table 11. Binding activity of antibodies

[0402] Antibody IMAB362ch1901ch1902Emax1.1751.3991.272EC50(nM)0.1080.0980.074

[0403] Table 12. Binding activity of mAb1901 humanized antibody

[0404] Antibody EmaxEC50(nM)IMAB3621.1150.086h1901-21.0390.076h1901-31.10550.22h1901-40.9860.201h1901-60.9370.091h1901-70.9210.166h1901-81.0470.091h1901-111.440.076h1901-121.220.116

[0405] Table 13. Binding activity of mAb1902 humanized antibody

[0406] Antibody Emax EC50 (nM) IMAB362 0.88 0.187 h1902-10.87 0.113 h1902-20.88 0.107 h1902-30.84 0.175 h1902-40.82 0.087 h1902-50.9 0.098 h1902-60.78 0.141 h1902-70.75 0.121 h1902-80.89 0.132 h1902-90.75 0.137 h1902-100.89 0.133

[0407] Test Example 2: Antibody Cell Level Binding Experiment

[0408] NUGC4 cells stably expressing Claudin18.2 were prepared into 1×10 6 / ml cell suspension, 100μl / well was added to a 96-well round-bottom plate (Corning, 3795). After centrifugation to remove the supernatant, 50μl / well of different concentrations of the Claudin18.2 antibody to be tested diluted with FACS buffer was added and incubated in a 4°C refrigerator in the dark for 1 hour. After centrifugation and washing three times at 300g with FACS buffer, the working concentration of Alexa Fluor 488-coated anti-human IgG (H+L) (invitrogen, A-11013) was added and incubated in a 4°C refrigerator in the dark for 40 minutes. After centrifugation and washing three times at 300g with FACS buffer, the geometric mean fluorescence intensity was detected on a BD FACS CantoII flow cytometer, and the EC50 value of the binding of the Claudin18.2 antibody to NUGC4 cells stably expressing Claudin18.2 was calculated. The results are shown in Figure 1.

[0409] Test Example 3: Antibody Internalization Experiment

[0410] The Claudin18.2 antibody to be tested, pre-labeled with DyLight 488 NHS Ester (thermofisher, 46403), was added to 1×10 6 NUGC4 cells stably expressing Claudin18.2 were incubated on ice for 1 hour in the dark. The cells were then washed three times with pre-chilled FACS buffer (PBS, pH 7.4, 2% fetal bovine serum) by centrifugation. The supernatant was removed, pre-warmed complete culture medium was added, and the cells were placed in a 37°C, 5% CO2 cell culture incubator. Cells were removed after 0, 0.5, 1, 2, and 4 hours, and stored on ice in the dark. After all samples were collected, the supernatant was removed by cold centrifugation at 300g. Elution buffer (0.05M glycine, pH 1.7, 0.1M sodium chloride) was added, and the cells were incubated at room temperature for 7 minutes. The cells were washed once with FACS buffer by centrifugation at 300g. The geometric mean fluorescence intensity was measured on a BD FACS CantoII flow cytometer, and the internalization efficiency of the Claudin18.2 antibody in NUGC4 cells stably expressing Claudin18.2 was calculated. The results (see Figure 2) show that the humanized antibody has good internalization efficiency.

[0411] Test Example 4: Determination of Antibody Affinity Based on Flow Cytometry

[0412] On the day of the experiment, HEK293 / hClaudin18.2 cells were collected and plated in a U-bottom 96-well plate at a rate of 1×10 cells per well. 5 to 2×10 5 cells. Add Claudin18.2 antibody with a starting concentration of 5 μg / ml and 2× gradient dilution (12 concentration points), incubate at 4°C for 1 hour, the positive control is IMAB362, and a negative control without antibody is set up. Centrifuge to remove the antibody, then add 100 μl / well FITC anti-human IgG Fc antibody (200×), incubate at 4°C in the dark for 30 minutes, wash twice with PBS + 2% FBS and prepare for flow cytometry detection. Start BD FACS CantoII, open BD FACSDiva software after preheating, set up a new experiment, detect HEK293 / hClaudin18.2 negative control sample, adjust FSC and SSC voltages to appropriate values ​​and save. According to Quantum TM According to the FITC-5 MESF Kit instructions, blank sample B and standard curve 1 were tested separately, and the FITC voltage was adjusted to an appropriate value and saved. The samples in the U-bottom 96-well plate were tested at the saved voltage and the data were recorded. The experimental data were analyzed using Flowjo software to obtain the Geo Mean value. TMThe MESF-Geo Mean standard curve was fitted according to the FITC-5MESF Kit instructions. The molar concentration of the Claudin18.2 antibody bound to HEK293 / hClaudin18.2 cells and the free antibody concentration were calculated based on the fluorescence values ​​of the FITC anti-human IgG Fc antibody. The antibody's Bmax and dissociation constant, KD, were calculated using Scatchard plotting. The results are shown in Table 14.

[0413] Table 14. Humanized antibody cell-level affinity

[0414] Antibody IMAB362h1901-11h1902-5KD (nM) 10.26.81.64

[0415] Test Example 5: Evaluation of the ADCC effect of antibodies

[0416] Digest various NUGC4 cells (high, medium, and low expression of Claudin18.2), centrifuge at 1000 rpm, resuspend and count. 5 Resuspend cells at a density of 100 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). Add 25 μl of cells per well of a 96-well plate (Corning, 3903) (7500 cells / well). Dilute the antibody in the above phenol red-free medium to a 3x dilution, and add 25 μl / well of the antibody to the cell plate. Incubate in a 37°C, 5% CO2 incubator for 0.5 hours.

[0417] Effector cells (FcrR3A-V158-NFAT-RE-Jurkat cells) were collected, centrifuged at 1000 rpm, and then resuspended and counted. 6 The cells were resuspended in phenol red-free RPMI 1640 supplemented with 10% FBS (New Zealand ultra-low IgG fetal bovine serum) at a density of 10 cells / ml, and 25 μl of cells (7.5×10 4 The cells were cultured in a 37°C, 5% CO2 incubator for 6 hours.

[0418] 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).

[0419] The results showed (see Table 15 and Figures 3A-3C) that antibodies h1901-11 and h1902-5 both exhibited strong ADCC activity in NUGC4 cells expressing Claudin18.2 to varying degrees: low (Figure 3A), medium (Figure 3B), and high (Figure 3C).

[0420] Table 15. IC50 (ng / ml) of ADCC effector units of antibodies in NUGC4 cells with different expression levels of Claudin18.2

[0421] Claudin18.2 expression level h1901-11 h1902-5 IMAB362 low expression 22.42 35.46 183.4 medium expression 15.35 30.00 210.4 high expression 26.17 32.16 132.6

[0422] Test Example 6: ADC Molecular Cell Activity Experiment

[0423] This experiment used the CellTiter-Glo Luminescence Cell Viability Assay to detect the killing effect of ADC molecules on human gastric cancer cell lines in vitro. On the first day, cells with low expression of NUGC4-claudin18.2, medium expression of NUGC4-claudin18.2, and high expression of NUGC4-claudin18.2 were collected and the density was adjusted to 2.5×10 4 90 μl / well of the diluted sample was added to a 96-well white, clear-bottom plate, for approximately 2,500 cells per well. Incubate overnight at 37°C in a 5% CO2 incubator. The next day, dilute the sample in a U-bottom 96-well plate, starting at 5 μM and performing a 4× serial dilution across nine concentrations. Add 10 μl / well of the diluted sample to the plate. Incubate at 37°C in a 5% CO2 incubator for 6 days. On the eighth day, remove the plate and add 50 μl / well of Cell Titer-Glo Reagent. Incubate at room temperature for 2 to 3 minutes. Luminescence readings were obtained using a PHERAstar FS microplate reader. Data were analyzed using GraphPad Prism software. See Table 16.

[0424] Table 16. ADC in vitro cell killing assay

[0425]

[0426] Test Example 7: In vivo efficacy evaluation of ADC molecules

[0427] Balb / c naked mice were subcutaneously inoculated with human gastric cancer cell NUGC4 (Claudin18.2 moderately expressed) cells (5×106 Containing 50% matrigel per mouse), the mice were divided into groups on day 0, 8 mice per group, for a total of 8 groups. The average tumor volume was approximately 84.41 mm 3 .

[0428] ADC was injected intraperitoneally for 3 times, with each mouse receiving 10 g / 0.1 ml of the drug according to body weight, on day 0, day 4, and day 11, respectively.

[0429] ADC was injected intraperitoneally on the day of grouping, for a total of 4 times with an interval of 5 days. Each animal was injected with 10g / 0.1ml according to body weight.

[0430] Tumor volume and body weight were measured twice a week and the data were recorded.

[0431] Excel 2003 statistical software was used: mean was calculated as avg; SD was calculated as STDEV; SEM was calculated as STDEV / SQRT; and P values ​​of intergroup differences were calculated as TTEST.

[0432] The formula for calculating tumor volume (V) is: V = 1 / 2 × L 长 ×L 短 2

[0433] Relative volume (RTV) = VT / V0

[0434] Tumor inhibition rate (%) = (CRTV-TRTV) / CRTV (%)

[0435] Where V0 and VT are the tumor volumes at the start of the experiment (day 0, the first dose), and at the end of the experiment, respectively. CRTV and TRTV are the relative tumor volumes 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.

[0436] Table 17. ADC tumor inhibition experimental results

[0437]

[0438] vs blank control group: **p<0.01.

[0439] 2. Preparation

[0440] The equipment used in the preparation and testing process and the calculation method of the results are as follows:

[0441] SEC size exclusion chromatography:

[0442] An analytical method that separates solutes based on the relative relationship between the pore size of the gel and the coil size of the polymer sample molecules.

[0443] SEC% (SEC monomer content percentage) = 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).

[0444] SEC instrument: Agilent 1260; columns: waters, XBrige SEC (300×7.8 mm 3.5 μm).

[0445] CE capillary gel electrophoresis:

[0446] A method of electrophoresis in which gel is transferred to a capillary tube as a supporting medium and samples are separated according to their molecular weight at a certain voltage.

[0447] Reduction CE purity percentage = A main peak / A total * 100% (A main peak is the peak area of ​​the light chain main peak + the heavy chain main peak in the sample, and A total is the sum of all peak areas.

[0448] CE measurement instrument: Beckman model plus800.

[0449] Osmolality determination:

[0450] The freezing point method determines osmotic pressure based on the proportional relationship between freezing point depression and the molar concentration of a solution. It uses a highly sensitive temperature sensor to measure the freezing point of a solution and converts the electrical charge into osmotic pressure. Instrument manufacturer: Loser, model OM815.

[0451] Protein concentration determination:

[0452] Because the drug in the antibody drug conjugate absorbs at 280nm, the protein concentration is corrected using the following formula:

[0453] A280=Cd*ε280d+Cmab*ε280mab;

[0454] A370=Cd*ε370d;

[0455] Cd represents the drug concentration, Cmab represents the protein concentration, ε280d represents the drug extinction coefficient at 280 nm, ε280mab represents the protein extinction coefficient at 280 nm, and ε370d represents the drug extinction coefficient at 370 nm. ε280mab = 1.49 mg-1*cm-1*ml, ε280d = 5000 (drug molar extinction coefficient at 280 nm) / 1074.13 (drug molecular weight) = 4.65 mg-1*cm-1*mL, ε370d = 19000 (drug molar extinction coefficient at 370 nm) / 1074.13 (drug molecular weight) = 17.69 mg-1*cm-1*mL. The above extinction coefficients are mass extinction coefficients.

[0456] Protein concentration determination instrument: UV-visible spectrophotometer, model: Nano Drop oneC, optical path length: 1 mm.

[0457] Example 2-1: Screening of formulation buffer system and pH value

[0458] Formulations containing 20 mg / mL (protein concentration) of ADC-9 and the following different buffer systems, as well as 0.1 mg / mL polysorbate 80 (PS80) were prepared.

[0459] 1) 10 mM citric acid-sodium citrate (CA), pH 5.5

[0460] 2) 10 mM succinic acid-sodium succinate (SA), pH 5.0

[0461] 3) 10 mM succinic acid-sodium succinate, pH 5.5

[0462] 4) 10 mM histidine hydrochloride (His-HCl), pH 5.5

[0463] 5) 10 mM histidine hydrochloride, pH 6.0

[0464] 6) 10 mM histidine hydrochloride, pH 6.5

[0465] 7) 10 mM histidine-acetate (His-AA), pH 5.0

[0466] 8) 10 mM histidine-acetate, pH 5.5

[0467] 9) 10 mM phosphate (PB), pH 6.5.

[0468] Each formulation was filtered, filled, stoppered, and capped. Samples were taken for high temperature stability (40°C), shaking (25°C, 300 rpm), and appearance, SEC, and reduced CE. The results are shown in Table 18.

[0469] After shaking for 11 days, only samples 2), 3), 7), and 9) showed a clear appearance. After 15 days at 40°C, samples 1), 2), 3), 7), and 8) showed a clear appearance. This indicates that from an appearance perspective, the formulations of samples 2), 3), and 7) are superior.

[0470] After being placed at 40°C for 15 days, the samples were tested by SEC. The results showed that the monomer content of samples 4), 5), 6), 7), and 8) decreased by about 4%; the monomer content of other preparations decreased by 7%-10%.

[0471] After being placed at 40°C for 15 days, the samples were tested by reduction CE. The results showed that the main peak of samples 4), 5), 7), and 8) decreased by about 1%-2%, which was better than other samples.

[0472] Based on the above data, sample No. 7, i.e., 10 mM His-AA, pH 5.0 preparation, was superior to the other preparations in terms of appearance and various chemical test items. Therefore, 10 mM His-AA, pH 5.0 was selected as the final buffer.

[0473] Table 18. pH and buffer stability results

[0474]

[0475]

[0476] Note: “D” in the table means day, for example, D3 means 3 days, and so on; D0 means the beginning of the experiment, the same below.

[0477] Example 2-2: Screening of surfactant types and concentrations

[0478] Formulations containing different polysorbate grades and concentrations, 10 mM His-AA, pH 5.0 buffer, 80 mg / mL sucrose, and 20 mg / mL ADC-9 protein were prepared. Each formulation was filtered, filled, stoppered, and capped. The samples underwent high-temperature stability studies (40°C) and freeze-thaw studies. The freeze-thaw studies involved five freeze-thaw cycles (FT5C, 35°C-2 to 8°C) followed by three days at room temperature (25°C D3). Appearance, SEC, and reduced CE were evaluated. The specific formulation design is shown in Table 19.

[0479] The results are shown in Table 20. The experimental results show that the preparation using 0.2 mg / ml PS80 has the best appearance and chemical test items under various conditions.

[0480] Based on the above results, the type and concentration of surfactant were determined to be 0.2 mg / ml PS80.

[0481] Table 19. Polysorbate type and concentration screening preparations

[0482]

[0483] Note: PS20 stands for polysorbate 20.

[0484] Table 20. Polysorbate type screening results

[0485]

[0486]

[0487] Note: “M” in the table stands for month, M1 stands for 1 month, and so on, the same below.

[0488] Example 2-3: Sugar type screening

[0489] Formulations containing sucrose, trehalose, and mannitol were prepared, along with 10 mM His-AA (pH 5.0) buffer, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) of ADC-9. Each formulation was filtered, filled, stoppered, and capped. Samples were subjected to high-temperature stability studies (40°C), freeze-thaw cycles at -35°C / 4°C, and room temperature for 3 days. Appearance, SEC, and reduced CE were also evaluated.

[0490] The results are shown in Table 21. Under freeze-thaw conditions for different sugar formulations, the appearance of samples using sucrose was superior to that of samples using trehalose or mannitol. SEC analysis showed that samples using sucrose or trehalose were superior to those using mannitol. After one month at 40°C, the appearance of samples using sucrose was superior to that of samples using trehalose or mannitol. SEC and reduced CE analysis also showed that samples using sucrose were slightly superior to those using trehalose.

[0491] Table 21. Sugar type solution screening results

[0492]

[0493] Formulations containing sucrose, trehalose, and mannitol were prepared, along with 10 mM His-AA (pH 5.0) buffer, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) of ADC-9. Each formulation was filtered, filled, half-stoppered, lyophilized, stoppered, and capped. High-temperature stability studies (40°C) were performed to assess appearance, SEC, and reduced CE. For the lyophilization process, refer to lyophilization process parameters 1 in Table 22.

[0494] Table 22. Freeze-drying process parameters 1

[0495] Freeze-drying process parameters: set temperature (°C) set time (min) holding time (h) vacuum degree (Pa) pre-freeze 5101N / A pre-freeze -45502.5N / A

[0496] Primary drying-201204520 Secondary drying 256081

[0497] After being placed at 40°C for one month, the SEC test results (see Table 23) showed that the sample using sucrose was slightly better than the sample using trehalose, and even better than the sample using mannitol. The reduced CE test results (see Table 23) showed that the sample using sucrose was equivalent to the sample using trehalose, and was better than the sample using mannitol.

[0498] Table 23. Screening results of sugar type lyophilized agents

[0499]

[0500] Example 2-4: Experiment on optimizing sample appearance after freeze-drying

[0501] A stock solution was prepared with 10 mM His-AA, pH 5.0, 80 mg / mL sucrose, 0.2 mg / mL PS80, and 20 mg / mL (protein concentration) ADC-9. After filtration and filling, the solution was lyophilized according to lyophilization process parameter 1 (see Table 22). The appearance of the lyophilized sample was examined. The lyophilized sample was a smooth white powder cake with a slightly shrunken bottom edge.

[0502] The sugar concentration of the sample was further lowered to 60 mg / mL, and a stock solution was prepared with 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 filtration and filling, the solution was lyophilized according to lyophilization process parameter 1 in Table 22. After lyophilization, the surface of the powder cake was smooth and wrinkle-free, with a slight shrinkage at the bottom edge.

[0503] The sugar concentration was reduced to 40 mg / ml, but the osmotic pressure of the finished product was too low at this point, posing a risk of low osmotic pressure during clinical administration. To ensure the osmotic pressure of the finished product, the buffer ionic strength was increased to 30 mM. A stock solution was prepared using 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, lyophilization was continued according to lyophilization process parameter 1. After lyophilization, the surface of the sample powder cake was flat and without collapse, and the bottom edge of the powder cake was intact.

[0504] The results for the three formulations are shown in Table 24.

[0505] Table 24. Appearance of samples after lyophilization

[0506]

[0507]

[0508] Example 2-5: Sample stability after freeze-drying

[0509] The stock solution was prepared according to the formulation in Table 25, filtered and filled, and then freeze-dried according to freeze-drying process parameter 1 (see Table 22). The freeze-dried sample was placed under 40°C M1 conditions and then re-dissolved to detect its stability changes.

[0510] Stability results are shown in Table 25. The lyophilized sample of Formulation 3 showed a 3.7% decrease in reduced CE under 40°C M1 conditions. The other two formulations showed no significant changes in any of the chemical analysis parameters, demonstrating significantly better stability than Formulation 3. The pH of the original solution of Formulation 2 before lyophilization was 5.04, and the pH of the reconstituted solution after lyophilization was 5.27.

[0511] Table 25. Formulation stability results

[0512]

[0513] Example 2-6: Stability of lyophilized formulation solution

[0514] The formulations were prepared according to the formulation in Table 26. After filtration, filling, stoppering, and capping, a -35°C / 4°C freeze-thaw cycle was performed. The samples were placed at room temperature for 3 days, shaken for 11 days, and subjected to a high temperature stability study (40°C). The changes in sample appearance, SEC, and reduced CE under the corresponding conditions were examined.

[0515] The stability results are shown in Table 26. After reducing the sucrose concentration and increasing the buffer ionic strength, there was no significant difference between the appearance and purity changes of Preparation No. 2 and Preparation No. 1. The reduction in CE under high temperature conditions was slightly better than that of Preparation No. 1.

[0516] Table 26. Formulation stability results

[0517]

Claims

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 comprises a heavy chain variable region and a light chain variable region, in: i) the heavy chain variable region has HCDR1, HCDR2 and HCDR3 having the same amino acid sequence as the heavy chain variable region as shown in SEQ ID NO: 5, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 having the same amino acid sequence as the light chain variable region as 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.

2. The pharmaceutical composition according to claim 1, wherein the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, in: iii) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20, respectively; or iv) the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively; Preferably, the anti-Claudin18.2 antibody comprises a heavy chain variable region and a light chain variable region, wherein: v) the heavy chain variable region is shown in SEQ ID NO: 31 and the light chain variable region is shown in SEQ ID NO: 29; or vi) the heavy chain variable region is as shown in SEQ ID NO: 26 and the light chain variable region is as shown in SEQ ID NO: 23; More preferably, the anti-Claudin18.2 antibody comprises: vii) the heavy chain set forth in SEQ ID NO:49, and the light chain set forth in SEQ ID NO:47; or viii) the heavy chain represented by SEQ ID NO:44, and the light chain represented by SEQ ID NO:

41.

3. The pharmaceutical composition according to claim 1 or 2, wherein the anti-Claudin18.2 antibody drug conjugate has a structure as shown in the general formula (Pc-LYD): in: 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 are the same or different and are each independently selected from a hydrogen atom, a deuterium atom, a halogen, an alkyl group, a haloalkyl group, a deuterated alkyl group, an alkoxy group, a hydroxyl group, an amino group, a cyano group, a nitro group, a hydroxyalkyl group, a cycloalkyl group and a heterocyclic group; Or, R a and R b Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; R 1 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; R 2 is selected from the group consisting of a hydrogen atom, a halogen, a haloalkyl group, a deuterated alkyl group, a cycloalkyl group, a cycloalkylalkyl group, an alkoxyalkyl group, a heterocyclic group, an aryl group, and a heteroaryl group; Or, R 1 and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; Or, R a and R 2 Together with the carbon atom to which it is attached, it forms a cycloalkyl or heterocyclyl group; m is an integer from 0 to 4; n is 1 to 10, and n is a decimal or an integer; L is the joint unit; Pc is anti-Claudin18.2 antibody; Preferably, the anti-Claudin18.2 antibody-drug conjugate has a structure as shown in the following formula: in: n is 2 to 8, and n is a decimal or an integer; Pc is an anti-Claudin18.2 antibody.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pharmaceutical composition further comprises a surfactant; Preferably, the surfactant is polysorbate; More preferably, the surfactant is polysorbate 80 or polysorbate 20; Most preferably, the surfactant is polysorbate 80.

5. The pharmaceutical composition according to claim 4, wherein the surfactant concentration is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL to 0.2 mg / mL, more preferably 0.2 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the composition further comprises sugar; Preferably, the sugar is selected from sucrose, mannitol and trehalose; More preferably, the sugar is sucrose.

7. The pharmaceutical composition according to claim 6, wherein the sugar concentration is 20 mg / mL to 100 mg / mL, preferably 40 mg / mL to 80 mg / mL, more preferably 40 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the anti-Claudin18.2 antibody drug conjugate has a concentration of 1 mg / mL to 100 mg / mL in terms of protein concentration, preferably 10 mg / mL to 30 mg / mL in terms of protein concentration, and more preferably 20 mg / mL in terms of protein concentration.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the concentration of the buffer is 5 mM to 50 mM, preferably 10 mM to 30 mM, more preferably 30 mM.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the pH of the pharmaceutical composition is 5.0-6.5, preferably 5.0-5.5, more preferably 5.0-5.

3.

11. The pharmaceutical composition according to any one of claims 1 to 10, comprising the following components: (a) 10 mg / mL to 30 mg / mL of the anti-Claudin18.2 antibody drug conjugate in terms of protein concentration, (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; the pH of the pharmaceutical composition is 5.0-5.5; Preferably, the pharmaceutical composition comprises the following components: (a) 20 mg / mL of the anti-Claudin18.2 antibody-drug conjugate in terms of protein concentration, (b) 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL of sucrose, and (d) 30 mM histidine-acetate buffer, wherein the pH of the pharmaceutical composition is 5.0-5.

3.

12. A pharmaceutical composition comprising: Anti-Claudin18.2 antibody drug conjugate at a protein concentration of 20 mg / mL, 0.2mg / mL polysorbate 80, 40 mg / mL sucrose, and 30 mM histidine-acetate buffer; The pH of the pharmaceutical composition is 5.0 to 5.3; The anti-Claudin18.2 antibody drug conjugate has a structure as shown in the following formula: in: n is 2 to 8, and n is a decimal or an integer; Pc is an anti-Claudin18.2 antibody comprising a heavy chain as shown in SEQ ID NO:49, and a light chain as shown in SEQ ID NO:

47.

13. A lyophilized preparation containing an antibody drug conjugate, Features The preparation can be reconstituted to form the pharmaceutical composition according to any one of claims 1 to 12.

14. A method for preparing a lyophilized preparation containing an antibody drug conjugate, comprising the step of freeze-drying the pharmaceutical composition according to any one of claims 1 to 12.

15. A lyophilized preparation containing an antibody drug conjugate, the preparation being obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 12.

16. A reconstitution solution containing an antibody drug conjugate, Features The reconstituted solution is prepared by reconstituted the lyophilized preparation according to claim 13 or 15; Preferably, the reconstitution solution comprises the following components: (a) 10 mg / mL to 30 mg / mL of the anti-Claudin18.2 antibody drug conjugate in terms of protein concentration, (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; the pH of the reconstituted solution is about 5.0-5.5; More preferably, the pharmaceutical composition comprises the following components: (a) 20 mg / mL of the anti-Claudin18.2 antibody drug conjugate in terms of protein concentration, (b) 0.2 mg / mL of polysorbate 80, (c) 40 mg / mL of sucrose, and (d) 30 mM histidine-acetate buffer, the pH of the reconstituted solution is 5.0-5.

3.

17. A product comprising a container containing the pharmaceutical composition according to any one of claims 1 to 12, the lyophilized preparation according to claim 13 or 15, or the reconstituted solution according to claim 16.

18. A method for treating a tumor or cancer, comprising administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 1 to 12, or the lyophilized preparation according to claim 13 or 15, or the reconstituted solution according to claim 16, or the product according to claim 17; in, The tumor or cancer is preferably selected from the group consisting of: squamous cell carcinoma of the head and neck, cancer of the head and neck, brain cancer, glioma, glioblastoma multiforme, neuroblastoma, cancer of the central nervous system, neuroendocrine tumors, pharyngeal cancer, nasopharyngeal cancer, esophageal cancer, thyroid cancer, malignant pleural mesothelioma, lung cancer, breast cancer, liver cancer, hepatoma, 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, Krukenberg's tumor, myeloproliferative neoplasms, squamous cell carcinoma, Ewing's sarcoma, systemic light chain amyloidosis and Merkel cell carcinoma; More preferably, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, primary 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 the group consisting of non-small cell lung cancer and small cell lung cancer; the leukemia is selected from the group consisting of chronic myeloid leukemia, acute myeloid leukemia, lymphocytic leukemia, lymphoblastic leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia and myeloid cell leukemia.