Multi-specific antibody pharmaceutical composition as well as preparation method and application thereof

Abstract

The invention relates to a multispecific antibody pharmaceutical composition as well as a preparation method and application thereof, and particularly provides a pharmaceutical composition which comprises a buffer solution and a multispecific antibody, the multispecific antibody comprises a first antigen binding domain targeting CD3, a second antigen binding domain targeting MSLN and a third antigen binding domain targeting PD-L1; the pH value of the pharmaceutical composition is 4.5 to 7.0. The pharmaceutical composition can be a liquid preparation or a freeze-dried preparation, has high stability, and can effectively maintain the physicochemical properties and biological activity stability of the multispecific antibody. The invention also provides application of the pharmaceutical composition in preparation of drugs for prevention and / or treatment and / or neoadjuvant treatment and / or adjuvant treatment of diseases.

Description

[0001] This application claims priority to Chinese patent application 202410795405.2, filed on June 19, 2024. This application incorporates the entire text of the aforementioned Chinese patent application. TECHNICAL FIELD

[0002] The present application relates to the field of pharmaceutical compositions, in particular to a multispecific antibody pharmaceutical composition targeting CD3, MSLN and PD-L1, a preparation method thereof and uses thereof. BACKGROUND

[0003] Mesothelin (MSLN) is a cell surface glycoprotein encoded by the MSLN gene. MSLN is highly expressed in mesothelioma, ovarian cancer, lung cancer, esophageal cancer, pancreatic cancer, gastric cancer, cholangiocarcinoma, endometrial cancer, thymic carcinoma, colon cancer and breast cancer, etc. compared to normal tissues, and its abnormal expression plays an important role in tumor cell proliferation, adhesion and drug resistance. PD-L1, also known as B7-H1 or CD274, is the first characterized functional ligand of the coinhibitory programmed death receptor 1 (PD-1). Studies have found that PD-L1 is highly expressed in different types of tumors, including melanoma, ovarian cancer, lung cancer and renal cancer. PD-L1 plays a key role in preventing autoimmunity in normal tissues and maintaining peripheral tolerance. Tumor cells can use the PD-1 / PD-L1 signaling pathway to evade anti-tumor immune responses and eventually spread and metastasize. Therefore, blocking the PD-1 / PD-L1 signaling pathway can activate the endogenous anti-tumor immune response, thereby exerting a therapeutic effect on tumors.

[0004] Currently, T cell engage (TCE) drugs based on CD3 bispecific antibodies for the treatment of solid tumors have received extensive attention. TCE contains two domains, one domain binds to CD3 of T cells, and the other domain targets and binds to cancer cells. The molecular design is used to help T cells recognize cancer cells and fully activate, release cytokines such as TNFα, perforin, granzyme B, IFN-γ, etc., and kill cancer cells. MSLN is a TAA of solid tumors, and the related multi-drug is still under research, among which HPN536 has a faster clinical progress. However, in clinical applications, TCE treatment of solid tumors is not effective, there is a risk of CRS, or T cell overactivation leads to T cell dysfunction, etc. Therefore, there is a need in the art to develop T cell activation multispecific antibody drugs with better performance.

[0005] However, multispecific antibodies are prone to degradation, aggregation or undesired chemical modification due to their large molecular weight and complex structure, thus becoming unstable. In order to make multispecific antibodies suitable for administration and maintain stability during storage and use, and to achieve better effects, it is particularly important to study stable multispecific antibody drug formulations. SUMMARY

[0006] The present application provides a multispecific pharmaceutical composition with high stability, which can effectively maintain the physicochemical properties and biological activity stability of the multispecific antibody. The following aspects are provided:

[0007] In a first aspect, the present application provides a pharmaceutical composition comprising:

[0008] (1) a buffer;

[0009] (2) a multispecific antibody comprising a first antigen binding domain targeting CD3, a second antigen binding domain targeting MSLN, and a third antigen binding domain targeting PD-L1; wherein:

[0010] (i) the first antigen binding domain comprises a VH and a VL, wherein the VH comprises HCDR1, HCDR2 and HCDR3 contained in the VH shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2 and LCDR3 contained in the VL shown in SEQ ID NO: 2; and / or,

[0011] (ii) the second antigen binding domain comprises a VHH, wherein the VHH comprises CDR1, CDR2 and CDR3 contained in the VHH shown in SEQ ID NO: 3; and / or,

[0012] (iii) the third antigen binding domain comprises a VHH, wherein the VHH comprises CDR1, CDR2 and CDR3 contained in the VHH shown in SEQ ID NO: 4;

[0013] The pH of the pharmaceutical composition is 4.5-7.0;

[0014] Preferably, the CDRs are defined according to the numbering system of Kabat, IMGT, Chothia, Contact, AbM or a combination thereof.

[0015] In some embodiments, the buffer is selected from an acetate buffer, a citrate buffer, a succinate buffer, a histidine buffer, a glycine buffer, or a phosphate buffer; in some preferred embodiments, the acetate buffer is selected from an acetic acid-sodium acetate buffer, the citrate buffer is selected from a citric acid-sodium citrate buffer, the succinate buffer is selected from a succinic acid-sodium succinate buffer, the histidine buffer is selected from a histidine-histidine hydrochloride buffer, and the phosphate buffer is selected from a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0016] In some embodiments, the concentration of the buffer is 5-80 mM, such as 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or any sub-range or numerical point therein. In some preferred embodiments, the concentration of the buffer is 10-60 mM (e.g., 10, 20, 40, or 60 mM).

[0017] In some embodiments, the pH of the buffer is 4.5-6.5, such as pH 4.5, pH 5.0, pH 5.5, pH 6.0, pH 6.5, or any sub-range or numerical point therein.

[0018] In some embodiments, the pharmaceutical composition further comprises a stabilizer selected from at least one of a disaccharide, a sugar alcohol, or a salt; in some preferred embodiments, the disaccharide is sucrose or trehalose; in some preferred embodiments, the sugar alcohol is sorbitol; and in some preferred embodiments, the salt is NaCl.

[0019] In some embodiments, the concentration of the stabilizer is 10-150 mg / mL, such as 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, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, or any sub-range or numerical point therein. In some preferred embodiments, the concentration of the stabilizer is 40-120 mg / mL (e.g., 40, 50, 60, 70, 80, or 120 mg / mL).

[0020] In some embodiments, the pharmaceutical composition further comprises a surfactant selected from a non-ionic surfactant. In some preferred embodiments, the non-ionic surfactant is selected from a polysorbate (e.g., polysorbate 20 or polysorbate 80) or a poloxamer (e.g., poloxamer 188).

[0021] In some embodiments, the concentration of the surfactant is 0.01-2 mg / mL, e.g., 0.01 mg / mL, 0.05 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or any sub-range or numerical point therein. In some preferred embodiments, the concentration of the surfactant is 0.05-0.5 mg / mL (e.g., 0.05, 0.2, or 0.5 mg / mL).

[0022] In some embodiments, the pharmaceutical composition further comprises a metal ion chelator and / or an antioxidant; specifically, during the production and storage of the antibody, metal ions in the container can migrate into the solution and catalyze oxidation, so a metal ion chelator is added to chelate the metal ions; an antioxidant can also be added to protect the antibody by preferential oxidation; preferably, the metal ion chelator is disodium ethylenediaminetetraacetate; preferably, the antioxidant is methionine.

[0023] In some embodiments, the concentration of the metal ion chelator is 0.01-0.7 mg / mL, e.g., 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, or any sub-range or numerical point therein. In some preferred embodiments, the concentration of the metal ion chelator is 0.02-0.2 mg / mL (e.g., 0.02 mg / mL).

[0024] In some embodiments, the concentration of the antioxidant is 0.01-0.5%, e.g., 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or any sub-range or numerical point therein. In some preferred embodiments, the concentration of the antioxidant is 0.2%.

[0025] In some embodiments, the pharmaceutical composition can also be protected by nitrogen, the purpose of which is to reduce oxidation by reducing the oxygen content of the solution.

[0026] In some embodiments, the concentration of the multispecific antibody is 5-40 mg / mL, e.g., 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, or any sub-range or numerical point therein. In some preferred embodiments, the concentration of the multispecific antibody is 10-20 mg / mL (e.g., 10 or 20 mg / mL).

[0027] In some embodiments, the VH of the first antigen binding domain comprises a HCDR1 set out in SEQ ID NO: 10, a HCDR2 set out in SEQ ID NO: 11, and a HCDR3 set out in SEQ ID NO: 12; the VL comprises a LCDR1 set out in SEQ ID NO: 13, a LCDR2 set out in SEQ ID NO: 14, and a LCDR3 set out in SEQ ID NO: 15; and / or,

[0028] the VHH of the second antigen binding domain comprises a CDR1 set out in SEQ ID NO: 16, a CDR2 set out in SEQ ID NO: 17, and a CDR3 set out in SEQ ID NO: 18; and / or,

[0029] the VHH of the third antigen binding domain comprises a CDR1 set out in SEQ ID NO: 19, a CDR2 set out in SEQ ID NO: 20, and a CDR3 set out in SEQ ID NO: 21.

[0030] In some embodiments, the multispecific antibody comprises:

[0031] (i) the first antigen binding domain comprises a VH sequence set out in SEQ ID NO: 1, and a VL sequence set out in SEQ ID NO: 2; and / or,

[0032] (ii) the second antigen binding domain comprises a VHH sequence set out in SEQ ID NO: 3; and / or,

[0033] (iii) the third antigen binding domain comprises a VHH sequence set out in SEQ ID NO: 4;

[0034] Alternatively, the multispecific antibody comprises:

[0035] (I) the first antigen binding domain comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VH sequence set out in SEQ ID NO: 1, and a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a VL sequence set out in SEQ ID NO: 2; and / or,

[0036] (II) the second antigen binding domain comprises a VHH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the VHH sequence of SEQ ID NO: 3; and / or,

[0037] (III) the third antigen binding domain comprises a VHH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the VHH sequence of SEQ ID NO: 4.

[0038] In some embodiments, the multispecific antibody comprises:

[0039] (i) a first peptide chain having the structure of [VL]-[CL],

[0040] (ii) a second peptide chain having the structure of [VH]-[CH]-[L1]-[VHH1], and

[0041] (iii) a third peptide chain having the structure of [VHH1]-[L2]-[Fc monomer]-[L1]-[VHH2];

[0042] wherein the VL comprises the sequence of SEQ ID NO: 2, the CL comprises the sequence of SEQ ID NO: 7; the VH comprises the sequence of SEQ ID NO: 1, the CH comprises the sequence of SEQ ID NO: 8, the VHH1 comprises the sequence of SEQ ID NO: 3; the Fc monomer comprises the sequence of SEQ ID NO: 9, the VHH2 comprises the sequence of SEQ ID NO: 4; the L1, L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycines and / or one or more serines (e.g. a peptide linker of the form (G4S)n), further preferably L1 is the sequence of SEQ ID NO: 5 and L2 is the sequence of SEQ ID NO: 6.

[0043] In some embodiments, the pharmaceutical composition comprises any one of the following formulations:

[0044] (1) 20 mg / mL multispecific antibody, 20 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 4.5 ± 0.3, for example 4.5;

[0045] (2) 20 mg / mL multispecific antibody, 20 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 5.0 ± 0.3, for example 5.0;

[0046] (3) 20 mg / mL multispecific antibody, 20 mM citrate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 5.0 ± 0.3, for example 5.0;

[0047] (4) 20 mg / mL multispecific antibody, 20 mM histidine buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 5.0 ± 0.3, for example 5.0;

[0048] (5) 20 mg / mL multispecific antibody, 20 mM succinate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 5.0 ± 0.3, for example 5.0;

[0049] (6) 20 mg / mL multispecific antibody, 20 mM citrate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 6.0 ± 0.3, for example 6.0;

[0050] (7) 20 mg / mL multispecific antibody, 20 mM histidine buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 6.0 ± 0.3, for example 6.0;

[0051] (8) 20 mg / mL multispecific antibody, 20 mM succinate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 6.0 ± 0.3, e.g. 6.0;

[0052] (9) 20 mg / mL multispecific antibody, 10 mM phosphate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 6.0 ± 0.3, e.g. 6.0;

[0053] (10) 20 mg / mL multispecific antibody, 10 mM phosphate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 5.0-7.0, further preferably 7.0 ± 0.3, e.g. 7.0;

[0054] (11) 20 mg / mL multispecific antibody, 20 mM citrate buffer, 60 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 5.5 ± 0.3, e.g. 5.5;

[0055] (12) 20 mg / mL multispecific antibody, 20 mM histidine buffer, 60 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5-7.0, preferably 4.5-6.5, further preferably 5.5 ± 0.3, e.g. 5.5;

[0056] (13) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 4.5; wherein the buffer is 20-60 mM acetate buffer, preferably 20 mM acetate buffer;

[0057] (14) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 4.5; wherein the buffer is 20-60 mM acetate buffer, preferably 40 mM acetate buffer;

[0058] (15) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 10 mM acetate buffer;

[0059] (16) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 20 mM acetate buffer;

[0060] (17) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.5; wherein the buffer is 20-60 mM acetate buffer, preferably 20 mM acetate buffer;

[0061] (18) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 40 mM acetate buffer;

[0062] (19) 10 mg / mL multispecific antibody, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 60 mM acetate buffer;

[0063] (20) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 5.5-6.5, preferably 5.5 ± 0.3, for example 5.5;

[0064] (21) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 5.5-6.5, preferably 6.0 ± 0.3, for example 6.0;

[0065] (22) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 5.5-6.5, preferably 6.5 ± 0.3, for example 6.5;

[0066] (23) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 80-120 mg / mL sucrose, further preferably 120 mg / mL sucrose;

[0067] (24) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 0.7 mg / mL of a metal ion chelator selected from disodium ethylenediaminetetraacetate;

[0068] (25) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 0.2% of an antioxidant selected from methionine;

[0069] (26) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition is further protected by nitrogen;

[0070] (27) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is polysorbate 20 or poloxamer 188; preferably 0.2 mg / mL of polysorbate 20;

[0071] (28) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is polysorbate 20 or poloxamer 188; preferably 0.2 mg / mL of poloxamer 188;

[0072] (29) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the stabilizer is selected from trehalose or sorbitol; preferably 80 mg / mL of trehalose;

[0073] (30) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the stabilizer is selected from trehalose or sorbitol; preferably 50 mg / mL of sorbitol;

[0074] (31) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is 0.05-0.5 mg / mL of polysorbate 80; preferably 0.05 mg / mL of polysorbate 80;

[0075] (32) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is 0.05-0.5 mg / mL polysorbate 80; preferably 0.5 mg / mL polysorbate 80;

[0076] (33) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is sucrose or trehalose; preferably 60 mg / mL sucrose;

[0077] (34) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is sucrose or trehalose; preferably 60 mg / mL trehalose;

[0078] (35) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 40-70 mg / mL sucrose; further preferably 40 mg / mL sucrose;

[0079] (36) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 40-70 mg / mL sucrose; further preferably 50 mg / mL sucrose;

[0080] (37) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 40-70 mg / mL sucrose; further preferably 70 mg / mL sucrose;

[0081] (38) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 0.02 mg / mL metal ion chelator selected from disodium ethylenediaminetetraacetate;

[0082] or,

[0083] (39) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 200 mM salt selected from NaCl.

[0084] In some embodiments, the pharmaceutical composition is selected from a liquid formulation or a lyophilized formulation.

[0085] In a second aspect, the present application provides a method for preparing a lyophilized formulation, comprising the step of freeze-drying the pharmaceutical composition of the first aspect.

[0086] In some embodiments, the step of freeze-drying comprises pre-freezing, primary drying and secondary drying; wherein the temperature of the pre-freezing is -80 to -30℃, the transition time is 40 to 60 min, and the holding time is 120 to 180 min.

[0087] And / or, the temperature of the primary drying is -40 to -15℃, the transition time is 50 to 150 min, the holding time is 500 to 3000 min, and the pressure is 5 to 25 pa.

[0088] And / or, the temperature of the secondary drying is 15 to 40℃, the transition time is 100 to 150 min, the holding time is 500 to 800 min, and the pressure is 0.5 to 3 pa.

[0089] In some preferred embodiments, the temperature of the primary drying is -31 to -23℃ (e.g., -31℃, -28℃, -25℃ or -23℃), the holding time is 720 to 1300 min (e.g., 720 min, 900 min or 1300 min), and the pressure is 10 to 20 pa (e.g., 10 pa, 15 pa or 20 pa); in some preferred embodiments, the temperature of the secondary drying is 22 to 35℃ (e.g., 22℃, 25℃, 30℃ or 35℃).

[0090] In some embodiments, the pre-freezing step comprises an annealing process. In some preferred embodiments, the pre-freezing step comprises: first cooling to 3 to 8℃, the transition time is 3 to 8 min, and the holding time is 20 to 40 min; then cooling to -80 to -30℃, the transition time is 40 to 60 min, and the holding time is 120 to 180 min; then annealing to -8 to -18℃, the annealing warming-up time is 20 to 40 min, and the annealing holding time is 90 to 420 min; and finally cooling to -80 to -30℃, the transition time is 100 to 140 min, and the holding time is 200 to 300 min.

[0091] In some preferred embodiments, the temperature of the annealing process is -13℃, and the holding time is 150 to 360 min (e.g., 150 min or 360 min).

[0092] In a third aspect, the present application provides a lyophilized formulation prepared by the method of the second aspect.

[0093] In a fourth aspect, the present application provides a reconstituted solution, which is prepared by reconstituting the lyophilized preparation of the third aspect.

[0094] In a fifth aspect, the present application provides an article of manufacture comprising a container containing the pharmaceutical composition of the first aspect or the lyophilized preparation of the third aspect or the reconstituted solution of the fourth aspect.

[0095] In a sixth aspect, the present application provides use of the pharmaceutical composition of the first aspect or the lyophilized preparation of the third aspect or the reconstituted solution of the fourth aspect or the article of manufacture of the fifth aspect in the preparation of a medicament for the prevention and / or treatment and / or neoadjuvant therapy and / or adjuvant therapy of a disease.

[0096] In some embodiments, the disease is a tumor; in some preferred embodiments, the tumor is a solid tumor or a hematological tumor; in some more preferred embodiments, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, cholangiocarcinoma, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer or colorectal cancer; the hematological tumor comprises acute myeloid leukemia.

[0097] In some embodiments, the antibody in solution must have sufficient chemical and physical stability. Oxidation, deamidation, and hydrolysis are examples of chemical stability problems that antibodies can have in a formulation. Aggregation and gel formation are examples of physical stability problems that antibodies can have in a formulation. A pharmaceutical formulation of an antibody is considered stable if the extent of degradation, modification, aggregation, loss of biological activity, etc. of the antibody therein is acceptably controlled and does not increase unacceptably over time.

[0098] In some embodiments, the multispecific antibody of the present application is a CD3 / MSLN / PD-L1 trispecific antibody described in international patent application PCT / CN2023 / 139722, which is incorporated by reference in its entirety herein.

[0099] It should be understood that, within the scope of the present application, each of the technical features described above and the technical features specifically described hereinafter (such as the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they are not listed one by one here.

[0100] Definitions of terms

[0101] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present application, the following terms are defined below.

[0102] As used herein, the term "and / or" means any one of the items, or any two or more of the items, optionally in combination.

[0103] As used herein, the term "comprising" or "including" means including the recited elements or steps, but not excluding any other elements or steps. When used herein, the term "comprising" or "including" also contemplates embodiments consisting of the recited elements or steps in combination with any additional elements or steps. For example, when referring to an antibody variable region "comprising" a particular sequence, an antibody variable region consisting of the particular sequence is also contemplated.

[0104] The term "antibody", as used herein, refers to a molecule derived from an immunoglobulin that is capable of specifically binding to a target antigen through at least one antigen-combining site located in its variable region. When the term "antibody" is used, it is intended to encompass not only intact antibodies, but also antigen-binding fragments of antibodies that are capable of specifically binding to a target antigen. An "intact antibody" typically comprises two pairs of polypeptide chains (each pair having one light (LC) and one heavy (HC) chain). The light chains can be classified as kappa (kappa) and lambda (lambda) light chains. The heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the isotype of an antibody as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable region and the constant region are joined by a "J" region of about 12 or more amino acids, and the heavy chain further comprises a "D" region of about 3 or more amino acids. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is comprised of one domain, CL. The constant domains are not directly involved in binding of an antibody to an antigen, but exhibit various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair form the antigen binding site.

[0105] The term "complementarity determining region" or "CDR" as used herein refers to amino acid residues in the variable region of an antibody that are responsible for antigen binding. There are three CDRs in each of the variable regions of the heavy and light chains, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, e.g., as defined by the Rabat numbering system (Rabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86:9268-9272), or the Contact numbering system (MacCallum, R.M., Martin, A.C.R., & Thornton, J.M. (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.). For a given antibody, one of skill in the art will readily be able to identify the CDRs defined by each numbering system. And, the correspondence between different numbering systems is well known to one of skill in the art.

[0106] In the present application, the CDRs contained by the antibody or antigen-binding fragment thereof of the present application can be determined according to various numbering systems known in the art, for example, by the Kabat, Chothia, IMGT, AbM or Contact numbering system. In certain embodiments, the CDRs contained by the anti-CD3 antibody or antigen-binding fragment thereof involved in the present application are determined by the Kabat numbering system. In certain embodiments, the CDRs contained by the anti-MSLN or PD-L1 single-domain antibody or antigen-binding fragment thereof involved in the present application are determined by the Kabat, Chothia, IMGT, AbM or Contact numbering system.

[0107] As used herein, the term "single-domain antibody (sdAb)" has the meaning generally understood by those skilled in the art, which refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), typically derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of 4 framework regions and 3 complementarity determining regions, with the structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as it substantially retains antigen binding and specificity. A single-domain antibody is also known as a nanobody, and the two are used interchangeably. In the present text, the term "antigen-binding fragment" of a single-domain antibody refers to a polypeptide comprising a fragment of a single-domain antibody that retains the ability to specifically bind the same antigen to which the single-domain antibody binds, and / or competes with the single-domain antibody for specific binding to the antigen. An antigen-binding fragment of a single-domain antibody of the present application can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of a single-domain antibody of the present application. In some embodiments, the "antigen-binding fragment" of the single-domain antibody can be truncated at the N- or C-terminus to include only part of FR1 and / or FR4, or lack one or both of those framework regions, as long as it substantially retains antigen binding and specificity, as compared to the full-length single-domain antibody.

[0108] As used herein, the term "pharmaceutical composition" or "formulation" means a mixture of one or more of the antibodies described herein with other components such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to maintain the stability of the active ingredients of the antibodies, facilitate administration to an organism, and facilitate absorption of the active ingredients to exert a biological activity. In some embodiments, a pharmaceutical composition of the present application comprises a multispecific antibody. In some embodiments, a pharmaceutical composition of the present application is in solution or solid form. The solution form of the pharmaceutical composition described in the present application is water if not otherwise specified. The solid form of the pharmaceutical composition described in the present application is a liquid formulation obtained by a freeze-drying step if not otherwise specified.

[0109] As used herein, the term "liquid formulation" refers to a formulation in a liquid state and is not intended to refer to a reconstituted lyophilized formulation. The liquid formulation of the present application is stable upon storage and its stability is independent of lyophilization (or other state-changing methods such as spray-drying).

[0110] As used herein, the term "lyophilized formulation" refers to a formulation or pharmaceutical composition obtained after a freeze-drying step of a liquid or solution formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%. The lyophilized formulation can be obtained by freeze-drying a pharmaceutical composition or a liquid or solution formulation. In some embodiments, the freeze-drying step of the present application comprises pre-freezing, primary drying, and secondary drying.

[0111] As used herein, the term "annealing" is a heat treatment process in the pre-freezing step, which refers to raising the frozen sample to a certain temperature and keeping it at this temperature for a period of time before re-cooling to the pre-freezing final temperature. The core purpose is to achieve the reorganization and optimization of ice crystals in the material through temperature regulation, thereby improving the efficiency of subsequent drying and the quality of the finished product.

[0112] As used herein, the term "buffer" refers to a buffer that tolerates pH changes through the action of its acid-base conjugate components. Examples of buffers that control the pH in the appropriate range include acetate, succinate, gluconate, glycine, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, and other organic acid buffers. Among them,

[0113] "Acetate buffer" is a buffer that includes acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like. In some embodiments, the acetate buffer of the present application is selected from acetic acid-sodium acetate buffer.

[0114] A "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. In some embodiments, the citrate buffer of the present application is selected from a citric acid-sodium citrate buffer.

[0115] 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. In some embodiments, the succinate buffer of the present application is selected from a succinic acid-sodium succinate buffer.

[0116] A "histidine buffer" is a buffer comprising histidine ions. Examples of histidine buffers include histidine-histidine hydrochloride, histidine-acetic acid, histidine-phosphate, histidine-sulfate, and the like. In some embodiments of the present application, the histidine buffer is selected from a histidine-histidine hydrochloride buffer.

[0117] A "phosphate buffer" is a buffer comprising 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. In some embodiments, the phosphate buffer of the present application is selected from a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0118] As used herein, the term "stabilizer" means a pharmaceutically acceptable excipient that protects an active pharmaceutical ingredient and / or a formulation from chemical and / or physical degradation during manufacture, storage, and application. Suitable stabilizers for use in the present application can function as, for example, viscosity enhancers, fillers, solubilizers, and the like. The stabilizer can be ionic or non-ionic. For example, the stabilizer can be a non-ionic stabilizer, such as a sugar. For sugars as stabilizers, these include, but are not limited to, monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as sucrose, trehalose, lactose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and sugar alcohols, such as sorbitol, mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and the like, and combinations thereof. For ionic stabilizers, these include salts, such as NaCl. In some embodiments, the stabilizer of the present application is selected from at least one of a disaccharide, a sugar alcohol, and a salt; wherein the disaccharide is preferably sucrose or trehalose; the sugar alcohol is preferably sorbitol; and the salt is preferably NaCl.

[0119] As used herein, the term "surfactant" generally includes an agent that protects a protein, e.g., an antibody, from air / solution interface-induced stresses, solution / surface-induced stresses to reduce aggregation of the antibody or to minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, non-ionic surfactants such as polyoxyethylene sorbitan fatty acid esters (e.g., polysorbate 20 and polysorbate 80), polyoxyethylene-polyoxypropylene copolymers (e.g., poloxamer, Pluronic). In some embodiments, the surfactant of the present application is selected from polysorbate 20, polysorbate 80 or poloxamer 188.

[0120] Other contemplated excipients that can be utilized in the pharmaceutical compositions of the present application include, for example, metal ion chelators, antioxidants, flavoring agents, antimicrobial agents, sweeteners, antistatic agents, gelatin, and the like. Among them, in the process of antibody production and preservation, metal ions in the container can migrate into the solution and catalyze oxidation, so a metal ion chelator can be added to chelate metal ions; an antioxidant can also be added to protect the antibody by preferential oxidation. In some embodiments, the pharmaceutical composition described in the present application comprises a metal ion chelator (e.g., disodium ethylenediaminetetraacetate) and / or an antioxidant (e.g., methionine).

[0121] Beneficial effects

[0122] The present application carries out formulation research on CD3 / MSLN / PD-L1 trispecific antibodies, and develops a pharmaceutical preparation comprising CD3 / MSLN / PD-L1 trispecific antibodies or antigen binding fragments thereof, which has high stability and can effectively maintain the physicochemical properties and biological activity stability of CD3 / MSLN / PD-L1 trispecific antibodies. BRIEF DESCRIPTION OF DRAWINGS

[0123] Figure 1 The structural schematic diagram of the CD3-PDL1-MSLN trispecific antibody constructed in Example 1 is shown;

[0124] Figure 2 The binding activity of the 5Y3-16 antibody to human MC38 / MSLN cells in Example 2 is shown;

[0125] Figure 3 The binding activity of the 5Y3-16 antibody to CHO-hPDL1 cells in Example 2 is shown;

[0126] Figure 4 The blocking activity of the 5Y3-16 antibody on the binding of human PD1 to human PD-L1 in Example 2 is shown; Figure 5A 、 5B The in vivo killing activity and safety of the 5Y3-16 antibody in Example 2 are shown.

[0127] Sequence information

[0128] The description of the sequences involved in the present application is provided in the following table.

[0129] Table 1: Sequence information

[0130]

[0131] DETAILED DESCRIPTION

[0132] The embodiments of the present application will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present application, and are not limited to the scope of the present application. According to the following detailed description of the preferred embodiments and the drawings, the various purposes and advantages of the present application will become apparent to those skilled in the art.

[0133] The present application will now be described with reference to the following examples, which are intended to illustrate the present application (but not to limit the present application). The present application will now be described with reference to the following examples, which are intended to illustrate the present application (but not to limit the present application).

[0134] Those skilled in the art know that the examples describe the present application by way of example, and are not intended to limit the scope of the present application claimed. The experimental methods in the examples, unless otherwise specified, are conventional methods. The specific conditions not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if not specified by the manufacturer, are conventional products that can be obtained by purchase.

[0135] Example 1: Preparation of CD3-PDL1-MSLN trispecific antibody

[0136] The CD3-PDL1-MSLN trispecific antibody is a multispecific antibody targeting CD3, MSLN and PD-L1 invented by the present applicant, and the preparation of the antibody is described in the PCT invention application patent (publication number WO2024131751A1, "Antibodies and uses thereof", application date 2023 / 12 / 19), the entire contents of which are incorporated herein by reference. Among them, the antibody targeting CD3 is from the patent application WO2023125611A1, the VH sequence of which is shown as SEQ ID NO: 1, and the HCDR1-HCDR3 of VH are shown as SEQ ID NOs: 10-12, respectively; the VL sequence of which is shown as SEQ ID NO: 2, and the LCDR1-LCDR3 of VL are shown as SEQ ID NOs: 13-15, respectively. The preparation of the antibody is as follows:

[0137] 1. Preparation of antibody targeting MSLN

[0138] The llama was immunized with MSLN recombinant protein (Kaiyuan Biotech, MSL-HM280) as immunogen, and finally screened for the llama nanobody 3C6 with good human, monkey and mouse cross-binding activity. The humanized antibody 3C6hu of 3C6 was further constructed, and the VHH sequence of 3C6hu is shown in SEQ ID NO: 3, and the CDR1-CDR3 defined by IMGT are shown in SEQ ID NOs: 16-18, respectively.

[0139] 2. Preparation of antibodies targeting PD-L1

[0140] The llama was immunized with PD-L1 recombinant protein (Kaiyuan Biotech, PDL-HM210) as immunogen, and finally screened for the llama nanobody 260-7-B11 with good human and monkey cross-binding activity. The humanized antibody 7B11_hu of 260-7-B11 was further constructed, and the VHH sequence of 7B11_hu is shown in SEQ ID NO: 4, and the CDR1-CDR3 defined by IMGT are shown in SEQ ID NOs: 19-21, respectively.

[0141] 3. Preparation of CD3-PDL1-MSLN trispecific antibodies

[0142] The applicant named the invented CD3-PDL1-MSLN trispecific antibodies as 5Y3-16, and the structure of 5Y3-16 is shown in Figure 1 which consists of three chains:

[0143] 1. Light chain, consisting of humanized CD3 antibody light chain variable region (SEQ ID NO: 2) and light chain CL1 constant region (SEQ ID NO: 7);

[0144] 2. Heavy chain 1, sequentially consisting of humanized CD3 heavy chain variable region (SEQ ID NO: 1), C-terminal of IgG1 mutant 1 (SEQ ID NO: 8) connected to humanized MSLN single domain antibody 3C6hu (SEQ ID NO: 3) by Linker-1 (SEQ ID NO: 5);

[0145] 3. Heavy chain 2, sequentially consisting of 3C6hu (SEQ ID NO: 3) connected to N-terminal of IgG1 mutant 2-Fc (SEQ ID NO: 9) by Linker-2 (SEQ ID NO: 6), C-terminal of IgG1 mutant 2-Fc connected to humanized PD-L1 single domain antibody 7B11_hu (SEQ ID NO: 4) by Linker-1 (SEQ ID NO: 5).

[0146] The nucleic acid sequence encoding the 5Y3-16 polypeptide chain was constructed into the PTT5 plasmid vector (Ubio, lot: VT2202), and enough plasmid was extracted for standby. The plasmid of the 5Y3-16 antibody fragment was mixed at a ratio of heavy chain 1 : heavy chain 2 : light chain = 1 : 1 : 1, transfected into CHO-S cells (Gibco A29127), cultured, and harvested when the cell viability was < 80%; the antibody was purified for standby.

[0147] Example 2: Verification of the activity of the CD3-PDL1-MSLN multispecific antibody

[0148] 2.1 Flow cytometry detection of the binding of 5Y3-16 to human Jurkat cells (company: ATCC; item number: TIB-152), and the results showed that 5Y3-16 could bind to CD3-expressing Jurkat cells with an EC 50 Binding to human Jurkat cells, i.e., 5Y3-16 can bind to CD3-expressing Jurkat cells.

[0149] 2.2 Flow cytometry detection of the binding of 5Y3-16 to human MC38 / MSLN cells

[0150] The human MSLN (huMSLN) full-length sequence (cDNA purchased from Sinobiological, HG13128-UT) was cloned into a lentiviral vector, and the constructed lentiviral plasmid and packaging plasmid were co-transfected into HEK293T cells (thermo K1649B). The cell supernatant was collected at 48 h and 72 h, respectively, and then added to MC38 cells (Nanjing Kebai, item number CBP60825). After 24 h, 4 μg / ml puromycin was added for screening. The high huMSLN-expressing single cells were sorted by a cell sorter (Sony, LE-SH800SBP), and finally the high huMSLN-expressing stably transfected cell monoclonal MC38 / MSLN was obtained. Flow cytometry detection of the binding of 5Y3-16 to human MC38 / MSLN cells showed that 5Y3-16 could bind to MSLN-expressing cells, as shown in Figure 2

[0151] 2.3 Flow cytometry detection of the binding of 5Y3-16 to CHO-hPDL1 cells

[0152] The huPDL1 sequence (NCBI: Accession# NP _ ​054862.1) was cloned into lentivirus vector (plvx-IRES-puro, psPAX2, pMD2G), the constructed lentivirus plasmid and packaging plasmid were co-transfected into HEK293T cells, and the cell supernatant collected at 48 h was added into CHO cells (Thremo, R80007), and 4 μg / m1 puromycin was added at 24 h for screening. The single cells expressing huPDL1 were sorted by cell sorter (Sony, LESH800SBP), and finally the stable CHO-huPDL1 cells were obtained. The binding of 5Y3-16 to CHO-hPDL1 cells was detected by flow cytometry, and the results are shown in Figure 3 Figure 2A, which shows that 5Y3-16 can bind to cells expressing PD-L1.

[0153] 2.4 Flow cytometry detection of 5Y3-16 blocking human PD1 binding to human PD-L1

[0154] CHO-huPDL1 cells were taken, and gradient-diluted 5Y3-16 and human PD-1-mFc (ACRO, PD1-H5255) protein premix were added, followed by the addition of PE-labeled goat anti-mouse IgG Fc fluorescent secondary antibody, and flow cytometry was used for detection. The results are shown in Figure 4 Figure 2B, which shows that 5Y3-16 can effectively block the binding of human PD1 to human PD-L1.

[0155] 2.5 Dynamic affinity detection of 5Y3-16

[0156] The antibody was immobilized on an HC200M chip containing anti-human IgG Fc secondary antibody, and gradient-diluted human CD3εγ (company: aerobiosystems, product number: CDG-H52W5), human MSLN-Fc (Kaiyou Bio, MSL-HM280), and human PD-L1 protein (ACRO, PD1-H5258) were flowed through the chip. The results show that 5Y3-16 binds to human CD3εγ, human MSLN, and human PD-L1 with KD of 4.64E-07 M, 7.98E-09 M, and 1.21E-10 M, respectively, i.e., 5Y3-16 has good affinity to human CD3εγ, human MSLN, and human PD-L1.

[0157] 2.6 Effect of 5Y3-16 on PBMC immune reconstituted mouse subcutaneous HCC1806 xenograft tumor model

[0158] Select 6-8 weeks old female NOG-dKO (NOG-MHCI / II-2KO Mice) mice (Beijing Vantoll Life-Science Technology Co., Ltd.), each mouse intraperitoneally injected with 5 x 10^7 PBMC cells, and 7 days later each mouse subcutaneously inoculated with 3 x 10^6 HCC1806 cells (ATCC, CRL-2335, human breast cancer cells). When the tumors grew to an average of about 80 mm 3 When the tumors grew to an average of about 80 mm Figure 5A As shown in the results, the 5Y3-16 antibody has good efficacy and the in vivo efficacy shows dose correlation, and the tumor inhibition rates of the three dosages of 0.05 mg / kg, 0.25 mg / kg and 1 mg / kg are 81%, 101% and 106%, respectively. As shown in the results, the tumor inhibition rates of the three dosages of 0.05 mg / kg, 0.25 mg / kg and 1 mg / kg are 81%, 101% and 106%, respectively. Figure 5B As shown in the results, the tumor inhibition rates of the three dosages of 0.05 mg / kg, 0.25 mg / kg and 1 mg / kg are 81%, 101% and 106%, respectively.

[0159] Example 3: Investigation of the thermal stability of CD3 / MSLN / PD-L1 trispecific antibody preparation

[0160] According to the prescription information in Table 2, 5Y3-16 antibody preparations with an antibody concentration of 20 mg / mL were prepared. The antibody preparations were filtered with a microporous filter membrane, sampled, and the thermal stability of 5Y3-16 in each group of prescriptions was determined. Among them, the acetate buffer system in the example is acetic acid-sodium acetate buffer, the citrate buffer is citric acid-sodium citrate buffer, the succinate buffer is succinic acid-sodium succinate buffer, the histidine salt buffer is histidine-histidine hydrochloride buffer, and the phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

[0161] The Tm and Tagg values were determined by a protein stability detector (uncle). The changes in the endogenous fluorescence emission properties of aromatic amino acids in the antibody molecule were detected by full-wavelength fluorescence detection, and then the antibody denaturation curve was obtained, and the thermal denaturation temperature Tm value was quantitatively calculated. The antibody aggregation stability was detected by static light scattering technology, and the increase in scattering light signal caused by aggregation was determined to determine the protein aggregation initiation temperature Tagg value.

[0162] Table 2: Prescription information table for determination of Tm and Tagg values

[0163]

[0164] Table 3: Determination results of Tm and Tagg values

[0165] Inspection F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 Tml(°C) 63.0 61.6 53.4 58.4 57.5 55.6 61.5 57.2 58.8 59.9 54.0 59.5 Tm2(°C) 72.3 69.1 59.7 69.6 64.3 61.8 69.1 64.3 65.4 71.5 60.3 68.8 Tonset(°C) 56.7 54.0 48.7 50.9 51.4 52.2 54.7 53.2 53.8 52.6 49.8 52.5 Tagg(°C) ＞70.0 ＞70.0 53.6 66.5 57.9 56.7 60.7 58.4 59.8 59.9 55.0 58.4

[0166] Note: "Tm value" refers to the thermal denaturation temperature of the antibody, i.e. the temperature at which half of the protein is unfolded, at which the spatial structure of the antibody is destroyed, Tml and Tm2 are two thermal denaturation temperatures detected by the antibody in the thermal stability test. "Tonset value" refers to the temperature at which the antibody begins to unfold. "Tagg value" refers to the initial temperature at which the antibody forms aggregates. Therefore, the higher the Tm value, Tonset value, and Tagg value, the higher the thermal stability of the antibody.

[0167] The results are shown in Table 3, which shows that the suitable pH range of the 5Y3-16 antibody is 4.5-7.0, and the Tm of the 5Y3-16 antibody is >53°C and the Tagg is >53°C in the pH range of 4.5-7.0 in the 5 buffer systems (acetate buffer system, citrate buffer system, histidine buffer system, succinate buffer system, and phosphate buffer system), indicating good thermal stability.

[0168] Example 4: Investigation of the effects of pH and buffer type

[0169] According to the prescription information in Table 4, 5Y3-16 antibody formulations with a concentration of 20 mg / mL were prepared. The drug solution was filtered with a microporous filter membrane, and then was divided into aliquots for stability investigation. The stability investigation conditions were 40°C for 2 weeks, 5°C for 4 weeks, -20°C to room temperature for 5 freeze-thaw cycles, and -80°C to room temperature for 5 freeze-thaw cycles, respectively.

[0170] Table 4: Prescription information for pH and buffer type investigation

[0171]

[0172] Table 5: Results of pH and buffer type investigation

[0173]

[0174]

[0175] Note: 1) T0 represents the initial 0 point; 2) FT represents freeze-thaw, for example, -20°C to room temperature, FT5 represents 5 freeze-thaw cycles between -20°C and room temperature; 3) W represents weeks, for example, 40°C for 2 weeks means storage at 40°C for 2 weeks.

[0176] The results of the pH and buffer type screening are shown in Table 5, which shows that:

[0177] 1) Freeze-thaw: After 5 freeze-thaw cycles at -20°C to room temperature and 5 freeze-thaw cycles at -80°C to room temperature, respectively, the appearance of each prescription in Table 4 remained colorless and clear without visible foreign matter. In terms of DLS, SEC, and nr-CE-SDS, there was no significant change compared with T0. This indicates that the 5Y3-16 antibody can maintain stability after freeze-thaw in the prescriptions in Table 4.

[0178] 2) 5°C 4 weeks: After 4 weeks storage at 5°C, the appearance of each formulation in Table 4 remained colorless and clear with no visible particulate. There were no significant changes in DLS, SEC and nr-CE-SDS compared to TO. This indicated that 5Y3-16 antibody could be stable in the formulations in Table 4 for 4 weeks at 2-8°C.

[0179] 3) 40°C 2 weeks: After 2 weeks storage at 40°C, the appearance of each formulation in Table 4 remained colorless and clear with no visible particulate. There was a significant increase in antibody particle size in formulation F3 in DLS compared to TO. There were no significant changes in all other formulations compared to TO. In SEC, the purity of the main peak decreased by ~16% in formulation F3; the purity of the main peak decreased by ~6% in formulation F5; the purity of the main peak decreased by <5% in all other formulations. In nr-CE-SDS, the purity of the main peak decreased by 5% in formulation F3; the purity of the main peak decreased by ~6% in formulation F10; the purity of the main peak decreased by <5% in all other formulations.

[0180] In summary, 5Y3-16 antibody was stable in 20 mM acetate buffer (pH range 4.5-5.0), 20 mM histidine buffer (pH range 5.0-6.0), 20 mM citrate buffer (pH 6.0), 20 mM succinate buffer (pH range 5.0-6.0) and 10 mM phosphate buffer (pH range 6.0-7.0). 5Y3-16 antibody was relatively less stable in 20 mM citrate buffer (pH 5.0). In the same buffer system, the purity of the main peak decreased less in the formulations with pH 6.0 than in the formulations with pH 5.0 (histidine salt buffer F4 decreased by ~3.9%, F7 decreased by ~1.8%; succinate buffer F5 decreased by ~6.2%, F8 decreased by ~3.9%) after 2 weeks storage at 40°C, so pH 6.0 was slightly better than pH 5.0.

[0181] Example 5: Investigation of the effect of pH and buffer concentration

[0182] Formulations F13-F22 in Table 6 were used to prepare 5Y3-16 antibody formulations with a concentration of 10 mg / mL. The solutions were filtered through a microporous membrane and were aliquoted into 3 mL vials for stability investigation. The stability investigation conditions were 40°C for 2 weeks and -40°C to room temperature for 5 freeze-thaw cycles.

[0183] Table 6: Formulation information for investigation of pH and buffer concentration

[0184]

[0185] Table 7: Results of investigation of pH and buffer concentration

[0186]

[0187]

[0188] Note: 1) T0 represents initial 0 point; 2) FT5 represents freeze-thaw 5 times; 3) w represents week.

[0189] The pH / buffer system concentration screening results are shown in Table 7, and the results show that:

[0190] 1) Freeze-thaw: After 5 times of freeze-thaw from -40℃ to room temperature, the appearance of the 10 formulations in Table 6 remained colorless and clear without visible foreign matter. There was no significant change in insoluble particles, SEC and nr-CE-SDS compared with T0. It showed that the 5Y3-16 antibody could maintain stability after freeze-thaw in the formulations in Table 6.

[0191] 2) 40℃ 2 weeks: After 2 weeks at 40℃, the appearance of the 10 formulations in Table 6 remained colorless and clear without visible foreign matter. In terms of insoluble particles, all formulations met the quality requirements of the pharmacopoeia. In terms of SEC, the purity of the main peak of the antibody in all formulations decreased by <5%; among them, in the acetate system, the purity of the main peak of the antibody showed a downward trend with the increase of acetate concentration at pH 5.0. In terms of nr-CE-SDS, the purity of the main peak of each formulation decreased by <5%, and there was no significant difference between groups.

[0192] In summary, the stability of 5Y3-16 antibody was good in the acetate system (10, 20, 40, 60 mM, pH range 4.5-5.5) and the histidine salt system (20 mM, pH range 5.5-6.5).

[0193] Example 6: Investigation of the influence of stabilizers and surfactants

[0194] According to the F15, F23-F32, F39-F40 formulation information in Table 8, 5Y3-16 antibody formulations with an antibody concentration of 10 mg / mL were prepared. The filtrate was divided into 3 mL vials and subjected to stability investigation. The stability investigation conditions were 40℃ 2 weeks and -40℃ to room temperature freeze-thaw 5 times, respectively.

[0195] Among them, the addition method of 0.2% methionine is to first prepare a high-concentration methionine aqueous solution and then add it to the antibody solution to achieve the final target concentration; the addition method of disodium ethylenediaminetetraacetate is the same. The addition method of nitrogen protection is to fill an appropriate amount of nitrogen into the antibody formulation container and solution to reduce the oxygen content.

[0196] Table 8: Formulation information table for stabilizer and surfactant investigation

[0197]

[0198]

[0199] Note: N / A means 'no data here'.

[0200] Table 9: Stabilizer and surfactant investigation results

[0201]

[0202] Note: 1) T0 means initial 0 point; 2) FT5 means freeze-thaw 5 times; 3) W means week; 4) N / A means 'no data here'.

[0203] The stabilizer and surfactant investigation results are shown in Table 9, and the results show that:

[0204] 1) Stabilizer: Sucrose, trehalose and sorbitol have good stability effects on 5Y3-16 antibody. The 80 mg / mL sucrose concentration has the same stability effect as the 120 mg / mL sucrose concentration. After adding the metal ion chelator disodium ethylenediaminetetraacetate or the antioxidant methionine or nitrogen protection, the SEC and ar-CE purities do not decrease by more than 3% under the condition of 40°C for 2W, and the preparations in each group are stable. After adding sodium chloride, the SEC purity decreases by about 10% under the condition of 40°C for 2W, indicating that it induces aggregation of 5Y3-16 antibody.

[0205] 2) Surfactant: Polysorbate 20, polysorbate 80 and poloxamer 188 have good stability effects on 5Y3-16 antibody. There is no significant difference among the preparations containing 0.05 mg / mL-0.5 mg / mL of polysorbate 80, and the SEC and ar-CE purities do not decrease by more than 3%, and the preparations in each group are relatively stable.

[0206] Example 7: Freeze-drying tolerance investigation of CD3 / MSLN / PD-L1 trispecific antibody preparation

[0207] According to the prescription information of F11 and F12 in Table 10, 5Y3-16 antibody preparations with a concentration of 20 mg / mL were prepared, the liquid was filtered, and 1 mL of the liquid was divided into 3 mL of a vial, and after half-plugging, it was loaded into a freeze-drying box, and the freeze-drying was carried out according to the steps in Table 11. After pre-freezing, primary drying and secondary drying, the freeze-drying program was ended, the plug was pressed, and the cap was covered.

[0208] Table 10: Prescription information table of freeze-dried preparation

[0209]

[0210] Table 11: Freeze-drying steps

[0211] Freeze-drying process parameters Set temperature Transition time Hold time Vacuum degree Pre-freezing -45℃ 50 min 150 min N / A Primary drying -28℃ 60 min 1300 min 15 Pa Secondary drying 25℃ 120 min 600 min 1 Pa

[0212] Note: N / A means 'not applicable'.

[0213] Table 12: Comparison of mass before and after lyophilization

[0214]

[0215] Note: N / A means 'not applicable'.

[0216] The results are shown in Table 12. The lyophilized samples after reconstitution were compared with the original solution before lyophilization. The results showed that the reconstituted solution could maintain good performance of the liquid preparation. After the lyophilized powder was placed at 40°C for one month, the samples in each group were quickly reconstituted, and after reconstitution, the appearance was good, the protein concentration did not change significantly, the average particle size did not change significantly, the SEC and nr-CE purity did not change significantly, and the insoluble particles did not increase significantly, i.e. the 5Y3-16 antibody remained stable after reconstitution, and the lyophilization resistance of the antibody protein in the prescription was good.

[0217] Example 8: Investigation of the type of lyoprotectant

[0218] According to the prescription information of F33 and F34 in Table 13, 5Y3-16 antibody preparations with an antibody concentration of 10 mg / mL were prepared, and after the drug solution was divided, the Dongfulong lyophilizer was used to perform pre-freezing, primary drying, and secondary drying steps according to Table 11.

[0219] Among them, the binding activity of PD-L1 target, MSLN target and CD3 target was determined by ELISA method to determine the antigen binding capacity of the antibody. The four-parameter fitting was performed on the concentration of the antibody to be tested and the absorbance value to obtain the inverse "S" shaped curve, and the EC 50 value and the relative binding activity percentage compared with the reference were used to evaluate the antigen binding capacity of the antibody.

[0220] Table 13: Prescription information table for investigation of the type of lyoprotectant

[0221]

[0222] Table 14: Investigation results of the type of lyoprotectant

[0223]

[0224]

[0225] Note: N / A means 'not applicable'.

[0226] The results are shown in Table 14. The results showed that the stability of sucrose and trehalose was equivalent. After the lyophilized powder was placed at 40°C for one month and reconstituted, the stability and activity of the antibody were equivalent to those of the liquid preparation before lyophilization.

[0227] Example 9: Investigation of lyoprotectant concentration influence

[0228] According to the prescription information of F33, F36-F38 in Table 15, each antibody preparation with a 5Y3-16 antibody concentration of 10 mg / mL was prepared, and after the drug solution was aliquoted, the pre-freezing, primary drying, and secondary drying steps in Table 11 were performed for lyophilization.

[0229] Among them, the binding activity of PD-L1 target, MSLN target and CD3 target was determined by ELISA method to determine the antigen binding capacity of the antibody.

[0230] Table 15: Prescription information table for lyoprotectant concentration screening

[0231]

[0232] Table 16: Lyoprotectant concentration screening results

[0233]

[0234]

[0235] Note: N / A means 'no here'.

[0236] The results are shown in Table 16, which show that 40 mg / mL to 70 mg / mL of sucrose can maintain good stability of 5Y3-16 antibody in the lyophilized powder, and the reconstituted solution maintains good performance of the liquid preparation and the binding capacity of the antibody to the antigen.

[0237] Example 10: Investigation of lyophilization process parameters

[0238] According to the prescription information of F33 in Table 13, an antibody preparation of 5Y3-16 antibody was prepared. After the drug solution was aliquoted, the pre-freezing, primary drying pressure, primary drying temperature, and secondary drying temperature were investigated according to the lyophilization curves 1-10 in Table 17.

[0239] Table 17: Lyophilization process parameter investigation curves

[0240]

[0241]

[0242] Note: N / A means 'no here'.

[0243] Table 18: Investigation results of primary drying pressure, primary drying temperature, and secondary drying temperature lyophilization process parameters

[0244]

[0245]

[0246] Note: N / A means 'not applicable'.

[0247] Table 19: Pre-freeze lyophilization process parameter investigation results

[0248]

[0249] Note: N / A means 'not applicable'.

[0250] The primary drying pressure, primary drying temperature, and secondary drying temperature lyophilization process investigation results are shown in Table 18. The results show that within the primary drying pressure range of 10 Pa to 20 Pa, the primary drying temperature range of -31 °C to -23 °C, and the secondary drying temperature range of 22 °C to 35 °C, all qualified lyophilized products can be prepared, and the reconstituted solutions all maintain good performance of the liquid preparation.

[0251] The pre-freeze lyophilization process parameter investigation results are shown in Table 19. The results show that after adding the annealing step, there is no significant difference between the mass items after lyophilization and the original liquid, and the appearance is better, and the slight bottom shrinkage is obviously improved.

[0252] Although the specific embodiments of the present application have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details in light of the overall teachings of the disclosure, and such modifications and changes are intended to be within the scope of the present application. The entire scope of the present application is given by the appended claims and any equivalents thereof.

Claims

1. A pharmaceutical composition comprising: (1) Buffer solution; (2) A multispecific antibody comprising a first antigen-binding domain targeting CD3, a second antigen-binding domain targeting MSLN, and a third antigen-binding domain targeting PD-L1; wherein: (i) The first antigen-binding domain comprises VH and VL, wherein the VH comprises HCDR1, HCDR2, and HCDR3 contained in the VH shown in SEQ ID NO: 1, and the VL comprises LCDR1, LCDR2, and LCDR3 contained in the VL shown in SEQ ID NO: 2; and / or, (ii) The second antigen-binding domain comprises VHH, wherein the VHH comprises CDR1, CDR2, and CDR3 contained in the VHH shown in SEQ ID NO: 3; and / or, (iii) The third antigen-binding domain comprises VHH, wherein the VHH comprises CDR1, CDR2 and CDR3 contained in the VHH shown in SEQ ID NO: 4; The pH of the pharmaceutical composition is 4.5–7.0; Preferably, the CDR is defined according to a numbering system of Kabat, IMGT, Chothia, Contact, AbM, or a combination thereof.

2. The pharmaceutical composition of claim 1, wherein the buffer solution is selected from acetate buffer, citrate buffer, succinate buffer, histidine buffer, glycine buffer, or phosphate buffer; Preferably, the acetate buffer is selected from acetate-sodium acetate buffer, the citrate buffer is selected from citrate-sodium citrate buffer, the succinate buffer is selected from succinate-sodium succinate buffer, the histidine buffer is selected from histidine-histidine hydrochloride buffer, and the phosphate buffer is selected from disodium hydrogen phosphate-sodium dihydrogen phosphate buffer.

3. The pharmaceutical composition of claim 1 or 2, wherein the concentration of the buffer solution is 5-80 mM; Preferably, the concentration of the buffer solution is 10 to 60 mM (e.g., 10, 20, 40 or 60 mM).

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the pH of the buffer solution is 4.5 to 6.5; preferably, the pH of the buffer solution is 5.0 to 6.

0.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the pharmaceutical composition further comprises a stabilizer selected from at least one of disaccharides, sugar alcohols, and salts; Preferably, the disaccharide is sucrose or trehalose; Preferably, the sugar alcohol is sorbitol; Preferably, the salt is NaCl.

6. The pharmaceutical composition of claim 5, wherein the concentration of the stabilizer is 10-150 mg / mL; Preferably, the concentration of the stabilizer is 40–120 mg / mL (e.g., 40, 50, 60, 70, 80 or 120 mg / mL).

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pharmaceutical composition further comprises a surfactant selected from nonionic surfactants; Preferably, the nonionic surfactant is selected from polysorbates (e.g., polysorbate 20 or polysorbate 80) or poloxamer (e.g., poloxamer 188).

8. The pharmaceutical composition of claim 7, wherein the concentration of the surfactant is 0.01 to 2 mg / mL; preferably, the concentration of the surfactant is 0.05 to 0.5 mg / mL (e.g., 0.05, 0.2, or 0.5 mg / mL).

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the pharmaceutical composition further comprises a metal ion chelating agent and / or an antioxidant; Preferably, the metal ion chelating agent is disodium ethylenediaminetetraacetate; Preferably, the antioxidant is methionine.

10. The pharmaceutical composition of claim 9, wherein the concentration of the metal ion chelating agent is 0.01–0.7 mg / mL; Preferably, the concentration of the metal ion chelating agent is 0.02–0.2 mg / mL (e.g., 0.02 mg / mL).

11. The pharmaceutical composition of claim 9, wherein the concentration of the antioxidant is 0.01 to 0.5%; Preferably, the concentration of the antioxidant is 0.2%.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the pharmaceutical composition is further protected by nitrogen.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the concentration of the multispecific antibody is 5 to 40 mg / mL; Preferably, the concentration of the multispecific antibody is 10–20 mg / mL (e.g., 10 or 20 mg / mL).

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the VH of the first antigen-binding domain comprises HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, and HCDR3 shown in SEQ ID NO: 12; and the VL comprises LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15; and / or, The VHH of the second antigen-binding domain includes CDR1 shown in SEQ ID NO: 16, CDR2 shown in SEQ ID NO: 17, and CDR3 shown in SEQ ID NO: 18; and / or, The VHH of the third antigen-binding domain includes CDR1 shown in SEQ ID NO: 19, CDR2 shown in SEQ ID NO: 20, and CDR3 shown in SEQ ID NO:

21.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein, The multispecific antibody comprises: (i) The first antigen-binding domain comprises: the VH sequence shown in SEQ ID NO: 1, and the VL sequence shown in SEQ ID NO: 2; and / or, (ii) the second antigen-binding domain comprises the VHH sequence shown in SEQ ID NO: 3; and / or, (iii) The third antigen-binding domain comprises the VHH sequence shown in SEQ ID NO: 4; Optionally, the multispecific antibody comprises: (I) The first antigen-binding domain comprises: a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the VH sequence shown in SEQ ID NO: 1; and a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the VL sequence shown in SEQ ID NO: 2; and / or, (II) The second antigen-binding domain comprises: a VHH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the VHH sequence shown in SEQ ID NO: 3; and / or, (III) The third antigen-binding domain comprises a VHH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the VHH sequence shown in SEQ ID NO:

4.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the multispecific antibody comprises: (i) A first peptide chain having the structure shown in [VL]-[CL] (ii) A second peptide chain having the structure shown in [VH]-[CH]-[L1]-[VHH1], and (iii) A third peptide chain having the structure shown in [VHH1]-[L2]-[Fc monomer]-[L1]-[VHH2]; in, The VL contains the sequence shown in SEQ ID NO: 2, the CL contains the sequence shown in SEQ ID NO: 7; the VH contains the sequence shown in SEQ ID NO: 1, the CH contains the sequence shown in SEQ ID NO: 8, and the VHH1 contains the sequence shown in SEQ ID NO: 3; the Fc monomer contains the sequence shown in SEQ ID NO: 9, and the VHH2 contains the sequence shown in SEQ ID NO: 4; L1 and L2 are peptide linkers, preferably each independently selected from peptide linkers containing one or more glycines and / or one or more serines (e.g., the peptide linker shown in (G4S)n), and more preferably, L1 is the sequence shown in SEQ ID NO: 5 and L2 is the sequence shown in SEQ ID NO:

6.

17. The pharmaceutical composition according to any one of claims 1-16, comprising any one of the following formulations: (1) 20 mg / mL of multispecific antibody, 20 mM acetate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 4.5 ± 0.3, for example 4.5; (2) 20 mg / mL of multispecific antibody, 20 mM acetate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 5.0 ± 0.3, for example 5.0; (3) 20 mg / mL of multispecific antibody, 20 mM citrate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 5.0 ± 0.3, for example 5.0; (4) 20 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 5.0 ± 0.3, for example 5.0; (5) 20 mg / mL of multispecific antibody, 20 mM succinate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 5.0 ± 0.3, for example 5.0; (6) 20 mg / mL of multispecific antibody, 20 mM citrate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 6.0 ± 0.3, for example 6.0; (7) 20 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 6.0 ± 0.3, for example 6.0; (8) 20 mg / mL of multispecific antibody, 20 mM succinate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 6.0 ± 0.3, for example 6.0; (9) 20 mg / mL of multispecific antibody, 10 mM phosphate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 6.0 ± 0.3, for example 6.0; (10) 20 mg / mL of multispecific antibody, 10 mM phosphate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 5.0 to 7.0, more preferably 7.0 ± 0.3, for example 7.0; (11) 20 mg / mL of multispecific antibody, 20 mM citrate buffer, 60 mg / mL sucrose, 0.2 mg / mL polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 5.5 ± 0.3, for example 5.5; (12) 20 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 60 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 4.5 to 7.0, preferably 4.5 to 6.5, more preferably 5.5 ± 0.3, for example 5.5; (13) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 4.5; wherein the buffer is 20-60 mM acetate buffer, preferably 20 mM acetate buffer; (14) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 4.5; wherein the buffer is 20-60 mM acetate buffer, preferably 40 mM acetate buffer; (15) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 10 mM acetate buffer; (16) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 20 mM acetate buffer; (17) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.5; wherein the buffer is 20-60 mM acetate buffer, preferably 20 mM acetate buffer; (18) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 40 mM acetate buffer; (19) 10 mg / mL of multispecific antibody, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the buffer is 20-60 mM acetate buffer, preferably 60 mM acetate buffer; (20) 10 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 5.5 to 6.5, preferably 5.5 ± 0.3, for example 5.5; (21) 10 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 5.5 to 6.5, preferably 6.0 ± 0.3, for example 6.0; (22) 10 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80; wherein the pH of the pharmaceutical composition is 5.5 to 6.5, preferably 6.5 ± 0.3, for example 6.5; (23) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the stabilizer is 40-120 mg / mL of sucrose, preferably 80-120 mg / mL of sucrose, and more preferably 120 mg / mL of sucrose; (24) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 0.7 mg / mL of a metal ion chelating agent selected from disodium ethylenediaminetetraacetate; (25) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 0.2% antioxidant selected from methionine; (26) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition is also protected by nitrogen. (27) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is polysorbate 20 or poloxamer 188; preferably 0.2 mg / mL polysorbate 20; (28) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is polysorbate 20 or poloxamer 188; preferably 0.2 mg / mL poloxamer 188; (29) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the stabilizer is selected from trehalose or sorbitol; preferably 80 mg / mL trehalose; (30) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the stabilizer is selected from trehalose or sorbitol; preferably 50 mg / mL sorbitol; (31) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is 0.05-0.5 mg / mL polysorbate 80; preferably 0.05 mg / mL polysorbate 80; (32) 10 mg / mL multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, pH 5.0; wherein the surfactant is 0.05-0.5 mg / mL polysorbate 80; preferably 0.5 mg / mL polysorbate 80; (33) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is sucrose or trehalose; preferably 60 mg / mL sucrose; (34) 10 mg / mL multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is sucrose or trehalose; preferably 60 mg / mL trehalose; (35) 10 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 40-70 mg / mL sucrose; more preferably 40 mg / mL sucrose; (36) 10 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 40-70 mg / mL sucrose; more preferably 50 mg / mL sucrose; (37) 10 mg / mL of multispecific antibody, 20 mM histidine salt buffer, 0.2 mg / mL polysorbate 80, pH 6.0; wherein the stabilizer is 40-120 mg / mL sucrose, preferably 40-70 mg / mL sucrose; more preferably 70 mg / mL sucrose; (38) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL of sucrose, 0.2 mg / mL of polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 0.02 mg / mL of a metal ion chelating agent selected from disodium ethylenediaminetetraacetate; or, (39) 10 mg / mL of multispecific antibody, 10 mM acetate buffer, 80 mg / mL sucrose, 0.2 mg / mL polysorbate 80, pH 5.0; wherein the pharmaceutical composition further comprises 200 mM salt selected from NaCl.

18. The pharmaceutical composition according to any one of claims 1 to 17, wherein the pharmaceutical composition is selected from liquid formulations or lyophilized formulations.

19. A method for preparing a lyophilized formulation, comprising the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 18.

20. The method of claim 19, wherein the freeze-drying step comprises pre-freezing, primary drying, and secondary drying; wherein, The pre-freezing temperature is -80 to -30°C, the transition time is 40 to 60 minutes, and the holding time is 120 to 180 minutes; And / or, the temperature of the primary drying is -40 to -15°C, the transition time is 50 to 150 min, the holding time is 500 to 3000 min, and the pressure is 5 to 25 Pa; Preferably, the temperature of the primary drying is -31 to -23°C (e.g., -31°C, -28°C, -25°C or -23°C), the holding time is 720 to 1300 min (e.g., 720 min, 900 min or 1300 min), and the pressure is 10 to 20 Pa (e.g., 10 Pa, 15 Pa or 20 Pa); And / or, the secondary drying temperature is 15-40°C, the transition time is 100-150 min, the holding time is 500-800 min, and the pressure is 0.5-3 Pa; Preferably, the temperature of the secondary drying is 22-35°C (e.g., 22°C, 25°C, 30°C or 35°C).

21. The method of claim 20, wherein the pre-freezing step comprises an annealing process; Preferably, the pre-freezing step includes: first cooling to 3-8°C, with a transition time of 3-8 min and a holding time of 20-40 min; then cooling to -80--30°C, with a transition time of 40-60 min and a holding time of 120-180 min; then annealing to -8°C--18°C, with an annealing heating time of 20-40 min and an annealing holding time of 90-420 min; and finally cooling to -80--30°C, with a transition time of 100-140 min and a holding time of 200-300 min. More preferably, the annealing temperature is -13°C, and the holding time is 150 to 360 minutes (e.g., 150 minutes or 360 minutes).

22. A lyophilized formulation prepared by the method according to any one of claims 19 to 21.

23. A reconstituted solution prepared by reconstituted lyophilized formulation of claim 22.

24. An article comprising a container containing a pharmaceutical composition as claimed in any one of claims 1 to 18, a lyophilized formulation as claimed in claim 22, or a reconstituted solution as claimed in claim 23.

25. Use of the pharmaceutical composition of any one of claims 1 to 18, the lyophilized formulation of claim 22, the reconstituted solution of claim 23, or the article of claim 24 in the preparation of a medicament for the prevention and / or treatment and / or neoadjuvant and / or adjuvant treatment of diseases.

26. The use as described in claim 25, characterized in that, The disease is a tumor; preferably, the tumor is a solid tumor or a hematologic tumor; more preferably, the solid tumor is selected from mesothelioma, ovarian cancer, pancreatic cancer, cervical cancer, breast cancer, prostate cancer, bile duct cancer, colorectal cancer, gastric cancer, fallopian tube cancer, lung cancer, or colorectal cancer; the hematologic tumor includes acute myeloid leukemia.

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