Formulations containing anti-Claudin18.2 and CD3 bispecific antibodies and methods for preparing and using same

Abstract

The present invention relates to formulations comprising anti-CLDN18.2 / CD3, in particular pharmaceutical formulations comprising anti-CLDN18.2 / CD3, a buffer, a stabilizer and a surfactant. Furthermore, the present invention relates to the use of these formulations for the treatment or prevention of diseases.

Description

[Technical field]

[0001] The present invention relates to the field of anti-Claudin18.2 and CD3 bispecific antibody formulations. More specifically, the present invention relates to pharmaceutical formulations comprising bispecific antibodies, in particular stable liquid formulations, lyophilized formulations and reconstituted stable liquid formulations, as well as methods for preparing said pharmaceutical formulations, and therapeutic and / or prophylactic uses of said pharmaceutical formulations. [Background technology]

[0002] Drug stability is one of the important indicators for ensuring the efficacy and safety of drugs. Obtaining a good formulation is an important condition for ensuring that the efficacy and safety of drugs are maintained during storage. However, due to the complexity of bispecific antibodies themselves and their degradation pathways, it is currently not possible to predict the formulation conditions required for optimizing antibody stability. In particular, different antibodies usually have very different structures and CDR sequences, and these sequence differences will lead to different antibodies having different stability characteristics in solution. Therefore, based on the strict requirements for safety and efficacy of human antibodies, it is necessary to individually optimize the optimal formulation for each antibody.

[0003] Claudin 18 isotype 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker whose expression in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa, but is not expressed in the gastric stem cell region. CLDN18.2 is expressed in a significant number of primary gastric cancers and maintains its expression level in gastric metastatic cancer tissues. In addition to gastric cancer, CLDN18.2 is also expressed in pancreatic cancer, making it an ideal target molecule for treating these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as new targeted therapy for advanced gastric cancer. J Hematol Oncol 10, 105 (2017). https: / / doi.org / 10.1186 / s13045-017-0473-4).

[0004] CD3 is a homodimeric or heterodimeric antigen that binds to the T cell receptor complex (TCR) and is expressed on T cells, and is necessary for T cell activation. Functional CD3 is formed by the association of two dimers among four different chains: ε, ζ, δ, and γ. CD3 dimer sequences include γ / ε, δ / ε, and ζ / ζ. Antibodies against CD3 have been shown to aggregate CD3 on T cells, leading to T cell activation in a manner that engages peptide-bearing MHC molecules with the TCR. Therefore, it has been proposed that anti-CD3 antibodies can be used for therapeutic purposes involving T cell activation. In addition, it has been proposed that bispecific antibodies that can bind to CD3 and target tumor surface antigens can link tumor cells and T cells, directly activating T cells and releasing granzymes, perforin, and cytokines to kill tumors, thereby achieving the therapeutic purpose of inhibiting tumors. Since CLDN18.2 is highly expressed in gastric cancer, pancreatic cancer, esophagogastric junction cancer, etc., therapeutic objectives can be achieved by developing a bispecific antibody that simultaneously binds to CD3 and Claudin18.2.

[0005] Although several bispecific antibody formulations have already been proposed, there remains a need in the art for new pharmaceutical formulations of bispecific antibodies that are sufficiently stable and suitable for administration to human subjects, and it would also be advantageous for such bispecific antibody formulations to have simplicity of formulation and ease of use. Summary of the Invention

[0006] The present invention satisfies the above-mentioned needs by providing a pharmaceutical formulation comprising an anti-Claudin18.2 and CD3 bispecific antibody. The antibody formulation of the present invention exhibits good stability against multiple stability-influencing factors (e.g., temperature, pH).

[0007] In one embodiment, the present invention therefore provides a liquid antibody formulation comprising (i) an anti-Claudin18.2 and CD3 bispecific antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, preferably further comprising a chelating agent.

[0008] In some embodiments, the present invention provides a bispecific antibody, wherein the antibody comprises two binding domains, wherein the first binding domain specifically binds to CLDN18.2 and the second binding domain specifically binds to CD3.

[0009] In a preferred embodiment, the first binding domain that specifically binds to CLDN18.2 of the present invention is fully human and / or the second binding domain is humanized.

[0010] In some embodiments, the bispecific antibody of the invention is an IgG-like structure.

[0011] In one aspect, the present invention relates to the following embodiments:

[0012] 1. A bispecific antibody, comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2 and the second antigen-binding domain specifically binds to CD3, wherein the first antigen-binding domain comprises three complementarity determining regions A1-HCDR1, A1-HCDR2 and A1-HCDR3 contained in A1-VH as set forth in SEQ ID NO:4 and three complementarity determining regions A1-LCDR1, A1-LCDR2 and A1-LCDR3 contained in VL as set forth in SEQ ID NO:9, and the second antigen-binding domain comprises three complementarity determining regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in A2-VH as set forth in SEQ ID NO:30, 22 or 32 and three complementarity determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as set forth in SEQ ID NO:27.

[0013] 2. The bispecific antibody according to embodiment 1, wherein: The first antigen-binding domain comprises A1-HCDR1, A1-HCDR2 and A1-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and A1-LCDR1, A1-LCDR2 and A1-LCDR3 represented by the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively; and The second antigen-binding domain comprises (i) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or (ii) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or (iii) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively.

[0014] 3. The antibody of embodiment 1 or 2, wherein the first antigen-binding domain comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO:4; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO:4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 4, preferably said amino acid changes do not occur in the CDR regions, and / or The light chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 9; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO:9; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 9, and preferably, said amino acid changes do not occur in the CDR regions.

[0015] 4. The antibody of any one of embodiments 1 to 3, wherein the second antigen-binding domain comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 30, 22, or 32; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32, preferably said amino acid changes do not occur in the CDR regions, and / or The light chain variable region is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 27; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 27; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 27, and preferably, said amino acid changes do not occur in the CDR regions.

[0016] 5. The antibody according to any one of embodiments 1 to 4, which further comprises a heavy chain constant region and / or a light chain constant region.

[0017] 6. The antibody according to any one of embodiments 1 to 5, which is a bispecific antibody with an IgG-like structure.

[0018] 7. The antibody according to any one of embodiments 1 to 6, wherein the heavy chain constant region of said antibody is derived from IgG1, IgG2, IgG3 or IgG4, preferably IgG1.

[0019] 8. The antibody according to any one of embodiments 1 to 6, which comprises two heavy chain constant regions, one of which is a heavy chain constant region A1-HC, which is a first antigen. join The heavy chain constant region A1-HC of the second antigen-binding domain is linked to the heavy chain variable region A2-VH of the first antigen-binding domain to form the heavy chain of the CD3-binding portion, and the other heavy chain constant region A2-HC of the second antigen-binding domain is linked to the heavy chain variable region A2-VH of the second antigen-binding domain to form the heavy chain of the CD3-binding portion, and also includes two light chain constant regions, one of which, A1-LC, binds to the first antigen. join It is linked to the light chain variable region A1-VL of the domain CLDN18. 2 The other light chain constant region A2-LC is linked to the light chain variable region A2-VL of the second antigen-binding domain to form the light chain of the CD3-binding portion.

[0020] 9. The antibody of embodiment 8, wherein A1-HC may be homologous or distinct from A2-HC, and / or A1-LC is homologous or distinct from A2-LC.

[0021] 10. The antibody of embodiment 8, wherein the A1-VH and A1-VL of the CLDN18.2 binding portion are fully human, and the A2-VH and A2-VL of the CD3 binding portion are humanized.

[0022] 11. The antibody of embodiment 8, wherein the heavy chain of the CLDN18.2 binding portion is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 37; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 37; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 37; and / or The light chain of the CLDN18.2 binding moiety is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 38; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 38; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 38.

[0023] 12. The antibody of embodiment 8 or 11, wherein: The heavy chain of the CD3 binding portion is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 41, 39 or 42; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 41, 39, or 42; or (iii) comprises or consists of an amino acid sequence having one or more (preferably no more than 20 or 10, more preferably no more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 41, 39 or 42; and / or The light chain of the CD3 binding portion is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 40; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 40; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 40.

[0024] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the invention bind to CLDN18.2 (e.g., human CLDN18.2) with high affinity. In some embodiments, the antibodies of the invention specifically bind to CLDN18.2 (e.g., human CLDN18.2) but do not bind to CLDN18.1 (e.g., human CLDN18.1). In some embodiments, the antibodies or antigen-binding fragments of the invention have a higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, e.g., Zolbetuximab (abbreviated as Zmab) antibodies. In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the invention bind to CD3 (e.g., human CD3 or cynomolgus CD3) with a desired (e.g., lower) affinity. In some embodiments, the antibodies of the invention can bind to both human CD3 and cynomolgus CD3. In some embodiments, the affinity of the antibodies is measured by biolayer interferometry or surface plasmon resonance.

[0025] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the invention are capable of binding to CLDN18.2 on the surface of tumor cells and to CD3 on the surface of effector cells, hi some embodiments, the binding is detected by flow cytometry.

[0026] In one embodiment, the anti-CLDN18.2 / CD3 protein in the liquid antibody formulation of the present invention has a concentration of about 0.1 mg / mL to 100 mg / mL, preferably about 0.1 mg / mL to 10 mg / mL, and more preferably about 0.1 mg / mL to 1 mg / mL, and in another embodiment, the anti-CLDN18.2 / CD3 in the liquid antibody formulation of the present invention has a concentration of about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL.

[0027] In some embodiments, the buffer used in the formulation of the present invention is a histidine buffer, particularly a buffer system consisting of histidine and histidine hydrochloride. In some embodiments, the histidine in the histidine buffer of the present invention is at a concentration of about 0.01 mg / mL to 10 mg / mL, particularly about 0.1 mg / mL to 1 mg / mL, for example, about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL or 1 mg / mL. In one embodiment, the formulation of the present invention contains about 0.18 mg / mL of histidine. In another embodiment, the histidine buffer used in the formulation of the present invention consists of about 0.18 mg / mL of histidine and about 1.84 mg / mL of histidine hydrochloride.

[0028] In one embodiment, the stabilizer in the liquid antibody formulation of the present invention has a concentration of about 10 mg / mL to 100 mg / mL. In one embodiment, the stabilizer in the liquid antibody formulation of the present invention has a concentration of about 20 mg / mL to 60 mg / mL, for example, about 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, or 60 mg / mL.

[0029] In one embodiment, the stabilizer is selected from polyols (e.g., sorbitol, mannitol, or a combination thereof), sugars (e.g., sucrose, trehalose, maltose, or a combination thereof), amino acids (e.g., arginine hydrochloride, methionine, glycine, proline, and combinations thereof or salts thereof), and any combination thereof. In one embodiment, the stabilizer comprises about 20 mg / mL to 80 mg / mL sorbitol, e.g., 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 70 mg / mL, or 80 mg / mL sorbitol. In another embodiment, the stabilizer comprises about 20 mg / mL to 60 mg / mL sucrose, e.g., 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, or 60 mg / mL sucrose. In a preferred embodiment, the stabilizer further comprises arginine, preferably arginine hydrochloride, at a concentration of about 25 mM to 200 mM, preferably about 50 mM to 150 mM, more preferably about 50 mM to 120 mM, and most preferably about 60 mM to 100 mM, for example, about 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, or 90 mM.

[0030] In a preferred embodiment, the stabilizer includes a combination of sorbitol and arginine, or a combination of sucrose and arginine. For example, a combination of about 20 mg / mL to 40 mg / mL sorbitol and about 50 mM to 100 mM arginine, or a combination of about 30 mg / mL to 60 mg / mL sucrose and about 50 mM to 100 mM arginine may be used.

[0031] In a preferred embodiment, the liquid antibody formulation of the invention contains about 30 mg / mL to 60 mg / mL (e.g., about 50 mg / mL) sorbitol, or about 20 mg / mL to 30 mg / mL (e.g., about 25 mg / mL) sorbitol and 80 mM to 90 mM (about 85 mM) arginine.

[0032] In one embodiment, the surfactant in the liquid antibody formulation of the present invention has a concentration of about 0.1 mg / mL to 1 mg / mL. In one embodiment, the surfactant in the liquid antibody formulation of the present invention has a concentration of about 0.2 mg / mL to 0.8 mg / mL, for example, about 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, or 0.8 mg / mL.

[0033] In one embodiment, the surfactant is a non-ionic surfactant. In one embodiment, the surfactant is selected from a polysorbate surfactant, a poloxamer, and a polyethylene glycol. In one embodiment, the surfactant is selected from a polysorbate surfactant. In one specific embodiment, the surfactant in the liquid antibody formulation of the invention is polysorbate-80.

[0034] Chelating agent concentration and type: In one embodiment, the chelating agent in the liquid antibody formulation of the present invention has a concentration of about 0.005 mg / mL to 0.05 mg / mL. In one embodiment, the chelating agent in the liquid antibody formulation of the present invention has a concentration of about 0.008 mg / mL to 0.018 mg / mL, for example, about 0.008 mg / mL, 0.009 mg / mL, 0.010 mg / mL, 0.012 mg / mL, 0.014 mg / mL, or 0.018 mg / mL.

[0035] In one embodiment, the chelating agent is a carboxylic acid type chelating agent. In one embodiment, the chelating agent is selected from edetate disodium, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, and dihydroxyethylglycine. In one embodiment, the chelating agent is selected from edetate disodium.

[0036] In one embodiment, the liquid formulation has a pH value of about 5.0 to 6.0. In some embodiments, the liquid formulation has a pH value of any value between about 5.0 and 6.0, such as about 5.0, 5.2, 5.4, 5.6, 5.8, 6.0. Preferably, the formulation has a pH of 5.2±0.2 or 5.5±0.2, preferably 5.2.

[0037] In one embodiment, the liquid antibody formulation of the invention comprises: (i) about 0.1 mg / mL to 100 mg / mL of anti-CLDN18.2 / CD3 protein; (ii) about 0.1 mg / mL to 2 mg / mL of histidine; (iii) about 30 mg / mL to 70 mg / mL sorbitol; (iv) about 0.1 mg / mL to 1 mg / mL polysorbate 80, and (v) optionally, about 0.008 mg / mL to 0.018 mg / mL of edetate disodium; wherein the liquid formulation has a pH of about 5.0 to 5.5, for example, about 5.2; For example, the liquid antibody formulation may be (i) about 0.1 mg / mL to 10 mg / mL, for example, 0.1 mg / mL to 1 mg / mL of anti-CLDN18.2 / CD3 protein; (ii) about 1.55 mg / mL histidine; (iii) about 40 mg / mL to 60 mg / mL sorbitol or sucrose, preferably 50 mg / mL sorbitol or 30 mg / mL to 50 mg / mL sucrose; (iv) about 0.2 mg / mL to 0.8 mg / mL, for example, 0.2 mg / mL to 0.6 mg / mL of polysorbate 80, and (v) optionally, about 0.008 mg / mL to 0.018 mg / mL, e.g., 0.01 mg / mL, of edetate disodium; wherein the liquid formulation has a pH of about 5.0 to 5.5, for example, about 5.2; Alternatively, the liquid antibody formulation comprises: (i) about 1 mg / mL of anti-CLDN18.2 / CD3 protein, about 1.55 mg / mL of histidine, about 50.00 mg / mL of sorbitol, and about 0.2 mg / mL of polysorbate 80, at a pH of about 5.2; or (ii) about 50 mg / mL anti-CLDN18.2 / CD3 protein, about 1.55 mg / mL histidine, about 50.00 mg / mL sorbitol, about 0.01 mg / mL edetate disodium, and about 0.2 mg / mL polysorbate 80, at a pH of about 5.2; or (iii) about 50 mg / mL anti-CLDN18.2 / CD3 protein, about 0.82 mg / mL sodium acetate, about 50.00 mg / mL sorbitol, and about 0.2 mg / mL polysorbate 80, at a pH of 5.2.

[0038] The liquid formulation of the present invention can be stably stored for a long period of time, for example, at least 24 months or more. In one embodiment, the liquid formulation of the present invention can be stored at about -80°C to about 45°C, for example, about -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C or about 45°C, for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or more, and is stable.

[0039] In one embodiment, the liquid formulation of the present invention can be stably stored for at least 24 months. In yet another embodiment, the liquid formulation of the present invention is stable at at least 40°C. In yet another embodiment, the liquid formulation of the present invention is stable at about 2°C to 8°C for at least 3 months, preferably at least 12 months, more preferably 24 months. In one embodiment, the liquid formulation of the present invention is stable at room temperature or, for example, at about 25°C for at least 2 months, preferably at least 3 months, more preferably 6 months. In a further embodiment, the liquid formulation of the present invention is stable at about 40°C for at least 2 weeks, preferably at least 1 month.

[0040] In one embodiment, the stability of the formulation can be indicated by detecting changes in the appearance, visible foreign matter, protein content, turbidity, purity, and / or charge variants of the formulation. In one embodiment, the stability of the liquid formulation of the present invention can be detected in a forced high temperature stress experiment, for example after storage at 40°C ± 2°C for at least 1 week, 2 weeks, or preferably 1 month, or in an accelerated experiment, for example after storage at 25°C ± 2°C for at least 1 month or 2 months, or in a long-term experiment, for example after storage at 5°C ± 3°C for at least 2 months or 3 months, or in a shaking experiment (for example, shaking at room temperature, protected from light, at 650 r / min for 5 days), and / or in a freeze-thaw experiment (for example, freeze-thawing six times at -30°C / room temperature). In one embodiment, the stability of the liquid formulation of the present invention is detected relative to an initial value, for example the initial value on day 0 of storage, or the initial value before shaking or freeze-thawing experiments.

[0041] In one embodiment, the stability of the liquid formulation of the present invention is visually inspected after storage, or after a shaking experiment, or after a freeze-thaw experiment, where the liquid formulation of the present invention retains a clear to slightly opalescent appearance, is a colorless to pale yellow liquid, and is free of foreign matter. In one embodiment, the formulation is free of visible foreign matter when visually inspected with a transparency detector. In one embodiment, the stability of the liquid formulation of the present invention is inspected by measuring the change in protein content after storage, or after a shaking experiment, or after a freeze-thaw experiment, where, for example, when measured by ultraviolet spectrophotometry (UV), the rate of change in protein content is 20% or less, preferably 10% or less, for example 7% to 8%, more preferably 5%, 2% or 1% or less, relative to the initial value. In one embodiment, the stability of the liquid formulation of the present invention is inspected by measuring the change in turbidity of the liquid formulation of the present invention after storage, or after a shaking experiment, or after a freeze-thaw experiment, where, for example, OD 350mmWhen detected by the method, the change value is 0.06 or less, preferably 0.05 or less, more preferably 0.04 or less, 0.02 or less, relative to the initial value. In one embodiment, the stability of the liquid formulation of the present invention is examined by measuring the purity change of the liquid formulation of the present invention after storage, or after a shaking experiment, or after a freeze-thaw experiment, where the change value of the purity of the monomer (or the change value of the main peak) is 10% or less, for example 5%, 4% or 3% or less, for example 2% or less, preferably 1% or less, relative to the initial value, when measured by volume exclusion high performance liquid chromatography (SEC-HPLC). In one embodiment, the stability of the liquid formulation of the present invention is examined by measuring the purity change of the liquid formulation of the present invention after storage, or after a shaking experiment, or after a freeze-thaw experiment, where the decrease in the change value of the purity of the monomer (or the change value of the main peak) is 10% or less, for example 5%, 4%, 3%, 2% or 1% or less, relative to the initial value, when measured by non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS). In one embodiment, the stability of the liquid formulation of the invention is detected by cation exchange high performance liquid chromatography (CEX-HPLC) after storage or after a shaking experiment or after a freeze-thaw experiment, in which the sum of the change values ​​of the antibody charge variants (major component, acidic component and basic component) is less than 50%, e.g. less than 40%, 30%, 20%, 10% or 5%, and / or the change value of the major component is less than 20%, 15%, 10%, 8% or 5%, relative to the initial value. In one embodiment, the stability of the liquid formulation of the present invention is detected by direct ELISA after storage, or after a shaking experiment, or after a freeze-thaw experiment, in which the relative binding activity of the antibody is 70% to 130%, for example, 70%, 80%, 90%, 93%, 95%, 98%, 100%, 103%, 105%, 108%, 110%, 115%, 120%, 125% or 130%, preferably 90% to 110%, compared to the initial value.

[0042] In one embodiment, the liquid formulation of the invention is stable after storage, e.g., at least 2 months at 25° C. or 1 month at 40° C.±2° C., preferably with a change in viscosity relative to the initial value on day 0 of storage. (i) the main peak has a change of less than 1% when measured by SEC-HPLC method, and / or the preparation has a purity of more than 96%, preferably more than 97%, 98%; (ii) the change in the main peak is less than 2% when measured by the non-reducing CE-SDS method, and / or the preparation has a purity of greater than 96%, preferably greater than 97%, 98%; (iii) when measured by CEX-HPLC, the total change in each component (major component, acidic component and basic component) of the anti-CLDN18.2 / CD3 protein in the preparation is 40% or less, and / or the change in the major component is 20% or less; For example, after storage at 40° C.±2° C. for one month, the total change is about 40% or less (e.g., 35%, 30%, 25%, 20%, 15%, or 10% or less), or the change in the main component is 20% or less (e.g., 15%, 12%, 10%, or 8% or less), or For example, after storage at 25° C. for 2 months, the total change is about 20% or less (e.g., 15%, 14%, 13%, or 12% or less), or the change in the main component is about 15% or less (e.g., 10%, 8%, 7%, 6%, or 5% or less); and (iv) the relative binding activity of the anti-CLDN18.2 / CD3 protein in the formulation is 70% to 130%, e.g., 90%, 93%, 95%, 98%, 100%, 103%, 105%, 108%, 110%, 115% or 120%, e.g., 90% to 110%, as measured by ELISA; In a preferred embodiment, the liquid formulations of the present invention are stable under shaking and / or repeated freeze-thaw cycles.

[0043] Preferably, the formulation is stable under shaking or repeated freezing and thawing, for example, after shaking at room temperature, protected from light, at 650 r / min for 5 days, or after six repeated freeze-thaw cycles at -30°C / room temperature, (i) the main peak has a change of less than 1% when measured by SEC-HPLC method, and / or the formulation has a purity of greater than 96%, preferably greater than 97%, 98% or 99%; (ii) the change in the main peak is less than 2% when measured by the non-reducing CE-SDS method, and / or the preparation has a purity of greater than 96%, preferably greater than 97%, 98%; (iii) When measured by CEX-HPLC, the total change in each component (major component, acidic component, and basic component) of the anti-CLDN18.2 / CD3 protein in the formulation is 2% or less; and (iv) the relative binding activity of the anti-CLDN18.2 / CD3 protein in the formulation is between 70% and 130%, e.g., between 90% and 110%, as measured by ELISA; In one aspect, the liquid formulation of the present invention is a pharmaceutical formulation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid formulation is an intravenous infusion.

[0044] In another aspect, the present invention provides a solid antibody formulation obtained by performing a solidification process on the liquid antibody formulation of the present invention. The solidification process is performed, for example, by a crystallization method, a spray drying method, or a freeze-drying method. In a preferred embodiment, the solid antibody formulation is, for example, in the form of a freeze-dried powder for injection. The solid antibody formulation can be reconstituted in a suitable solvent before use to form a reconstituted formulation of the present invention. The reconstituted formulation is also a liquid antibody formulation of the present invention. In one embodiment, the suitable solvent is selected from water for injection, organic solvent for injection, including, but not limited to, oil for injection, ethanol, propylene glycol, etc., or a combination thereof.

[0045] In one aspect, the invention provides a delivery device comprising a liquid or solid antibody formulation of the invention, hi one embodiment, the delivery device of the invention is provided in the form of a pre-filled syringe comprising the liquid or solid antibody formulation of the invention, for use in, e.g., intravenous, subcutaneous, intradermal or intramuscular injection, intravenous infusion.

[0046] In another aspect, the present invention provides a method of delivering an anti-CLDN18.2 / CD3 protein to a subject, such as a mammal, comprising administering to said subject a liquid or solid antibody formulation of the present invention, said delivery being performed by a delivery device, e.g., utilizing a pre-filled syringe.

[0047] In a further aspect, the present invention provides a liquid or solid antibody formulation of the present invention in a method for preventing or treating a tumor (e.g., carcinoma) in a subject, said method comprising administering to the subject an effective amount of an anti-CLDN18.2xCD3 bispecific antibody or a fragment thereof (preferably an antigen-binding fragment), pharmaceutical composition, pharmaceutical combination or drug kit of the present invention.

[0048] The invention further provides methods of administering to a subject a liquid or solid antibody formulation of the invention, or a delivery device (e.g., a pre-filled syringe) or drug comprising said liquid or solid antibody formulation.

[0049] Other embodiments of the present invention will become apparent upon reference to the following detailed description. [Brief description of the drawings]

[0050] Preferred embodiments of the invention, which are described in detail below, are better understood when read in conjunction with the following drawings: For the purposes of illustrating the invention, there are shown in the drawings embodiments which are presently preferred, but it should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0051] [Figure 1]FIG. 1 is a schematic diagram showing the structure of a bispecific antibody used in the examples. [Diagram 2] 1 shows that the bispecific antibody of the present invention specifically kills CLDN18.2-positive gastric cancer cells NUGC-4. [Diagram 3] 1 shows that the bispecific antibody specifically kills CLDN18.2-positive pancreatic cancer cells DAN-GCLDN18.2. [Figure 4] This shows that the bispecific antibody does not non-specifically kill cells negative for CLDN18.2 expression. [Diagram 5] 1 shows the in vivo efficacy results of bispecific antibodies in a humanized NUGC-4 gastric cancer model. [Figure 6] 1 shows in vivo efficacy results of bispecific antibodies in a DAN-G-CLDN18.2 pancreatic cancer humanized model. [Figure 7] PK of bispecific antibodies in mice. [Figure 8] The effect of changing the pH value in the formulation is shown. (A) is a trend graph of protein content (UV method), (B) is a trend graph of purity (SEC-HPLC method), (C) is a trend graph of purity (nrCE-SDS method), (D) is a trend graph of charge variants-acidic components (iCIEF method), (E) is a trend graph of charge variants-major components (iCIEF method), and (F) is a trend graph of charge variants-basic components (iCIEF method). [Figure 9] The effects of different formulation compositions are shown. (A) is a trend graph of protein content (UV method), (B) is a trend graph of purity (SEC-HPLC method), (C) is a trend graph of purity (nrCE-SDS method), (D) is a trend graph of charge variants-acidic components (iCIEF method), (E) is a trend graph of charge variants-major components (iCIEF method), and (F) is a trend graph of polysorbate 80 content (HPLC-FLD method). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Detailed Description of the Invention definition 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 purposes of the present invention, the following terms are defined below.

[0053] The term "about," when used in conjunction with a number or numerical value, is meant to cover a range of numbers or numerical values ​​that is 5% less than the number or numerical value specified as the lower limit and 5% more than the number or numerical value specified as the upper limit.

[0054] The term "and / or," when used in conjunction with two or more options, should be understood to refer to any one of the options or any two or more of the options.

[0055] As used herein, the term "comprise" or "comprise" means including the recited element, integer or step, but not excluding any other element, integer or step. When the term "comprise" or "comprise" is used herein, it also covers the case consisting of the recited element, integer or step, unless otherwise specified. For example, when an antibody variable region "comprising" a certain sequence is mentioned, it is also intended to cover an antibody variable region consisting of that specific sequence.

[0056] The term "CLAUDIN" or "CLDN" as used herein refers to the most important scaffold protein that determines the structure of tight junctions between cells, and is involved in adherens junctions and plays an important role in the metastasis and invasion of tumor cells. Claudin proteins are widely present in mammalian epithelial and endothelial cells, and are mainly distributed on the epithelial cell lateral and basal plasma membranes. Different Claudin proteins have specific expression in different tissues, where the Claudin18 (CLDN18) gene is located at 3q22.3, has a molecular weight of 24 kDa, has 261 amino acid residues, belongs to the Claudins superfamily, and its protein composition has two extracellular loops and four transmembrane domains. The two subtypes of human CLDN18 or Claudin18 protein are Claudin18.1 or CLDN18.1 (UniProt ID: P56856-1) and Claudin18.2 or CLDN18.2 (UniProt ID: P56856-2), respectively, and in the primary structure sequences of both proteins, only amino acid residues at certain positions from the N-terminal signal peptide to the extracellular loop 1 (Loop1) structure are different, especially on the extracellular loop 1, CLDN18.1 and CLDN18.2 differ by only eight amino acids. The interspecies sequence homology of the two subtype proteins of CLDN18 is also very high. Here, the extracellular loop 1 of CLDN18.2 has a perfect sequence match in different species such as human, mouse, and rhesus monkey, and the homology between human and mouse CLDN18.2 proteins reaches 84%, revealing that the CLDN18.2 protein sequence is highly conserved (O. Tureci et al., Gene 481:83-92, 2011). CLDN18.2 or any variants and isotypes thereof can be isolated from cells or tissues that naturally express them, or recombinantly produced by techniques well known in the art and / or described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0057] As used herein, the term "anti-CLDN18.2 antibody", "anti-CLDN18.2", "CLDN18.2 antibody" or "antibody that binds to CLDN18.2" refers to an antibody that binds to (human) CLDN18.2 with sufficient affinity, and can therefore be used as a therapeutic agent targeting (human) CLDN18.2. In one embodiment, the (human) CLDN18.2 antibody binds to (human) CLDN18.2 with high affinity in vitro or in vivo. In one embodiment, the (human) CLDN18.2 antibody does not bind to CLDN18.1. In one embodiment, the (human) CLDN18.2 antibody binds to cells expressing CLDN18.2 but not to cells expressing CLDN18.1. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biolayer interferometry (BLI), MSD assay or surface plasmon resonance (SPR) or flow cytometry.

[0058] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of the multi-molecule T cell receptor (TCR), and is composed of a homodimer or heterodimer formed by two of the four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3- ε (hCD3ε) comprises the amino acid sequence described in UniProtKB / Swiss-Prot:P07766.2. Human CD3-δ (hCD3δ) comprises the amino acid sequence described in UniProtKB / Swiss-Prot:P04234.1. In some embodiments, CD3 according to the present invention refers to CD3 from humans or cynomolgus monkeys.

[0059] The term "antibody that binds CD3" or "anti-CD3 antibody" as used herein includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit (e.g., ε, δ, γ or ζ), as well as antibodies and antigen-binding fragments thereof that specifically recognize and link to dimeric complexes that specifically recognize two CD3 subunits (e.g., γ / ε, δ / ε and ζ / ζ CD3 dimers). The antibodies and antigen-binding fragments of the invention can bind to soluble CD3, bound CD3 and / or CD3 expressed on the cell surface. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants, including, for example, alternative monomeric and dimeric CD3 structures that lack the transmembrane region or are not bound to cell membranes. The invention provides antibodies that can activate human and cynomolgus monkey T cells by binding to human and cynomolgus monkey CD3 with lower or undetectable binding affinity. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biolayer interferometry (BLI), MSD assay or surface plasmon resonance (SPR) or flow cytometry.

[0060] The term "cell surface expressed CD3" refers to one or more CD3 proteins that are expressed on the cell surface in vivo or in vitro such that at least a portion of the CD3 protein is exposed on the extracellular side of the cell membrane and an antigen-binding portion that is accessible to antibodies. "Cell surface expressed CD3" includes CD3 protein in the context of a functional T cell receptor contained in the cell membrane. The term "cell surface expressed CD3" includes CD3 protein expressed as part of a homodimer or heterodimer (e.g., δ / ε, γ / ε and ζ / ζ CD3 dimers) on the cell surface.

[0061] Effector cells include effector T cells (T lymphocytes), such as CD4+ T cells, CD8+ T cells, Th1, Th2 and regulatory T cells (Tregs). Effector cells may also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0062] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen-binding site and encompasses natural and artificial antibodies of various structures, including, but not limited to, intact antibodies and antigen-binding fragments of antibodies.

[0063] A "bispecific" antibody refers to an antibody having two antigen-binding sites, each of which binds to a different epitope of the same antigen or a different epitope of a different antigen. In one embodiment, a bispecific antibody has binding specificity for a first antigen and a second antigen.

[0064] Bispecific antibody forms include IgG-like and non-IgG-like antibodies (Fan et al., (2015) Journal of Hematology & Oncology .8:130). The most common type of IgG-like antibody contains two Fab regions and one Fc region, and the heavy and light chains of each Fab can be derived from separate monoclonal antibodies. Non-IgG-like bispecific antibodies lack the Fc region, and each antigen or target binding domain can be a Fab, a single-chain variable fragment (scFv), or a fusion protein that mimics the variable domains of two antibodies, with the different binding domains linked to each other by peptide linkers, chemical coupling, non-covalent linkages, or other methods. These forms include bispecific T-cell adaptors (BiTEs).

[0065] Bispecific antibodies of the invention can be prepared in any bispecific antibody format or technique. For example, an antibody or fragment thereof having a first antigen-binding specificity can be operatively linked (e.g., by chemical coupling, genetic fusion, non-covalent bonding or other methods) to one or more other molecular entities, such as another antibody or antibody fragment having a second antigen-binding specificity, to produce a bispecific antibody. Specific exemplary bispecific formats that can be used in the context of the present invention include scFv-based or biantibody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-entry holes), CrossMab, CrossFab, (SEED)body, Duobody, IgG1 / IgG2, dual acting Fab (DAF)-IgG and Mab. 2 This includes, but is not limited to, bispecific forms.

[0066] The term "linker" as used herein refers to any molecule capable of directly linking different portions of a bispecific antibody. Examples of linkers that establish covalent bonds between different antibody portions include peptide linkers and non-proteinaceous polymers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol, polypropylene glycol.

[0067] The term "peptide linker" according to the present invention refers to a sequence of amino acids, which links the amino acid sequence of a first part of an antibody to a second part of the antibody. For example, a peptide linker may link a first (variable and / or binding) domain of an antibody to a second (variable and / or binding) domain. For example, a peptide linker may also link one part of an antibody to another part of an antibody, for example, an antigen binding domain to an Fc domain or a fragment thereof. Preferably, said peptide linker has a length sufficient to link two entities such that they maintain a conformation relative to each other without interfering with the desired activity.

[0068] Peptide linkers may or may not primarily comprise the amino acid residues Gly, Ser, Ala, or Thr. Useful linkers include glycine-serine polymers, including, for example, (GS)n, (GSGGS)n, (GGGGS)n, (GGGS)n, and (GGGGS)nG, where n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, or 10). Useful linkers further include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0069] The term "valent" according to the present invention indicates the presence of a certain number of binding sites in an antibody molecule. Thus, the terms bivalent, trivalent, tetravalent indicate the presence of two, three or four binding sites, respectively, in an antibody construct. Bispecific antibodies according to the present invention are at least bivalent and may be multivalent, e.g. bivalent, trivalent, tetravalent or hexavalent.

[0070] The term "binding domain" as used herein refers to any portion of a bispecific antibody that binds to a particular target or antigen. A binding domain is an antigen-binding site. A binding domain may be, for example, an antibody, an immunoglobulin itself, or an antibody fragment. Such a binding domain may or may not have a tertiary structure independent of the rest of the BsAB, and may or may not bind to its target as a separate entity.

[0071] In certain exemplary embodiments of the present invention, each antigen-binding domain of a bispecific antibody comprises a heavy chain variable region VH and a light chain variable region VL. In a bispecific antibody comprising a first antigen-binding domain and a second antigen-binding domain, the VH, VL or CDR of the first antigen-binding domain may be designated with the prefix "A1", and the VH, VL or CDR of the second antigen-binding domain may be designated with the prefix "A2". For example, the heavy chain CDRs (HCDRs) of the first antigen-binding domain may be referred to herein as A1-HCDR1, A1-HCDR2 and A1-HCDR3, and the heavy chain CDRs of the second antigen-binding domain may be referred to herein as A2-HCDR1, A2-HCDR2 and A2-HCDR3. Similarly, the heavy chain variable region VH of the first antigen-binding domain is referred to herein as A1-VH, and the heavy chain variable region VH of the second antigen-binding domain is referred to herein as A2-VH. The light chain CDRs of the first antigen-binding domain ( L The light chain CDRs of the first antigen-binding domain may be referred to herein as A1-LCDR1, A1-LCDR2 and A1-LCDR3, and the light chain CDRs of the second antigen-binding domain may be referred to herein as A2-LCDR1, A2-LCDR2 and A2-LCDR3. Similarly, the light chain variable region VL of the first antigen-binding domain is referred to herein as A1-VL, and the light chain variable region VL of the second antigen-binding domain is referred to herein as A2-VL.

[0072] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0073] "Antigen-binding fragment" refers to a molecule distinct from an intact antibody, which contains a portion of an intact antibody and binds to the antigen that the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibody), linear antibody, single chain antibody (e.g., scFv), single domain antibody, e.g., VHH, bivalent antibody or fragment thereof, or camelid antibody.

[0074] The term "antigen" refers to a molecule that elicits an immune response. Such immune response may involve the production of antibodies or the activation of specific immune cells, or both. Those skilled in the art will appreciate that almost any macromolecule, including proteins or peptides, may serve as an antigen. Antigens may also be derived from recombinant or genomic DNA. As used herein, the term "epitope" refers to the portion of an antigen (e.g., CLDN18.2) that specifically interacts with an antibody molecule.

[0075] An "antibody that binds to the same or overlapping epitope" as a reference antibody is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% or more in a competitive assay; conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% or more in a competitive assay.

[0076] An antibody that binds to its antigen competitively with a reference antibody is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% or more in a competitive assay. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% or more in a competitive assay. To determine whether one antibody competes with another antibody, various competitive binding assays are available, such as solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), and sandwich competition assay.

[0077] An antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen is an antibody that inhibits the binding of the reference antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% or more. Conversely, the reference antibody inhibits the binding of the antibody to its antigen by 50%, 60%, 70%, 80%, 90% or 95% or more. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for measuring affinity are known in the art.

[0078] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that may have at least 50%, 60%, 70%, 80%, 90% or 95% or more of the binding affinity and / or specificity of the reference antibody, which may be measured by any method known in the art for measuring binding affinity and / or specificity.

[0079] "Complementarity determining regions", "CDR regions" or "CDRs" are the regions in an antibody variable domain that are hypervariable in sequence and structurally determined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to an antigen epitope. The CDRs of the heavy and light chains are usually called CDR1, CDR2 and CDR3 and are numbered sequentially from the N-terminus. The CDRs in the heavy chain variable domain of an antibody are called HCDR1, HCDR2 and HCDR3, and the CDRs in the light chain variable domain of an antibody are called LCDR1, LCDR2 and LCDR3. For a given light or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined by any one or a combination of a number of known antibody CDR assignment systems, including, for example, the Chothia system, which is based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al., (1989) Nature 342:877-883; Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), the Kabat system, which is based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., USDepartment of Health and Human Services, National Institutes of Health (1987)), the AbM (University of Bath), Contact (University College London), and the International ImMunoGeneTics (ImmunoGeneTics). The database (IMGT) (available on the World Wide Web at imgt.cines.fr / ) and a North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures.

[0080] For example, according to different CDR determination methods, the residues of each CDR are as follows:

[0081] [Table 1] A CDR may be determined by having the same Kabat numbering position as the sequence of a reference CDR (eg, any one of the exemplary CDRs of the invention).

[0082] Unless otherwise specified, in the present invention, the term "CDR" or "CDR sequence" covers CDR sequences determined in any one of the above ways.

[0083] Unless otherwise specified, in the present invention, when referring to residue positions in an antibody variable region (including heavy chain variable region residues and light chain variable region residues), it refers to numbered positions based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0084] In one embodiment, the CDRs of the heavy chain variable region of the antibody according to the present invention are determined according to the following rules.

[0085] VH CDR1 is determined according to the AbM rules, and VH CDR2 and VH CDR3 are both determined according to the Kabat rules.

[0086] In one embodiment, the CDRs of the light chain variable region of the antibody of the present invention are determined according to the Kabat rules.

[0087] In one embodiment, the heavy chain variable region CDRs of the antibody according to the present invention are determined according to the following rules: VH CDR1 is determined according to the AbM rules, VH CDR2 and VH CDR3 are both determined according to the Kabat rules, and the light chain variable region CDRs are determined according to the Kabat rules.

[0088] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained by different allocation systems may differ, i.e., there are differences in the CDR sequences of the variable regions of the same antibody defined by different allocation systems. Thus, when an antibody is defined by a specific CDR sequence defined in the present invention, the scope of said antibody also includes antibodies whose variable region sequences include said specific CDR sequences, but whose CDR boundaries differ from the specific CDR boundaries defined in the present invention because a different approach (e.g., rules or combinations of different allocation systems) is used.

[0089] Antibodies with different specificities (i.e., directed to different binding sites of different antigens) have different CDRs (in the same allocation system). However, even though CDRs differ between antibodies, there are a limited number of amino acid positions in the CDRs that are directly involved in binding to the antigen. The minimum overlapping region can be determined to provide a "minimum binding unit" for antigen binding by at least two of the Kabat, Chothia, AbM, Contact and North methods. The minimum binding unit may be a subpart of one of the CDRs. As known to those skilled in the art, the structure of the antibody and protein folding can determine the residues of the remaining part of the CDR sequence. Therefore, the present invention contemplates variants of any of the CDRs provided herein. For example, in a variant of one CDR, the amino acid residues of the minimum binding unit are kept unchanged, but the remaining CDR residues defined according to Kabat or Chothia may be replaced with conservative amino acid residues.

[0090] The term "Fc region" is used herein to define the constant regions of CH2 and CH3 of an immunoglobulin heavy chain, and includes native sequence Fc regions and variant Fc regions. Native Fc regions can bind to different Fc receptors on the surface of immune cells and trigger CDC / ADCC / ADCP effector functions. Such effector functions generally require the association of an Fc region with a binding domain (e.g., an antibody variable domain). In some embodiments, an Fc region is mutated to enhance its CDC / ADCC / ADCP effector function. In some embodiments, an Fc region is mutated to reduce or eliminate its CDC / ADCC / ADCP effector function.

[0091] "IgG-type antibody" refers to the IgG type to which the heavy chain constant region of the antibody belongs. All antibodies of the same type have the same heavy chain constant region, and antibodies of different types have different heavy chain constant regions. For example, an antibody of IgG4 type refers to an antibody whose heavy chain constant region is derived from IgG4, or an antibody of IgG1 type refers to an antibody whose heavy chain constant region is derived from IgG1.

[0092] A "humanized" antibody refers to an antibody that comprises amino acid residues from non-human CDRs and amino acid residues from human FRs. In some embodiments, a humanized antibody comprises at least one, and typically two, of substantially all of the variable domains, in which all or substantially all of the CDRs (e.g., CDRs) correspond to portions derived from a non-human antibody, and all or substantially all of the FRs correspond to portions derived from a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has been humanized.

[0093] "Human antibody," "fully human antibody" or "fully humanized antibody" may be used interchangeably and refer to an antibody produced from a human or human cell, or derived from a non-human source, and having an amino acid sequence that corresponds to that of an antibody encoded in a human antibody library or another human antibody. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0094] As used herein, the terms "binding" or "specifically binding" mean that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antigen-binding site to bind to a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other conventional binding assays known in the art, such as radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0095] The term "therapeutic agent" as used herein includes any substance effective in the treatment or prevention of tumors, such as cancer, including chemotherapeutic agents, cytokines, antiangiogenic agents, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0096] The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction.

[0097] A "chemotherapeutic agent" includes a chemical compound useful in the treatment of cancer or immune system disorders.

[0098] The term "small molecule drug" refers to a low molecular weight organic compound that can regulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, generally less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetics, and antibody mimetics. As therapeutic agents, small molecules are more cell permeable, more susceptible to degradation, and less likely to induce an immune response than large molecules.

[0099] The term "immunomodulatory agent" as used herein refers to a natural or synthetic active agent or agent that suppresses or modulates an immune response. The immune response may be a humoral or cellular response. Immunomodulatory agents include immunosuppressants. In some embodiments, immunomodulatory agents of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0100] The term "effective amount" refers to an amount or dosage that provides the desired effect in a patient in need of treatment or prevention after administration of one or more doses of an antibody or fragment or composition or combination of the invention to the patient.

[0101] A "therapeutically effective amount" refers to an amount that effectively achieves the desired therapeutic result at the required dosage for the required period of time. A therapeutically effective amount is also an amount in which any toxic or adverse effects of the antibody or antibody fragment or composition or combination are not offset by the beneficial effects of the treatment. Compared to untreated subjects, a "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 40%, and more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100%.

[0102] A "prophylactically effective amount" refers to an amount to effectively achieve a desired prophylactic result at a necessary dosage for a necessary period of time. Generally, the prophylactically effective amount will be less than the therapeutically effective amount, since a prophylactic dose is administered prior to or at an earlier stage of disease in a subject.

[0103] The sequence identity between sequences is calculated as follows.

[0104] To determine the identity percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison). In a preferred embodiment, the length of the reference sequence aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. If a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position.

[0105] A mathematical algorithm can realize the sequence comparison between two sequences and the calculation of the identity percentage. In one preferred embodiment, the identity percentage between two amino acid sequences is determined by the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm integrated in the GAP program of the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. In another preferred embodiment, the identity percentage between two nucleotide sequences is determined by the GAP program of the GCG software package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix, and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and the one that should be used unless otherwise specified) employs a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. Alternatively, the percentage identity between two amino acid or nucleotide sequences may be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) as integrated into the ALIGN program (version 2.0) utilizing a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4. Additionally or alternatively, the nucleic acid and protein sequences described herein can be used as "query sequences" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0106] As used herein, the term "hybridize under stringent conditions (e.g., low stringency, moderate stringency, high stringency, or very high stringency conditions)" describes the conditions of hybridization and washing. Guidance for carrying out hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, which is incorporated by reference. The references describe hydrated and non-hydrated methods, and either method can be utilized. Preferred hybridization conditions referred to herein are as follows: 1) low stringency hybridization conditions are 6X sodium chloride / sodium citrate (SSC) at about 45° C., followed by two washes in 0.2X SSC, 0.1% SDS at at least 50° C. (for low stringency conditions, the wash temperature can be increased to 55° C.); 2) moderate stringency hybridization conditions are 6X SSC at about 45° C., followed by one wash in 0.2X SSC, 0.1% SDS at 60° C. 1 or more washes at 65° C., 3) high stringency hybridization conditions are 6× SSC at about 45° C., followed by one or more washes at 0.2× SSC, 0.1% SDS at 65° C., and preferably, 4) very high stringency hybridization conditions are 0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washes at 0.2× SSC, 0.1% SDS at 65° C. Very high stringency conditions (4) are the preferred conditions and are the conditions to be utilized unless otherwise specified.

[0107] The term "anti-tumor effect" refers to a biological effect that can be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a decrease in tumor cell proliferation, or a decrease in tumor cell viability.

[0108] The terms "tumor" and "cancer" are used interchangeably herein and cover solid tumors and hematological tumors.

[0109] The terms "cancer" and "cancerous" refer to or describe a mammalian physiological disorder generally characterized by unregulated cell growth. In some embodiments, cancers suitable for treatment with the antibodies of the invention include gastric, pancreatic or gastroesophageal junction cancer, including metastatic forms of these cancers.

[0110] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells or tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0111] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete or incomplete)), excipient, vector, stabilizer, or the like, administered with an active substance.

[0112] The term "antibody formulation" refers to a preparation in a form in which the biological activity of an antibody as an active ingredient can be effectively exerted, and which does not contain other ingredients that have unacceptable toxicity to a subject to which the formulation is administered. Such antibody formulations are usually sterile. Antibody formulations usually contain a pharmaceutically acceptable excipient. A "pharmaceutically acceptable" excipient is a reagent that can be appropriately administered to a subject mammal so that an effective dose of the active ingredient used in the formulation can be delivered to the subject. The concentration of the excipient corresponds to the mode of administration, and may be, for example, a concentration acceptable for injection.

[0113] The term "anti-CLDN18.2 / CD3 formulation", also abbreviated as "antibody formulation of the present invention" herein, refers to a preparation containing anti-CLDN18.2 / CD3 protein as an active ingredient and pharma- ceutically acceptable excipients. After combining anti-CLDN18.2 / CD3 protein with pharma- ceutically acceptable excipients, the anti-CLDN18.2 / CD3 protein as an active ingredient is suitable for therapeutic or prophylactic administration to humans or non-human animals. The antibody formulation of the present invention may be prepared in a liquid formulation in an aqueous form, such as a ready-to-use drug-filled syringe, or in a lyophilized formulation that is reconstituted (i.e., redissolved) by dissolving and suspending in a physiologically acceptable solution immediately before use. In some embodiments, the anti-CLDN18.2 / CD3 protein formulation is in the form of a liquid formulation.

[0114] A "stable" antibody formulation refers to one in which the antibody in the formulation retains an acceptable degree of physical and / or chemical stability after storage under specified conditions, shaking, or repeated freeze-thawing. Although the antibody in the antibody formulation may not retain 100% of its chemical structure after storage, shaking, or repeated freeze-thawing, an antibody formulation is generally considered to be "stable" when about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the antibody structure or function is maintained. In some specific embodiments, the anti-CLDN18.2 / CD3 protein formulation of the present invention exhibits undetectable low antibody aggregation, degradation, or chemical modification during production, preparation, transportation, and long-term storage, thereby exhibiting high stability with very little or no loss of biological activity of the anti-CLDN18.2 / CD3 protein. In some embodiments, the anti-CLDN18.2 / CD3 protein formulation of the present invention substantially retains its physical or chemical stability after storage, shaking, and / or repeated freeze-thawing. Preferably, the liquid formulation of the present invention can be stable for at least 2 weeks at room temperature or 40° C., and / or for at least 2 months at 25° C., and / or for at least 24 months at 2° C. to 8° C. Preferably, the liquid formulation of the present invention can be stable after shaking at 650 r / min for 5 days at room temperature, protected from light, and / or after 1 to 6 freeze-thaw cycles at −30° C. / room temperature.

[0115] Several analytical techniques are known in the art that can be used to measure protein stability, see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured at a selected temperature and a selected storage time. For example, the storage time can be selected based on the expected shelf life of the formulation. Optionally, accelerated stability testing can be used. In some embodiments, stability testing is performed by subjecting the antibody formulation to various stress tests. These tests may represent harsh conditions that the prepared antibody formulation can encounter during manufacturing, storage or transportation, or may represent conditions that can accelerate the instability of the antibody in the antibody formulation during non-manufacturing, storage or transportation. For example, the prepared anti-CLDN18.2 / CD3 protein formulation can be filled into glass vials to confirm the stability of the antibody under high temperature stress. Further, for example, the prepared anti-CLDN18.2 / CD3 protein formulation may be filled into a glass vial and shaken at room temperature, protected from light, at 650 r / min for 5 days, and then the stability of the antibody may be examined. Further, for example, the prepared anti-CLDN18.2 / CD3 protein formulation may be filled into a glass vial and freeze-thawed once to six times at -30°C / room temperature, and then the stability of the antibody may be examined. In one embodiment, frozen storage at -30°C or lower for 1 day followed by thawing at room temperature constitutes one freeze-thaw cycle.

[0116] An antibody can be considered to have "retained its physical stability" in a formulation if the formulation shows no or very little aggregation, precipitation, turbidity, and / or denaturation after a period of storage, after a period of shaking, or after multiple freeze-thaw cycles. Aggregation of antibodies in a formulation can potentially increase the immune response of patients and cause safety issues. Therefore, it is necessary to minimize or prevent aggregation of antibodies in a formulation. Light scattering methods can be used to measure visible aggregates in a formulation. SEC-HPLC can be used to measure soluble aggregates in a formulation. Stability of a formulation can also be indicated by visually inspecting the appearance, color, and / or clarity of the formulation, or by measuring the turbidity of the formulation by the OD350nm method, or by measuring the purity of the formulation by the non-reducing CE-SDS method. In one embodiment, the stability of the formulation is measured by measuring the percentage of antibody monomer in the formulation after storage at a particular temperature for a particular period of time, after shaking, or after repeated freeze-thaw cycles, where the greater the percentage of antibody monomer in the formulation, the more stable the formulation.

[0117] An "acceptable" degree of physical stability can indicate that at least about 90% of the anti-CLDN18.2 / CD3 protein monomer is detectable in the formulation after storage at a particular temperature for a particular period of time, after shaking, or after repeated freeze-thaw cycles. In some embodiments, an acceptable degree of physical stability indicates at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the anti-CLDN18.2 / CD3 protein monomer after storage at a given temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more. When assessing physical stability, the particular temperature at which the pharmaceutical formulation is stored may be any one of temperatures between about −80° C. and about 45° C., for example, at about −80° C., about −30° C., about −20° C., about 0° C., about 4° C.-8° C., about 5° C., about 25° C., about 35° C., about 37° C., about 40° C., about 42° C., or about 45° C. For example, a pharmaceutical formulation is considered stable if at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the anti-CLDN18.2 / CD3 protein monomer is detected after storage at about 40° C.±2° C. for one month or four weeks. A pharmaceutical formulation is considered stable if at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the anti-CLDN18.2 / CD3 protein monomer is detectable after 2 months of storage at about 25° C. A pharmaceutical formulation is considered stable if at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the anti-CLDN18.2 / CD3 protein monomer is detectable after 9 months of storage at about 5° C.

[0118] If the antibody in the formulation does not show significant chemical changes after a certain period of storage, after a certain period of shaking, or after multiple freeze-thaw cycles, the antibody can be considered to "retain its chemical stability" in the formulation. Most of the chemical instability comes from the formation of covalently modified forms of the antibody (e.g., charge variants of the antibody). For example, aspartic acid isomerization, N- and C-terminal modifications can form basic variants, and deamidation, sialylation, and glycation can form acidic variants. Chemical stability can be evaluated by detecting and / or quantifying the chemically modified forms of the antibody. For example, charge variants of the antibody in the formulation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing (iCIEF). In one embodiment, the stability of the formulation is measured by measuring the change value of the percentage of charge variants of the antibody in the formulation after storage at a certain temperature for a certain period of time, or after shaking, or after multiple freeze-thaw cycles, where the smaller the change value, the higher the stability of the formulation.

[0119] An "acceptable" degree of chemical stability can indicate that the percentage change in the charge variants (e.g., main component or acidic component or basic component) in the formulation after storage for a specified period of time at a specified temperature, or after shaking for a period of time, or after multiple freeze-thaw cycles, is 40% or less, e.g., 30% or less, 20% or less, or the sum of the percentage change in the charge variants (main component, acidic component and basic component) is 60% or less, e.g., 50% or less, 30% or less. In some embodiments, after storage at a particular temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or more, an acceptable level of chemical stability can be demonstrated by a change in the percentage of the major charge variant of about 50%, 40%, 30%, 20% or 15% or less, or a change in the total percentage of the charge variants of about 60%, 50% or 30% or less. When evaluating chemical stability, the temperature at which the pharmaceutical formulation is stored may be any one of about -80°C to about 45°C, for example, about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C or about 45°C. For example, a pharmaceutical formulation may be considered stable if the percentage change in the major component charge variant is less than about 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% after 24 months of storage at 5° C. A pharmaceutical formulation may also be considered stable if the percentage change in the major component charge variant is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% after 2 months of storage at 25° C.A pharmaceutical formulation may be considered stable if the percentage change in the major charge variant is less than about 50%, 40%, 30%, 20%, 16%, 15%, 14%, 13%, 12%, 10%, 5% or 4% after storage at 40° C. for one month.

[0120] The term "lyophilized formulation" refers to a composition obtained or obtainable by the process of freeze-drying a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0121] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

[0122] The term "room temperature" as used in the specification refers to a temperature between 15°C and 30°C, preferably between 20°C and 27°C, and more preferably 25°C.

[0123] "Stress conditions" refers to an environment that is chemically and / or physically unfavorable to antibody protein, which can result in unacceptable antibody protein destabilization, such as high temperature, shaking, or freeze-thawing. "High temperature stress" refers to storing an antibody formulation at room temperature or higher (e.g., 40°C ± 2°C) for a certain period of time. The stability of an antibody formulation can be examined by a high temperature stress accelerated test.

[0124] As used herein, the term "parenteral administration" refers to modes of administration other than enteral and topical administration, usually by injection or infusion, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. In some embodiments, the stable anti-CLDN18.2 / CD3 protein formulation of the invention is parenterally administered to a subject. In one embodiment, the anti-CLDN18.2 / CD3 protein formulation of the invention is administered to a subject by subcutaneous, intradermal, intramuscular or intravenous injection.

[0125] I. Antibody Preparations The present invention provides a stable liquid antibody formulation comprising (i) an anti-CLDN18.2 / CD3 antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, and optionally a chelating agent. The antibody formulation has a pH of about 5.0 to 6.0. In a preferred embodiment, the liquid antibody formulation of the present invention is in the form of an injectable formulation.

[0126] (i) Anti-CLDN18.2 / CD3 antibody In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the invention bind to CLDN18.2 (e.g., human CLDN18.2) with high affinity. In some embodiments, the antibodies of the invention specifically bind to CLDN18.2 (e.g., human CLDN18.2) but do not bind to CLDN18.1 (e.g., human CLDN18.1). In some embodiments, the antibodies or antigen-binding fragments of the invention have a higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, e.g., Zolbetuximab (abbreviated as Zmab) antibodies. In some embodiments, the anti-CLDN18.2×CD3 bispecific antibodies of the invention bind to CD3 (e.g., human CD3 or cynomolgus CD3) with a desired (e.g., lower) affinity. In some embodiments, the antibodies of the invention can bind to both human CD3 and cynomolgus CD3. In some embodiments, the affinity of the antibodies is measured by biolayer interferometry or surface plasmon resonance.

[0127] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the invention are capable of binding to CLDN18.2 on the surface of tumor cells and to CD3 on the surface of effector cells. In some embodiments, the binding is detected by flow cytometry.

[0128] In some embodiments, a bispecific antibody of the invention comprises a first antigen-binding domain and a second antigen-binding domain, where the first antigen-binding domain specifically binds to CLDN18.2 and the second antigen-binding domain specifically binds to CD3.

[0129] In a preferred embodiment of the invention, the first antigen-binding domain comprises three complementarity determining regions (A1-HCDRs) from the heavy chain variable region: A1-HCDR1, A1-HCDR2 and A1-HCDR3.

[0130] In a preferred embodiment of the invention, the first antigen-binding domain comprises three complementarity determining regions (A1-LCDRs) from the light chain variable region, A1-LCDR1, A1-LCDR2 and A1-LCDR3.

[0131] In some embodiments, the first antigen-binding domain comprises three complementarity determining regions from a heavy chain variable region (A1-HCDRs) and three complementarity determining regions from a light chain variable region (A1-LCDRs).

[0132] In some embodiments, the first antigen binding domain comprises a heavy chain variable region (A1-VH). In some embodiments, the first antigen binding domain comprises a light chain variable region (A1-VL). In some embodiments, the first antigen binding domain comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity determining regions (A1-HCDR) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (A1-LCDR) LCDR1, LCDR2 and LCDR3 from the light chain variable region.

[0133] In some embodiments, the A1-VH is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO:4; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO:4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 4, and preferably, said amino acid changes do not occur in the CDR regions.

[0134] In some embodiments, A1-VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 9; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO:9; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 9, and preferably, said amino acid changes do not occur in the CDR regions.

[0135] In a preferred embodiment of the invention, the second antigen-binding domain comprises three complementarity determining regions (A2-HCDRs) from the heavy chain variable region: A2-HCDR1, A2-HCDR2 and A2-HCDR3.

[0136] In a preferred embodiment of the invention, the second antigen-binding domain comprises three complementarity determining regions (A2-LCDRs) from the light chain variable region, A2-LCDR1, A2-LCDR2 and A2-LCDR3.

[0137] In some embodiments, the second antigen-binding domain comprises three complementarity determining regions from a heavy chain variable region (A2-HCDRs) and three complementarity determining regions from a light chain variable region (A2-LCDRs).

[0138] In some embodiments, the second antigen binding domain comprises a heavy chain variable region (A2-VH). In some embodiments, the second antigen binding domain comprises a light chain variable region (A2-VL). In some embodiments, the second antigen binding domain comprises a heavy chain variable region and a light chain variable region. In some embodiments, the heavy chain variable region comprises three complementarity determining regions (A2-HCDR) HCDR1, HCDR2 and HCDR3 from the heavy chain variable region. In some embodiments, the light chain variable region comprises three complementarity determining regions (A2-LCDR) LCDR1, LCDR2 and LCDR3 from the light chain variable region.

[0139] In some embodiments, A2-VH is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 22, 30 or 32; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 22, 30, or 32; or (iii) comprises or consists of an amino acid sequence having one or more (preferably not more than 10, more preferably not more than 5, 4, 3, 2 or 1) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 22, 30 or 32, and preferably, said amino acid changes do not occur in the CDR regions.

[0140] In some embodiments, the A2-VL is (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 27; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 27; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 27, and preferably, said amino acid changes do not occur in the CDR regions.

[0141] In some embodiments, the three complementarity determining regions (A2-HCDRs) A2-HCDR1, A2-HCDR2 and A2-HCDR3 from the A2-VH of the present invention are (i) the three complementarity determining regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in the VH represented by SEQ ID NO: 22, 30 or 32; or (ii) A sequence that contains at least one and not more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in total in the three A2-HCDR regions relative to the sequence of (i).

[0142] In some embodiments, the three complementarity determining regions (A2-LCDRs) A2-LCDR1, A2-LCDR2 and A2-LCDR3 from the A2-VL of the present invention are (i) the three complementarity determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as set forth in SEQ ID NO: 27; or (ii) A sequence that contains at least one and not more than five, four, three, two or one amino acid change (preferably an amino acid substitution, preferably a conservative substitution) in total in the three A2-LCDR regions compared to the sequence in (i).

[0143] In some embodiments, A2-HCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 19, or A2-HCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 19.

[0144] In some embodiments, A2-HCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 20 or 31, or A2-HCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 20 or 31.

[0145] In some embodiments, A2-HCDR3 comprises or consists of the amino acid sequence of SEQ ID NO: 21 or 29, or A2-HCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 21 or 29.

[0146] In some embodiments, A2-LCDR1 comprises or consists of the amino acid sequence of SEQ ID NO: 24, or A2-LCDR1 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 24.

[0147] In some embodiments, A2-LCDR2 comprises or consists of the amino acid sequence of SEQ ID NO: 25, or A2-LCDR2 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 25.

[0148] In some embodiments, A2-LCDR3 comprises or consists of the amino acid sequence of SEQ ID NO:26, or A2-LCDR3 comprises an amino acid sequence having one, two or three changes (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO:26.

[0149] In some embodiments, the bispecific antibody of the invention further comprises a heavy chain constant region. In some embodiments, the bispecific antibody of the invention further comprises a light chain constant region. In some embodiments, the bispecific antibody of the invention further comprises a heavy chain constant region and a light chain constant region. In some embodiments, the bispecific antibody of the invention comprises two heavy chain constant regions, one of which, the heavy chain constant region A1-HC, binds to the first antigen. join In some embodiments, the bispecific antibody of the present invention comprises two light chain constant regions, one of which, the light chain constant region A1-LC, binds to the first antigen, the other of which, the light chain constant region A2-HC, binds to the heavy chain variable region A2-VH of the second antigen-binding domain to form the heavy chain of the CD3-binding portion. join The light chain constant region A1-VL of the first antigen-binding domain is linked to the light chain variable region A1-VL of the second antigen-binding domain to constitute the light chain of the CLDN18.2-binding portion, and the other light chain constant region A2-LC is linked to the light chain variable region A2-VL of the second antigen-binding domain to constitute the light chain of the CD3-binding portion. In one embodiment, the bispecific antibody of the present invention comprises A1-HC, A1-LC, A2-HC and A2-LC. In some embodiments, A1-HC may be homologous or different from A2-HC. In some embodiments, A1-LC may be homologous or different from A2-LC. In some embodiments, A1-HC is different from A2-HC and A1-LC is different from A2-LC.

[0150] In some embodiments, the heavy chain constant region HC of the invention is an IgG1, IgG2, IgG3 or IgG4 heavy chain constant region, preferably an IgG1 heavy chain constant region, such as a wild-type IgG1 heavy chain constant region or a mutated IgG1 heavy chain constant region (e.g., IgG1LALA). In some embodiments, the antibody light chain constant region LC of the invention is a lambda or Kappa light chain constant region.

[0151] In some preferred embodiments, the heavy chain constant region H of the present invention is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 5 or 23; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 5 or 23; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or less than 10, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 5 or 23.

[0152] In some preferred embodiments, the heavy chain constant region H of the present invention is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 33 or 34; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 33 or 34; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 33 or 34.

[0153] In some embodiments, the antibody light chain constant region LC of the invention is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 10 or 28; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 10 or 28; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or less than 10, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 10 or 28.

[0154] In some specific embodiments of the invention, the bispecific antibody of the invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein: The first antigen-binding domain specifically binds to CLDN18.2, and the second antigen-binding domain specifically binds to CD3, wherein the first antigen-binding domain comprises three complementarity determining regions A1-HCDR1, A1-HCDR2 and A1-HCDR3 contained in A1-VH shown in SEQ ID NO:4, and three complementarity determining regions A1-LCDR1, A1-LCDR2 and A1-LCDR3 contained in VL shown in SEQ ID NO:9; The second antigen-binding domain comprises the three complementarity determining regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in A2-VH as shown in SEQ ID NO: 22, 30 or 32, and the three complementarity determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as shown in SEQ ID NO: 27.

[0155] In some specific embodiments of the invention, the bispecific antibody of the invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2 and the second antigen-binding domain specifically binds to CD3, wherein The first antigen-binding domain comprises A1-HCDR1, A1-HCDR2 and A1-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and A1-LCDR1, A1-LCDR2 and A1-LCDR3 represented by the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively; and The second antigen-binding domain comprises (i) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or (ii) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or (iii) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively.

[0156] In some specific embodiments of the invention, the bispecific antibody of the invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2 and the second antigen-binding domain specifically binds to CD3, wherein The first antigen-binding domain comprises an A1-VH comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:4 or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, and an A1-VL comprising or consisting of an amino acid sequence as set forth in SEQ ID NO:9 or an amino acid sequence having at least 90% identity thereto; The second antigen-binding domain comprises an A2-VH comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 22, 30 or 32, or an amino acid sequence having at least 90% identity thereto, and an A2-VL comprising or consisting of an amino acid sequence set forth in SEQ ID NO: 27, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0157] In some specific embodiments of the present invention, the bispecific antibody of the present invention is a bispecific antibody with an IgG-like structure, i.e., it comprises a heavy chain and a light chain of a CLDN18.2 binding portion and a heavy chain and a light chain of a CD3 binding portion. In some embodiments, the CH3 regions of the two heavy chains have a knob-into-hole sequence (Shane Atwell et al., Journal of Molecular Biology, 1997; A. Margaret Merchant et al., Nature Biotechnology, 1998), thus forming a knob-into-hole structure. In some embodiments, the structure of the bispecific antibody of the present invention is shown in FIG. 1.

[0158] In some embodiments, the heavy chain of the CLDN18.2 binding moiety is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 37; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 37; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 37.

[0159] In some embodiments, the light chain of the CLDN18.2 binding moiety is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 38; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 38; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 38.

[0160] In some embodiments, the heavy chain of the CD3 binding moiety is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 39, 41 or 42; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 39, 41, or 42; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 39, 41 or 42.

[0161] In some embodiments, the light chain of the CD3 binding moiety is (i) comprising or consisting of an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 40; (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 40; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably conservative amino acid substitutions) compared to an amino acid sequence selected from SEQ ID NO: 40.

[0162] In one embodiment of the present invention, the amino acid changes described herein include amino acid substitutions, insertions or deletions. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

[0163] In preferred embodiments, the amino acid changes described in the present invention occur in regions outside the CDRs (e.g., FRs). More preferably, the amino acid changes described in the present invention occur in regions outside the heavy chain variable region and / or outside the light chain variable region. In some embodiments, the amino acid changes described in the present invention occur in the Fc region of the antibody heavy chain constant region, and in preferred embodiments, the amino acid changes in the Fc region reduce or eliminate the ADCC and / or CDC effects of the antibody.

[0164] In some embodiments, substitution is conservative substitution.Conservative substitution refers to one amino acid being replaced by another amino acid of the same kind, for example, one acidic amino acid being replaced by another acidic amino acid, one basic amino acid being replaced by another basic amino acid, or one neutral amino acid being replaced by another neutral amino acid.Exemplary substitutions are as shown in the table below.

[0165] [Table 2] In some embodiments, the substitutions are made in the CDR regions of the antibody. Generally, the resulting variant has a modification (e.g., improvement) in certain biological properties (e.g., improved affinity) relative to the parent antibody and / or has substantially retained certain biological properties of the parent antibody. An exemplary substitution variant is an affinity matured antibody.

[0166] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to produce an Fc region variant that alters one or more functional properties of the antibody, e.g., serum half-life, complement fixation, complement dependent cytotoxicity, Fc receptor binding, and / or antibody dependent cellular cytotoxicity. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) that contains an amino acid change (e.g., a substitution) at one or more amino acid positions.

[0167] In certain embodiments, it may be necessary to produce cysteine ​​engineered antibodies, such as "ThioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues.

[0168] In some embodiments, the antibodies provided herein can be further modified to include other non-protein moieties that are known and readily available in the art. Moieties suitable for inducing antibody activity include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerin), polyvinyl alcohol, and mixtures thereof.

[0169] In some embodiments, in a bispecific antibody of the invention, a first antigen-binding domain that specifically binds to CLDN18.2, or a heavy and light chain combination, is fully human, and a second antigen-binding domain that binds to CD3, or a heavy and light chain combination, is humanized.

[0170] In one preferred embodiment, the anti-CLDN18.2 / CD3 bispecific antibody in the antibody formulation of the present invention is the anti-CLDN18.2 / CD3 bispecific antibody disclosed in PCT Application No. PCT / CN2021 / 121286 (International Filing Date: September 28, 2021).

[0171] The amount of antibody or antigen-binding fragment thereof included in the antibody formulation of the invention can vary depending on the particular desired properties of the formulation, the particular environment, and the particular purpose for which the formulation is used. In some embodiments, the antibody formulation is a liquid formulation and may contain about 0.1 mg / mL to 100 mg / mL, preferably about 0.1 mg / mL to 10 mg / mL, for example, about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, or 5 mg / mL of anti-CLDN18.2 / CD3 bispecific antibody.

[0172] (ii) Buffer A buffer is a reagent capable of maintaining the pH of a solution within an acceptable range. In some embodiments, the buffer used in the formulation of the present invention can control the pH of the formulation of the present invention to a pH range of about 5.0 to 6.0, for example, a pH of about 5.0 to 5.5. In some specific embodiments, the antibody formulation of the present invention has a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7 or 5.8. For example, the antibody formulation of the present invention has a pH of 5.2±0.2 or 5.5±0.2, preferably 5.2.

[0173] In some embodiments, the formulations of the invention comprise a buffer system selected from a histidine-histidine hydrochloride buffer system, a citric acid-sodium citrate buffer system, an acetic acid-sodium acetate buffer system, and a phosphate buffer system, preferably a histidine-histidine hydrochloride buffer system.

[0174] In some embodiments, the buffer used in the formulation of the present invention is a histidine buffer, particularly a buffer system consisting of histidine and histidine hydrochloride. In one embodiment, the histidine in the histidine buffer of the present invention is about 0.01 mg / mL to 10 mg / mL. 、 In particular, the concentration is about 0.1 mg / mL to 1 mg / mL, for example, about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL or 1 mg / mL. In one embodiment, the formulation of the present invention contains about 0.18 mg / mL histidine. In another embodiment, the histidine buffer used in the formulation of the present invention consists of, for example, about 0.18 mg / mL histidine and about 1.84 mg / mL histidine hydrochloride.

[0175] (iii) Stabilizers The suitable stabilizer used in the present invention may be selected from sugars, polyols, amino acids and combinations thereof.The sugars as stabilizers include, but are not limited to, sucrose, trehalose, maltose and combinations thereof.The polyols as stabilizers include, but are not limited to, sorbitol, mannitol and combinations thereof.The amino acids as stabilizers include, but are not limited to, arginine, arginine hydrochloride, methionine, glycine, proline and combinations thereof.

[0176] For example, in some embodiments, the stabilizer comprises one or more selected from the following:

[0177] a polyol selected from sorbitol, mannitol or a combination thereof, about 10 mg / mL to 100 mg / mL, preferably 20 mg / mL to 40 mg / mL, for example 25 mg / mL, or 40 mg / mL to 60 mg / mL, for example 50 mg / mL, - a sugar selected from sucrose, trehalose, maltose or a combination thereof, for example at about 10 mg / mL to 100 mg / mL, preferably 30 mg / mL to 60 mg / mL, for example 40 mg / mL, - for example 20 mM to 200 mM, such as about 50 mM to 110 mM, for example 70 mM to 100 mM, in particular about 80 mM to 90 mM of an amino acid selected from arginine hydrochloride, methionine, glycine, proline and combinations thereof.

[0178] In one embodiment, the liquid formulation of the present invention contains sucrose as a stabilizer. The amount of sucrose in the liquid formulation of the present invention may be about 10 mg / mL to 100 mg / mL, preferably 30 mg / mL to 60 mg / mL, for example 40 mg / mL.

[0179] In one embodiment, the liquid formulation of the present invention comprises sorbitol as a stabilizer. The amount of sorbitol in the liquid formulation of the present invention may be about 10 mg / mL to 100 mg / mL, preferably 20 mg / mL to 40 mg / mL, for example 25 mg / mL, or 40 mg / mL to 60 mg / mL, for example 50 mg / mL.

[0180] In one embodiment, the liquid formulation of the present invention comprises arginine as a stabilizer. The amount of arginine in the liquid formulation of the present invention may be about 50 mM to 110 mM, for example 70 mM to 100 mM, particularly about 80 mM to 90 mM, for example about 17.91 mg / mL of arginine hydrochloride.

[0181] In one embodiment, the liquid formulation of the present invention comprises a combination of sorbitol and arginine as a stabilizer. In the combination, sorbitol may be present in an amount of about 10 mg / mL to 60 mg / mL, preferably 15 mg / mL to 40 mg / mL, for example, 20 mg / mL to 35 mg / mL, for example, about 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL or 30 mg / mL. In the combination, arginine may be present in an amount of about 70 mM to 100 mM, particularly about 85 mM. Preferably, the liquid formulation of the present invention comprises about 20 mg / mL to 30 mg / mL of sorbitol and about 15 mg / mL to 20 mg / mL of arginine hydrochloride. More preferably, the liquid formulation of the present invention comprises about 25 mg / mL sorbitol and about 17.91 mg / mL arginine hydrochloride.

[0182] In one embodiment, the liquid formulation of the present invention comprises a combination of sucrose and arginine as a stabilizer. In the combination, sucrose may be present in an amount of about 10 mg / mL to 60 mg / mL, preferably 20 mg / mL to 50 mg / mL, for example, 30 mg / mL to 40 mg / mL, for example, about 30 mg / mL, 32 mg / mL, 34 mg / mL, 36 mg / mL, 38 mg / mL, 40 mg / mL, 42 mg / mL, 44 mg / mL, 46 mg / mL, 48 mg / mL or 50 mg / mL. In the combination, arginine may be present in an amount of about 70 mM to 100 mM, particularly about 85 mM. Preferably, the liquid formulation of the present invention comprises about 30 mg / mL to 50 mg / mL sucrose and about 15 mg / mL to 20 mg / mL arginine hydrochloride. More preferably, the liquid formulation of the present invention comprises about 40 mg / mL sucrose and about 17.91 mg / mL arginine hydrochloride.

[0183] In one embodiment, the liquid formulation of the present invention contains sorbitol as a single stabilizer. In this embodiment, the amount of sorbitol in the liquid formulation of the present invention may be about 30 mg / mL to 70 mg / mL, for example, 40 mg / mL to 60 mg / mL. For example, sorbitol may be present in an amount of about 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL or 70 mg / mL, preferably about 50 mg / mL.

[0184] (iv) Surfactant As used herein, the term "surfactant" refers to organic substances that have an amphiphilic structure, i.e., they consist of groups with opposite solubility tendencies, usually an oil-soluble hydrocarbon chain and a water-soluble ionic group.

[0185] In one embodiment, the surfactant in the liquid formulation of the present invention is a non-ionic surfactant, such as an alkyl poly(oxylane). Specific non-ionic surfactants that can be included in the formulation of the present invention include polysorbates, such as polysorbate-20, polysorbate-80, polysorbate-60 or polysorbate-40, and poloxamers. In one preferred embodiment, the liquid formulation of the present invention includes polysorbate-80 as the surfactant.

[0186] In some embodiments, surfactants that can be used in the liquid formulations of the present invention include, but are not limited to, polysorbate surfactants (e.g., polysorbate 80, polysorbate 20), poloxamers, and polyethylene glycols.

[0187] The amount of surfactant included in the antibody formulation of the present invention can vary depending on the particular desired properties of the formulation, the particular environment, and the particular purpose for which the formulation is used. In some preferred embodiments, the formulation may contain about 0.01 mg / mL to 5 mg / mL, preferably about 0.1 mg / mL to 1 mg / mL, for example about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, or 1.0 mg / mL of a surfactant, particularly polysorbate-80, preferably about 0.2 mg / mL of polysorbate-80.

[0188] (v) Chelating agent As used herein, the term "chelating agent" means capable of forming a chelate with a central atom, and the formation of the chelate greatly enhances the stability of the complex.

[0189] In one embodiment, the chelator in the liquid formulation of the present invention is a carboxylic acid type chelator. In one embodiment, the chelator is selected from edetate disodium, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, and dihydroxyethylglycine. In one embodiment, the chelator is selected from edetate disodium.

[0190] In one embodiment, the chelating agent in the liquid antibody formulation of the present invention has a concentration of about 0.005 mg / mL to 0.05 mg / mL. In one embodiment, the chelating agent in the liquid antibody formulation of the present invention has a concentration of about 0.008 mg / mL to 0.018 mg / mL, for example, about 0.008 mg / mL, 0.009 mg / mL, 0.010 mg / mL, 0.012 mg / mL, 0.014 mg / mL, or 0.018 mg / mL, preferably 0.01 mg / mL.

[0191] (vi) Other excipients The antibody liquid formulations of the invention optionally include other excipients, such as, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, etc. These excipients and other known drug excipients and / or additives used in the formulations of the invention are known in the art, such as those listed in The Handbook of Pharmaceutical Excipients, 4th edition, edited by Rowe et al., American Pharmaceuticals Association (2003) and Remington: the Science and Practice of Pharmacy, 21st edition, edited by Gennaro, Lippincott Williams & Wilkins (2005).

[0192] II. Preparation of Formulations The present invention provides a stable formulation comprising an anti-CLDN18.2 / CD3 antibody. The anti-CLDN18.2 / CD3 antibody used in the formulation of the present invention can be prepared by known techniques for producing antibodies in the art. For example, the antibody can be prepared recombinantly. In one preferred embodiment, the antibody of the present invention is prepared recombinantly in 293 cells or CHO cells.

[0193] Currently, antibodies are widely applied as active ingredients in medicines. Techniques for purifying therapeutic antibodies to medicinal levels are known in the art. For example, Tugcu et al. (Maximizing productivity of chromatography steps for purification of monoclonal antibodies, Biotechnology and Bioengineering 99 (2008) 599-613.) describe a three-column purification method for monoclonal antibodies that utilizes ion exchange chromatography (anion IEX and / or cation CEX chromatography) after a protein A capture step. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553-566) describe a two-column purification method that utilizes weakly partitioning anion exchange resins after protein A affinity chromatography.

[0194] In general, recombinantly produced antibodies can be purified by conventional purification methods to provide pharmaceutical substances with sufficient repeatability and suitable purity for the preparation of antibody formulations. For example, after the antibody is secreted from recombinant expression cells into the medium, the supernatant from the expression system can be concentrated by commercially available protein concentration filters, such as Amicon ultrafiltration devices. The antibody can then be purified by methods such as chromatography, dialysis and affinity purification. Protein A is applied as an affinity ligand to purify IgG1, IgG2 and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, may also be used. After obtaining an antibody of sufficient purity, a formulation containing the antibody can be prepared by methods known in the art.

[0195] For example, the preparation can be carried out by the steps of (1) centrifuging the fermentation liquid after the end of fermentation to obtain a supernatant and removing impurities such as cells, (2) capturing the antibodies by affinity chromatography (for example, a protein A column with specific affinity for IgG1, IgG2 and IgG4 type antibodies), (3) inactivating the virus, (4) removing impurities in the protein by purification (typically CEX cation exchange chromatography can be adopted), (5) filtering the virus (to reduce the virus titer by, for example, 4 log10 or more), and (6) ultrafiltration / permeation filtration (which can be used to replace the protein with a formulation buffer that contributes to its stability and concentrate it to a suitable concentration for injection). For example, see B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48-57.

[0196] III. Analytical methods for formulations Stability studies of biological products generally include real-time stability studies under actual storage conditions (long-term stability studies), accelerated stability studies and forced condition test studies. Stability studies should explore and optimize the study conditions based on the research objectives and the characteristics of the product itself, and formulate stability study plans such as long-term, accelerated and / or forced condition tests for various influencing factors (e.g., temperature, repeated freezing and thawing, vibration, etc.). Accelerated and forced condition tests are useful for understanding the stability status of products in short-term deviations from storage conditions and in extreme cases, as well as providing supporting data for determining shelf life and storage conditions.

[0197] During the process of storage, shaking or repeated freezing and thawing of antibody formulations, the antibody may aggregate, decompose or be chemically modified, leading to antibody heterogeneity (including size heterogeneity and charge heterogeneity) and aggregates and fragments, etc., which may affect the quality of the antibody formulation. Therefore, it is necessary to monitor the stability of antibody formulations.

[0198] In this field, several methods are known that can be used to detect the stability of antibody formulations. For example, methods such as reduced CE-SDS and non-reduced CE-SDS with SEC-HPLC can be used to analyze the purity of antibody formulations and evaluate the level of antibody aggregation, and methods such as ion exchange chromatography (IEX) can be used to analyze charge variants in antibody formulations. The stability of a formulation can be quickly determined by visually detecting the appearance of the formulation. The change in turbidity of the formulation is measured by the OD 350nm The changes in protein content in the formulation can be detected by ultraviolet spectrophotometry (UV) which can provide information about the amount of soluble and insoluble aggregates.

[0199] The non-reduced CE-SDS method is a method for measuring antibody purity using a capillary as a separation path. In CE-SDS, protein migration is driven by the surface charge due to SDS binding, which is directly proportional to the molecular weight of the protein. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in the molecular sieve gel matrix of the capillary. This method is widely applied to monitor the purity of denatured intact antibodies. Generally, in the non-reduced CE-SDS method, the test sample is mixed with SDS sample buffer and iodoacetamide. The mixture may then be incubated at 68°C to 72°C for about 10 to 15 minutes, and the supernatant, which is centrifuged after cooling to room temperature, is used for analysis. Protein migration is detected by an ultraviolet detector to obtain an electropherogram. The purity of the antibody preparation can be calculated as the percentage of the peak area of ​​the IgG main peak to the sum of all peak areas. For further description of the CE-SDS method, see, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

[0200] Volume Exclusion High Performance Liquid Chromatography, or SEC-HPLC, is another important method used for antibody standard and quality control. The method separates molecules mainly based on the difference in molecular size or hydrodynamic radius. SEC-HPLC can separate antibodies into three major forms: high molecular weight form (HMMS), main peak (mainly antibody monomer) and low molecular weight form (LMMS). The purity of the antibody can be calculated as the percentage of the main peak area relative to the sum of all peak areas in the chromatogram. SEC-HPLC can measure the percentage of antibody monomer in the formulation product and give information on the content of soluble aggregates and shear products. For further descriptions of SEC-HPLC methods, see, e.g., J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994), J. Pharm. Bio. Anal., 15:1928 (1997), J. Pharm. Bio. Anal., 14:1133-1140 (1986). Further, reference may be made to, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusion ultra-high performance liquid chromatography (SE-UHPLC), Journal of Pharmaceutical and Biomedical Analysis (2015), http: / / dx.doi.org / 10.1016 / j.jpba.2015.02.032, and Alexandre Goyon et al., Protocols for the analytical characterization of therapeutic monoclonal antibodies. I- Non-denaturing chromatographic techniques, Journal of Chromatography, http: / / dx.doi.org / 10.1016 / j.jchromb.2017.05.010.

[0201] Accelerated stability studies can be used to test the stability characteristics of products, and contribute to the screening of stable pharmaceutical formulation forms. For example, accelerated stability studies can be performed by placing formulation samples at elevated temperatures, such as about 40°C ± 2°C, 25°C ± 2°C. In addition, the stability characteristics of products can be detected by performing shaking experiments or repeated freeze-thaw experiments. For example, shaking experiments are performed at room temperature, protected from light, and shaken at 650 r / min for 1 to 5 days. For example, a product can be frozen at -30°C or below for 1 day and then thawed at room temperature, which constitutes one freeze-thaw cycle, and repeated freeze-thaw experiments can be performed, where one to six repeated freeze-thaw cycles can be performed. Detection indicators of product stability can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC method, non-reduced CE-SDS method) and charge variants (iCIEF method, CEX-HPLC method). In addition, the efficacy or biological activity of antibodies can be detected. For example, the binding ability of an antibody and its antigen molecule (TIGIT) in a formulation can be detected. Various methods are known to those skilled in the art that can be used to quantitate the specific binding of an antibody to an antigen, for example immunoassay tests, ELISAs, and the like.

[0202] Explanation of Abbreviations [Table 3] EXAMPLES

[0203] Working Example The anti-CLDN18.2 / CD3 bispecific antibody is an antibody developed in-house by Cinda Bio-Pharmaceutical (Suzhou) Co., Ltd. and is disclosed in PCT Application No. PCT / CN2021 / 121286.

[0204] In order to develop an injection formulation applicable to long-term stable storage of the fully human antibody and simple and easy to use, the effects of different pH values, different stabilizer and surfactant contents on the mass of the antibody protein were investigated through forced stability experiments at 40°C and accelerated stability experiments at 25°C, and finally a formulation beneficial to the stability was screened. The materials and methods used in the entire research process are as follows:

[0205] material and method 1.1. Materials used in the study of the formulations of the present invention

[0206] [Table 4]

[0207] 1.2. Apparatus and Equipment Used in Studying the Formulations of the Invention

[0208] [Table 5]

[0209] 1.3. Test items and methods for formulation stability Throughout the research, the detection items mainly included: (1) detecting the appearance and the presence or absence of visible foreign matter; (2) measuring the protein content in the preparation by ultraviolet light (UV method); (3) detecting the turbidity of the preparation by OD350nm method; (4) measuring the purity of the antibody preparation by volume exclusion high performance liquid chromatography (SEC-HPLC) and expressing it as a percentage of the area of ​​the main peak in the total area of ​​all peaks; (5) measuring the purity of the antibody preparation by non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS) and expressing it as a percentage of the area of ​​the main peak in the total area of ​​all peaks; (6) measuring the charge variants in the antibody preparation by CEX-HPLC method and expressing it as the percentage of the main component, acidic component and basic component; (7) measuring the relative binding activity of the anti-CLDN18.2 / CD3 antibody to the antigen in the antibody preparation by direct ELISA method; and (8) measuring the content of polysorbate 80 in the antibody preparation by HPLC-FLD.

[0210] Visible foreign object detection According to the method described in the National Pharmacopoeia Commission, Pharmacopoeia of the People's Republic of China (2015 edition, Four Parts General Principle 0904 "Testing Method for Visible Foreign Substances", Beijing: China Pharmaceutical Science and Technology Press, 2015), samples were tested for visible foreign bodies using a transparency detector (manufactured by Tianjin Tianda Tianfa, model no. YB-2) and an insoluble particle detector (manufactured by Tianjin Tianda Tianfa, model no. GWJ-8).

[0211] Protein content measurement The protein content in the samples was measured using an ultraviolet spectrophotometer (Shimadzu, Japan, model UV-1800) or a multichannel microspectrophotometer (Thermo, USA, model Nanodrop 8000).

[0212] Turbidity measurement The absorbance of the samples was measured at 350 nm using an ultraviolet spectrophotometer (Shimadzu, Japan, model number UV-1800) to determine the turbidity of the samples.

[0213] Purity (SEC-HPLC method) The separation was performed by a size exclusion chromatography column, and the mobile phase was a phosphate buffer solution (3.12 g of sodium dihydrogen phosphate dihydrate, 8.77 g of sodium chloride, and 34.84 g of arginine were weighed, dissolved in ultrapure water, and then the pH was adjusted to 6.8 with hydrochloric acid to a constant volume of 1000 mL). The chromatography column protection solution was 0.05% (w / v) NaN3, the injection amount was 50 μL, the flow rate was 0.5 mL / min, the collection time was 30 minutes, the column temperature was 25 °C, and the detection wavelength was 280 nm. The sample waiting for measurement was diluted to 2 mg / mL with ultrapure water to prepare the test solution. The formulation buffer solution was taken and diluted in the same manner as above, and then used as the blank solution. 50 μL of each of the blank solution and the test solution were taken and injected into the liquid chromatograph, and detection was started.

[0214] Purity (non-reduced CE-SDS method) Detection was performed by capillary gel electrophoresis. The capillary was an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before electrophoresis, the capillary column was washed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi. The sample to be measured was diluted to 2.0 mg / mL with an appropriate amount of ultrapure water, and 50 μL of the diluted sample was placed in a 1.5 mL centrifuge tube. 45 μL of pH 6.5 sample buffer (0.32 g of citric acid monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate were weighed out and dissolved in 45 mL of ultrapure water, and the volume was adjusted to 50 mL to prepare a citric acid-phosphate buffer solution, and 200 μL of the buffer solution was precisely measured, and 80 μL of 10% (w / v) sodium dodecyl sulfate solution was added, and water was added until the total volume was 1 mL, and the mixture was mixed uniformly) and 1 μL of internal standard solution (10 kDa protein, 5 mg / mL) (Beckman Coulter, product number: 390953) and 5μL of 250mmol / L NEM solution (62mg of N-ethylmaleimide was weighed and dissolved in 2mL of ultrapure water) were added, mixed thoroughly and uniformly, heated at 70℃±2℃ for 10min±2min, cooled to room temperature, and transferred to a sample flask to prepare the test sample solution. The same volume of formulation buffer as the test sample was taken and operated in the same manner as above to prepare a blank solution. Sample injection conditions: -5kV 20s, separation voltage: -15kV 35min. The temperature of the capillary column was controlled at 25℃, and the detection wavelength was 220nm.

[0215] Charge variants (CEX-HPLC method) Detection was performed by cation exchange chromatography (CEX-HPLC method). Separation was performed using a MabPac SCX-10 strong cation exchange chromatography column. Mobile phase A was 10 mmol / L phosphate buffer (0.51 g NaH2PO4·2H2O, 2.40 g Na2HPO4·12H2O were weighed and dissolved in 800 mL of ultrapure water, the volume was adjusted to 1000 mL, and the solution was filtered through a Φ0.22 μm filter membrane), and mobile phase B was 10 mmol / L phosphate + 200 mmol / L sodium chloride buffer (0.51 g NaH2PO4·2H2O, 2.40 g Na2HPO4·12H2O, 11.69 g NaCl were weighed and dissolved in 800 mL of ultrapure water, the volume was adjusted to 1000 mL, and the solution was filtered through a Φ0.22 μm filter membrane). The sample was diluted with ultrapure water to 2.0 mg / mL to prepare the test solution. The formulation buffer was taken and diluted in the same manner as above to prepare the blank solution. 50 μL of each of the blank solution and test solution was taken and injected into the liquid chromatograph, with the flow rate of the mobile phase being 1.0 mL / min, the collection time being 35 minutes, the column temperature being 35°C, the detection wavelength being 280 nm, and the temperature of the sample plate being 10°C. The sample was injected and analyzed, and the contents of the main components, acidic components, and basic components were calculated by the area normalization method.

[0216] Polysorbate 80 content (HPLC-FLD method) Detection was performed by fluorescence detection chromatography (HPLC-FLD method). Isocratic elution was performed using 0.15 mol / L sodium chloride, 0.05 mol / L trishydroxymethylaminomethane, pH 8.0, 5% acetonitrile, 5.0 μmol / L N-phenyl-1-naphthylamine, and 15 ppm 23 polyoxyethylene fatty alcohol ether, with a flow rate of 1.5 mL / min, a collection time of 3 minutes, a pressure limit of 100 bar, an injection volume of 10 μL, a column temperature of 30°C, a detection wavelength of 350 nm for intense emission, and 420 nm for emission. The content of polysorbate 80 was calculated by the calibration curve method.

[0217] Example 1. Construction of stable expression cell line Preparation of cell lines overexpressing human CLDN18.2 A cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, hereinafter the same) was constructed using the Freedom® CHO-S® Reagent Kit (Invitrogen, A1369601) according to the manufacturer's instructions. First, the full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed in the vector pCHO1.0 to form a plasmid, and the constructed plasmid was introduced into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) by chemical transfection and electrical transfection, respectively, and the transfected cells were subjected to two rounds of press screening to obtain cell pools that expressed CLDN18.2, respectively. Next, cells that highly expressed CLDN18.2 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter), and monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 that stably expressed CLDN18.2 were obtained by the dilution method.

[0218] Preparation of cell lines overexpressing human CLDN18.1 A cell line stably expressing human Claudin18.1 (abbreviated as CLDN18.1, hereinafter the same) was constructed using the Freedom® CHO-S® Reagent Kit (Invitrogen, A1369601) according to the manufacturer's instructions. First, the full-length gene of human CLDN18.1 (UniProt ID: P56856-1) was constructed in the vector pCHO1.0 (Invitrogen, A1369601) to form a plasmid, and the constructed plasmid was introduced into CHO-S cells (Invitrogen, A1369601) by chemical transfection. Two rounds of press screening were performed on the transfected cells to obtain cell pools that expressed CLDN18.1, respectively. Next, cells that highly expressed CLDN18.1 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter), and a monoclonal cell line, CHO-hCLDN18.1, that stably expressed CLDN18.1 was obtained by the dilution method.

[0219] Construction of tumor cell lines overexpressing CLDN18.2 The full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was constructed in the vector pWPT-GFP (Addgene, 12255), replacing the GFP sequence therein, and transfected into HEK293T (ATCC, CRL-3216) cells together with the lentivirus packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259), and virus packaging was performed. Culture supernatants cultured for 48 and 72 hours were collected, respectively, and lentivirus was concentrated using PEG8000. The concentrated virus was transfected into pancreatic cancer DAN-G cells and gastric cancer KATO III cells, and then the cells that expressed CLDN18.2 were sorted by flow cytometry (MoFlo XDP, Beckman Coulter) to obtain the tumor cell lines DAN-G-hCLDN18.2 and KATO III-hCLDN18.2 stably transfected with CLDN18.2.

[0220] Example 2. Generation of CLDN18.2 monoclones In the present invention, the hybridoma technology was used to immunize mouse H2L2 fully human antibody gene recombinant mice (purchased from Harbour BioMed) with the cells (CHO-hCLDN18.2) obtained in Example 1. Then, the spleen cells of the mice were obtained and electrofused with myeloma cells. The supernatant was then collected and screened for hybridoma cells that specifically expressed anti-CLDN18.2 antibodies by flow cytometry (FACS), and the secreted antibodies did not bind to CLDN18.1. The cells (HEK293-hCLDN18.2) awaiting detection obtained in Example 1 were counted, and 1×10 6 The hybridoma cells were diluted to 100 cells / mL and added to a U-bottom 96-well plate at 100 μL / well. Centrifuged at 500 g for 5 min and the cell medium was removed. The supernatant from the hybridoma 96-well plate was added to the U-shaped plate, the cells were resuspended, and 100 μL was added per well and incubated on ice for 30 min. Centrifuged at 500 g for 5 min, the supernatant was removed, and the cells were washed once with PBS. 100 μL of anti-mouse Fab FITC-labeled secondary antibody (diluted 1:500 in PBS) was added per well, and 100 μL of anti-human Fab FITC-labeled secondary antibody was added as a positive control antibody. Incubated on ice for 30 min in the dark. Centrifuged at 500 g for 5 min, the supernatant was removed, and the cells were washed once with PBS. The cells were resuspended in 50 μL 1×PBS and detected on-board by FACS. The positive clones were rescreened for CHO-hCLDN18.1 as above. After two rounds of screening, a fully human antibody clone, HB37A6, was obtained.

[0221] Example 3. Preparation of recombinant CLDN18.2 monoclonal antibody The anti-CLDN18.2 monoclonal antibody HB37A6 (see CN202010570517.X) and the control antibody zolbetuximab (abbreviated as Zmab, sequence derived from INN117) were expressed in HEK293 cells (Invitrogen, A14527) in the form of full-length monoclonal antibodies.

[0222] First, an expression vector was constructed, and the heavy and light chain variable regions (see sequence information) of HB37A6 and the control antibody were placed at the N-terminus of the heavy chain constant region (SEQ ID NO: 5) and the light chain kappa constant region (SEQ ID NO: 10) of human IgG1, respectively. Then, a pcDNA3.1 expression vector with an N-terminal signal peptide was constructed to obtain a light and heavy chain expression vector. The obtained light and heavy chain expression vector was co-transfected with PEI (Polysciences Inc, 23966) into HEK293 cells, and after 7 days of culture, the medium supernatant was collected. The supernatant was purified by a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), and then ultrafiltered and replaced with PBS (Gibco, 70011-044), the concentration was detected by the A280 method, and the purity was measured by the SEC-HPLC method, and an antibody solution with a purity of more than 95% was obtained, and the recombinant CLDN18.2 monoclonal antibody HB37A6 was obtained.

[0223] The specific transfection and purification process is as follows.

[0224] Depending on the required transfection volume, passage Expi293 cells (Invitrogen, A14527) to a cell density of 1.5 × 10 the day before transfection. 6 The cell density was adjusted to approximately 3 × 10 cells / mL on the day of transfection. 6 cells / mL. 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) was taken as transfection buffer, and the appropriate plasmid was added to the transfected cells at 1.0 μg / mL and mixed uniformly. The appropriate polyethyleneimine (PEI) (Polysciences, 23966) was added to the plasmid (the ratio of plasmid to PEI is 1:3 for 293F cells), mixed uniformly, and then incubated at room temperature for 20 min to obtain the DNA / PEI mixture. The DNA / PEI mixture was gradually added to the cells while gently shaking the flask, and then placed in a 36.5°C, 8% CO2 incubator for culture, and after 7 days, the cell feed was obtained, and the cell supernatant was collected and purified.

[0225] The Protein A column (Hitrap Mabselect Sure, GE, 11-0034-95) used for purification was treated with 0.1 M NaOH for 2 h, and glass bottles, etc. were washed with distilled water and then dry roasted at 180 ° C for 4 h. Before purification, the collected cell feed solution was centrifuged at 4500 rpm for 30 min, and the supernatant was filtered through a 0.22 μm filter. The Protein A column was equilibrated with 10 column volumes of binding buffer (sodium phosphate 20 mM, NaCl 150 mM, PH 7.0). The filtered supernatant was added to the purification column, which was then equilibrated with 10 column volumes of binding buffer. 5 mL of elution buffer (citric acid + sodium citrate 0.1 M, pH 3.5) was added to collect the eluate, and 80 μL of Tris-HCl with a concentration of 2 M per mL of eluate was added. The collected antibodies were concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044) to detect the concentration.

[0226] Example 4 Preparation of anti-CD3 monoclonal antibody Mouse CD3 antibody sp34 (US Pat. No. 8,236,308, J. Immunol. Methods., 1994, 178:195) and anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, and HzSP34.97, which were humanized and affinity-optimized from sp34, were expressed in HEK293 cells (Invitrogen, A14527) in the form of full-length monoclonal antibodies.

[0227] Heavy chain variable regions of HzSP34.24, HzSP34.87, HzSP34.97, and sp34 AreaThe heavy and light chain variable regions (see sequence information) were placed at the N-terminus of the IgG1 heavy chain constant region (SEQ ID NO: 23) and the light chain lambda constant region (SEQ ID NO: 28). Then, a pcDNA3.1 expression vector with an N-terminal signal peptide was constructed to obtain a light and heavy chain expression vector. The obtained light and heavy chain expression vector was co-transfected with PEI (Polysciences Inc, 23966) into HEK293 cells, and after 7 days of culture, the medium supernatant was collected. The supernatant was purified by a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), and then ultrafiltered and exchanged with PBS (Gibco, 70011-044). The concentration was detected by the A280 method, and the purity was measured by the SEC-HPLC method, and an antibody solution with a purity of more than 95% was obtained, and the recombinant anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, HzSP34.97 and mouse CD3 antibody sp34 were obtained. For specific transfection and purification processes, see Example 3.

[0228] Example 5. Construction and preparation of bispecific antibodies According to the combinations in Table 2, anti-CLDN18.2 monoclonal antibody HB37A6 was combined with the sequences of the antigen-binding regions of three different anti-human CD3 monoclonal antibodies HzSP34.24, HzSP34.87 and HzSP34.97 to construct bispecific antibodies 030, 032 and 033 in the "1+1" format targeting CLDN18.2×CD3, respectively, and the antibody structure diagrams are shown in FIG. 1. Specifically, the antigen-binding domain specifically targeting CLDN18.2 has a heavy chain variable region sequence of SEQ ID NO: 4 and a light chain variable region sequence of SEQ ID NO: 9, and the antigen-binding domain specifically targeting CD3 has a heavy chain variable region sequence of SEQ ID NO: 22, SEQ ID NO: 30 and SEQ ID NO: 32 and a light chain variable region sequence of SEQ ID NO: 27. The Fc region of the antibody is selected from the IgG1 LALA sequence with a knob-into-hole structure (A. Margaret Merchant et al., Nature Biotechnology, 1998). Thus, the bispecific antibody CLDN18.2 terminal portion has the heavy chain represented by SEQ ID NO: 37 and the light chain represented by SEQ ID NO: 38. The bispecific antibody CD3 terminal portion has the heavy chain represented by SEQ ID NO: 39, 41 and 42, respectively, and the light chain represented by SEQ ID NO: 40.

[0229] The plasmid construction process of the bispecific antibody is as follows: CLDN18.2 heavy chain sequence (SEQ ID NO: 37), CLDN18.2 light chain sequence (SEQ ID NO: 38), CD3 heavy chain sequence (SEQ ID NO: 39, 41 and 42), CD3 light chain sequence (SEQ ID NO: 40) were inserted into the vector pcDNA3.1 (Invitrogen, V790-20) respectively to obtain the CLDN18.2 end heavy chain plasmid, light chain plasmid, CD3 end heavy chain plasmid and light chain plasmid respectively. Then, the CLDN18.2 end heavy chain plasmid and light chain plasmid and the CD3 end heavy chain plasmid and light chain plasmid were transiently transfected into Expi293 cells (Invitrogen, A14527) using PEI (Polysciences, 23966) respectively to express the CLDN18.2 end antibody molecule and the three CD3 end antibody molecules. After 7 days, the cell fermentation liquid was obtained, filtered and clarified, and then captured by Protein A column of Hitrap Mabselect Sure (GE Healthcare, 11-0034-95), respectively, to obtain CLDN18.2-terminal and CD3-terminal antibodies. After detecting the concentration by the A280 method, the antibodies of both ends were mixed in a molar ratio of 1:1. An appropriate amount of reducing agent GSH was added and reacted at room temperature overnight. The reducing agent was removed by ultrafiltration to stop the reaction. Then, it was precisely purified by MonoS cation exchange chromatography (GE Healthcare, 17-5168-01), and the A solution was 20 mM sodium phosphate buffer (pH 6.6), the B solution was 20 mM sodium phosphate buffer (pH 6.6) containing 1 M sodium chloride, and the elution gradient was 0% to 50% (30 column volumes). The eluted protein solution was ultrafiltered and exchanged with PBS (Gibco, 70011-044), and the molecular weight was measured by mass spectrometry and the purity was confirmed by SEC-HPLC. The obtained bispecific antibodies 030, 032 and 033 were used in the following examples.

[0230] [Table 6]

[0231] Example 6. Determining the affinity of bispecific antibodies The equilibrium dissociation constant (KD) of the bispecific antibody of the present invention binding to human CD3 protein was measured by biolayer interferometry (BLI). The affinity measurement by the BLI method was performed by a conventional method (Estep, P et al., High throughput solution based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013.5(2):P270-278).

[0232] Thirty minutes before the start of the experiment, a suitable number of AHC (18-5060, Fortebio) sensors were immersed in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween®-20) according to the number of samples. 100 μL of SD buffer, each bispecific antibody, and human CD3 protein (CT026H0323H, Beijing Yiqiao Shenzhou) were added to a 96-well black polystyrene half-volume microplate (Greiner, 675076), respectively. Detection was performed by Fortebio Octet Red96, and the plate was positioned according to the position of the sample, and the sensor position was selected. The instrument parameters were set as follows: Baseline, Loading~1nm, Baseline, Association and Dissociation steps were performed, and the execution time of each step was determined by the binding and dissociation rate of the sample, the rotation speed was 1000 rpm, and the temperature was 30°C. The KD value was analyzed using ForteBio analysis software.

[0233] The affinities of the bispecific antibodies are shown in Table 3. Here, the CD3 terminus of the 030 molecule has the highest affinity of 7.4 nM. The CD3 terminus of the 032 and 033 molecules have sequentially decreasing affinities of 89 nM and 440 nM, respectively.

[0234] [Table 7]

[0235] The equilibrium dissociation constant (KD) of binding to human CLDN18.2 was measured by surface plasmon resonance (SPR). Following the manufacturer's instructions, the antigen human Claudin18.2 (GenScrip, P50251802) was coupled to the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino coupling reagent kit (GE Healthcare, BR-1006-33). After coupling, 1 M ethanolamine was injected to seal the remaining activation sites. Following the manufacturer's instructions, the binding and dissociation of each bispecific antibody in the mobile phase with the chip surface antigen was detected by Biacore (GE Healthcare, T200) to obtain affinity and kinetic constants. Gradient diluted antibodies (0 nM to 100 nM, 2-fold diluted) were flowed over the chip surface in ascending order of concentration, with a binding time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated with 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). The data results were analyzed using the Biacore T200 analysis software with a 1:1 binding model. As a result, as shown in Table 4, the 030, 032 and 033 bispecific antibodies use the same clone HB37A6 at the CLDN18.2 end, and the affinity of the CLDN18.2 end is consistent, with a very strong affinity of 0.57 nM.

[0236] [Table 8]

[0237] Example 7. In vitro T cell killing experiments An in vitro T cell killing experiment was carried out for the bispecific antibodies 030, 032 and 033 obtained in Example 5. Human peripheral blood mononuclear cells (PBMCs, Allcells or Saily) were resuspended in complete medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03) and PBMCs were added at a concentration of 2 × 10 6 The concentration was adjusted to 1 / mL.

[0238] NUGC-4 or Claudin18.2-overexpressing DAN-G tumor target cells DAN-G-hCLDN18.2 and non-target cells L363 (DSMZ, ACC49) were labeled with Far-Red (Invitrogen) for 10 min, washed twice, and then resuspended in complete medium at a cell concentration of 2 × 10 5 The concentration was adjusted to 1 / mL.

[0239] PBMCs were mixed with bispecific antibodies 030, 032, and 033 (030 and 032 were initially at 1 nM, and 033 was initially at 400 nM, with each antibody diluted 5-fold to give a total of 10 concentration points) and incubated at 37°C for 30 min, followed by incubation with 50 μL of tumor target cells (1 × 10 4 1000 pieces) were added to 50 μL of PBMC effector cells. After 24 hours of incubation at 37°C, the cells were centrifuged and resuspended in propidium iodide (PI, Invitrogen) at a final concentration of 10 μg / mL. Far-Red and PI double-positive cells were detected by flow cytometry (BD, FACSCELESTA). The tumor target cell killing ratio was calculated using FACSDiva software (BD, Celestsa).

[0240] The results in Figure 2 show that the 030 and 032 molecules have very strong killing activity in gastric cancer cells NUGC-4, with EC50 values ​​of both less than 1 pM. Meanwhile, the results in Figure 3 show that in pancreatic cancer cells DAN-G-hCLDN18.2, which highly express CLDN18.2, the EC50 values ​​of both are less than 0.1 pM. In addition, the 033 molecule has weaker killing activity in the two tumor cell lines, weaker than 030 and 032, about 1000 times lower, but still can reach maximum killing (nearly 100% cell lysis). However, when the expression of CLDN18.2 was negative, none of the 030, 032 and 033 molecules had non-specific killing (Figure 4). This suggests that the 030, 032 and 033 molecules all showed tumor cell-specific killing that was dependent on the expression of CLDN18.2. Furthermore, the killing effect of the bispecific antibody of the present invention correlates with the abundance of CLDN18.2 on the cell surface; within a certain range of expression abundance, the higher the expression level of CLDN18.2 on the cell surface, the higher the killing effect.

[0241] Example 8. In vivo efficacy experiment - gastric cancer model This experiment investigated the antitumor effect of bispecific antibodies against NUGC-4 tumor-bearing NOG female mice. Forty-nine NOG female mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) were selected.

[0242] Resuscitate PBMC cells (Allcells), centrifuge the cells, and disperse the PBMC cells in PBS (1x) to a cell density of 2x10 7 A cell suspension with a total cell count of 4 × 10 PBMC cells was obtained. 6 The mice were injected intraorbitally at 100 / mouse.

[0243] NUGC-4 cells were subjected to normal resuscitation subculture and used for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed in PBS (1x) to a cell density of 6x10 7 cells / mL and mixed 1:1 with Matrigel to obtain a cell concentration of 3 × 10 7A cell suspension of 0.2 cells / mL was prepared. On day 3, 0.2 mL of the cell suspension was taken and subcutaneously inoculated into the right flank region of NOG humanized mice to establish a NUCG-4 tumor-bearing mouse model.

[0244] Seven days after cell seeding, the maximum width and length of the mouse tumor were measured with a caliper, and the tumor volume was calculated to be 53.35 mm 3 ~168.07mm 3 Mice with tumor volumes of 0.3 mg / kg or less were selected, and serpentine grouping (6 mice per group) was performed based on the tumor volumes of the mice. The bispecific antibodies 030, 032 and 033 of the present invention, and the negative control h-IgG (Equitech-Bio, lot number 160308-02) were intravenously injected into each mouse at doses of 0.3 mg / kg and 1 mg / kg, once a week for a total of 4 times, and the tumor volumes of the mice were measured twice a week. At the same time, the tumor inhibition rate (TGI%) was calculated, and the formula was: TGI% = 100% x (tumor volume of control group - tumor volume of treatment group) / (tumor volume of control group - tumor volume before administration of control group).

[0245] As shown in Figure 5, in a human gastric cancer NUCG-4 tumor-bearing mouse model, 030 and 032 could reach TGI 100% at a low dose of 0.3 mg / kg, and even reached 50% CR (complete remission) at a high dose of 1 mg / kg (3 out of 6 mice were completely tumor-free). Meanwhile, 033 molecule had little efficacy at low doses, and at a dose of 1 mg / kg, TGI could reach 20%. During the experimental period, mice in the experimental and control groups did not lose weight.

[0246] Example 9. In vivo efficacy experiment - pancreatic cancer model This experiment investigated the antitumor effect of bispecific antibodies against DAN-G-Claudin18.2 tumor-bearing female NOG mice. PBMC cells were injected intraorbitally into 49 NOG mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.), at a dose of 4 × 10 6 The inoculation volume was 200 μL / animal (Example 8This time was designated as day 0.

[0247] The human pancreatic cancer cells DAN-G-CLDN18.2 constructed in Example 1 were subjected to normal subculture for subsequent in vivo experiments. The cells were collected by centrifugation and dispersed in PBS (1x) until the cell density reached 10x10 6 The cell suspension and Matrigel gel were mixed in a 1:1 ratio to obtain a suspension with a cell concentration of 5 × 10 6 A cell suspension was prepared with a cell density of 100 / mL. On day 0, 0.2 mL of the cell suspension was subcutaneously inoculated into the right flank region of NOD-SCID mice to establish a CLDN18.2-overexpressing DNA-G pancreatic cancer humanized model.

[0248] Seven days after tumor cell inoculation, the maximum width and length of the mouse tumor were measured with a caliper, and the tumor volume was calculated. The mice were then grouped into serpentine groups based on their tumor volumes. 3 ~120.64mm 3 Mice within this range were divided into serpentine groups (6 mice per group) according to the size of their tumor volume.

[0249] Mice were intravenously injected with bispecific antibodies 030, 032 and 033 of the invention, and with a negative control h-IgG (Equitech-Bio, Lot No. 160308-02), throw The dose was 0.3mg / kg and 1mg / kg, once a week for a total of four times, and the tumor volume of the mice was measured twice a week. At the same time, the tumor inhibition rate (TGI%) was calculated using the following formula: TGI%=100%×(tumor volume of control group−tumor volume of treatment group) / (tumor volume of control group−tumor volume before administration of control group). As shown in FIG. 6, in the CLDN18.2 overexpressing DNA-G pancreatic cancer humanized model, 030 and 032 molecules could reach TGI100% at both two doses. Meanwhile, 033 molecule could also reach TGI42% (0.3 mg / kg) and TGI76% (1 mg / kg), respectively, which may be correlated with the high expression of CLDN18.2 in DNA-G-CLDN18.2 pancreatic cancer cells. During the experimental period, mice in the experimental and control groups did not lose weight.

[0250] Example 10. Mouse PK experiments In this study, 10mg / kg of 030, 032 and 033 were injected into female Balb / C mice (Weitong Lihua) via tail vein to investigate the pharmacokinetic properties in mice. After administration, blood was collected from the eyeball at 0.086hr, 0.5hr, 2hr, 6hr, 24hr, 48hr, 4days, 7days, 14days and 21days, respectively, and the blood was centrifuged at 4℃, 3000rpm for 10min to collect serum. The antibody content in serum was measured by ELISA, and the half-life of 030, 032 and 033 in mice was calculated.

[0251] As shown in Figure 7, the experimental results showed that 030, 032 and 033 all achieved the same PK as a normal monoclonal antibody with half-life in mice. Furthermore, it is demonstrated that the bispecific antibody constructed in the present invention does not affect the half-life of the antibody.

[0252] Example 11. pH Screening Experiments 1. Experimental procedure Buffer solutions containing 1.55 mg / mL histidine and 50.00 mg / mL sorbitol were prepared, and the pH was adjusted to 5.0, 5.5, 6.0, 6.5 and 7.0 with hydrochloric acid, respectively. The CLDN18.2 / CD3 protein was ultrafiltered and replaced with buffer solutions of different pH values, and the protein content was adjusted to about 1 mg / mL. Polysorbate 80 was added to a final concentration of 0.2 mg / mL, filtered, and dispensed into vials, plugged and capped. The stability of the above samples was examined under conditions of 40°C±2°C, and the specific method was presented. 5 As shown in.

[0253] [Table 9]

[0254] 2, Judgment criteria Based on the product recognition and the accuracy of the equipment and method, a judgment criterion was set that would ensure that the quality of the sample would not change when compared with the initial value.

[0255] [Table 10]

[0256] As can be seen from the results (as shown in FIG. 8), after 4 weeks at 40℃±2℃, the samples under each pH condition passed the appearance and visible foreign matter, and the protein content and pH did not change obviously. The purity (nrCE-SDS method) of the samples under each pH condition did not change obviously, and the purity (SEC-HPLC method) of the samples increased in all other samples except for the sample at pH 5.0, which did not change obviously. The charge variant-acidic components of all samples increased significantly, and with the increase in pH value, the acidic components increased more rapidly; the charge variant-main components of all samples decreased obviously, and with the increase in pH value, the main components decreased more rapidly; the charge variant-basic components of all samples decreased obviously, except for the sample at pH 5.0, which did not change obviously; the biological activities of anti-Claudin18.2 all met the criteria; and the biological activities of anti-CD3 terminal exceeded the judgment criteria in all other conditions except for the sample at pH 5.0, which met the criteria.

[0257] Taking into account the results of the biological activities of the charge mutants and the anti-CD3 terminus, and the pH fluctuation during actual production, a formulation determination experiment was carried out, finally determining pH 5.2.

[0258] Example 12. Formulation determination experiment This example mainly examines the effect of buffer systems (histidine and sodium acetate) and additives (sorbitol, arginine hydrochloride, and disodium edetate) on the stability of the bispecific antibody protein, and the detailed prescription information is shown in Table 7.

[0259] A buffer solution for each formulation was prepared according to Table 7, and the bispecific protein was ultrafiltered and replaced with the respective formulation solution. After replacement, the protein content of each formulation was adjusted to 1 mg / mL, polysorbate 80 was added to the final concentration of 0.2 mg / mL, filtered, dispensed into vials, and capped with a plug. The stability of the above samples was examined at 40°C ± 2°C.

[0260] [Table 11]

[0261] [Table 12]

[0262] The criteria are shown in Table 6, and as can be seen from the results (as shown in Figure 9), after being left at 40°C ± 2°C for 4 weeks, the samples of all the formulations passed the criteria for appearance and visible foreign matter, and the protein content and pH value did not change obviously. Except for the purity (SEC-HPLC method) of the samples of formulation 2 and formulation 5, which did not change obviously, the changes of the other formulations all exceeded the criteria. The changes in purity (nrCE-SDS method) of the samples of formulation 3 and formulation 4 exceeded the criteria, and the downward trend of formulation 1 and formulation 2 was consistent and slower than that of formulation 5. The changes in the charge variants of the samples of all the formulations were expressed as a significant increase in acidic components and a significant decrease in main components. The biological activity of the anti-Claudin18.2 end of all the formulations met the criteria, the biological activity of the anti-CD3 end of formulation 2 and formulation 5 met the criteria, and the other formulations all exceeded the criteria. Except for formulation 2, which did not change its polysorbate 80 content, all other formulations showed different degrees of reduction.

[0263] Combined with the results of Examples 11 and 12, Formulation 2 was selected as the final formulation of anti-CLDN18.2 / CD3 bispecific antibody. In order to avoid pH adjustment by hydrochloric acid during production, the buffer system was adjusted to histidine and histidine hydrochloride, i.e., the formulation of anti-CLDN18.2 / CD3 bispecific antibody was 1.0 mg / mL recombinant anti-differentiation antigen cluster 3 (CD3) and anti-tight junction protein 18.2 (Claudin18.2) bispecific antibody, 0.18 mg / mL histidine, 1.84 mg / mL histidine hydrochloride, 50.00 mg / mL sorbitol, 0.01 mg / mL edetate disodium and 0.2 mg / mL polysorbate 80, pH 5.2.

[0264] Although exemplary embodiments of the present invention have been described above, those skilled in the art should understand that these disclosures are merely exemplary and that various other substitutions, adaptations and modifications can be made within the scope of the present invention. Accordingly, the present invention is not limited to the specific embodiments set forth herein.

[0265] Sequence information

[0266] [Table 13-1] [Table 13-2] [Table 13-3] [Table 13-4] [Table 13-5] [Table 13-6] [Table 13-7] [Table 13-8] [Table 13-9]

Claims

1. (i) anti-CLDN18.2 / CD3 protein, (ii) a buffering agent; (iii) a stabilizer, and (iv) a surfactant, The anti-CLDN18.2 / CD3 bispecific antibody protein comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to CLDN18.2, and the second antigen-binding domain specifically binds to CD3, wherein the first antigen-binding domain is composed of three complementarity determining regions A1-HCDR1, A1-HCDR2 and A1-HCDR3 contained in A1-VH shown in SEQ ID NO: 4 and A1-HCDR3 shown in SEQ ID NO:

9. the first antigen-binding domain comprises three complementarity determining regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in A2-VH represented by SEQ ID NO: 30, 22 or 32, and three complementarity determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL represented by SEQ ID NO: 27; Preferably, the liquid antibody formulation has a pH of about 5.0 to 6.0, e.g., 5.2±0.2 or 5.5±0.2, preferably 5.

2. Liquid antibody formulation.

2. The anti-CLDN18.2 / CD3 protein in the liquid antibody formulation is at a concentration of about 0.1 mg / mL to 100 mg / mL, preferably about 0.1 mg / mL to 10 mg / mL, for example, about 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL or 5 mg / mL; The liquid antibody formulation of claim 1.

3. The liquid antibody formulation comprises a buffer selected from a histidine-histidine hydrochloride buffer system, a citric acid-sodium citrate buffer system, an acetic acid-sodium acetate buffer system, and a phosphate buffer system, preferably the buffer in the liquid antibody formulation is selected from histidine, histidine hydrochloride, and combinations thereof, preferably the buffer has a concentration of about 0.01 mg / mL to 10 mg / mL, preferably about 0.1 mg / mL to 1 mg / mL, for example, about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, or 1 mg / mL; The liquid antibody formulation according to claim 1 or 2.

4. the stabilizer is selected from polyols (e.g., sorbitol, mannitol, and combinations thereof), sugars (e.g., sucrose, trehalose, maltose, and combinations thereof), amino acids (e.g., arginine, arginine hydrochloride, methionine, glycine, proline, and combinations thereof), and any combination thereof; For example, the stabilizer is - a polyol selected from sorbitol, mannitol or a combination thereof, - a sugar selected from sucrose, trehalose, maltose or a combination thereof, and - one or more selected from the amino acids selected from arginine hydrochloride, methionine, glycine, proline and combinations thereof; The liquid antibody formulation according to claim 1 or 2.

5. The stabilizer is (i) a combination of about 20 mg / mL to 40 mg / mL sorbitol and about 50 mM to 100 mM arginine; or (ii) about 30 mg / mL to 60 mg / mL sucrose in combination with about 50 mM to 100 mM arginine; or (iii) about 30 mg / mL to 70 mg / mL sorbitol, preferably present in an amount of about 50 mg / mL; The liquid antibody formulation according to any one of claims 1 to 4.

6. The surfactant in the liquid antibody formulation is selected from a polysorbate surfactant, a poloxamer, a polyethylene glycol, or a combination thereof, and is preferably polysorbate-80. The liquid antibody formulation according to any one of claims 1 to 4.

7. The surfactant is at a concentration of about 0.01 mg / mL to 5 mg / mL, preferably about 0.1 mg / mL to 1 mg / mL, for example about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1.0 mg / mL, more preferably about 0.2 mg / mL; The liquid antibody formulation according to any one of claims 1 to 5.

8. further comprising a chelating agent, said chelating agent being selected from disodium edetate, nitrilotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid and dihydroxyethylglycine, preferably said chelating agent being selected from disodium edetate; Preferably, the chelating agent is at about 0.008 mg / mL to 0.018 mg / mL, for example, at about 0.008 mg / mL, 0.009 mg / mL, 0.010 mg / mL, 0.012 mg / mL, 0.014 mg / mL or 0.018 mg / mL, preferably 0.01 mg / mL; The liquid antibody formulation according to claim 7 .

9. In the anti-CLDN18.2 / CD3 protein, The first antigen-binding domain comprises A1-HCDR1, A1-HCDR2 and A1-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 1, 2 and 3, respectively, and A1-LCDR1, A1-LCDR2 and A1-LCDR3 represented by the amino acid sequences of SEQ ID NOs: 6, 7 and 8, respectively; and The second antigen-binding domain comprises: (i) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or (ii) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; or (iii) A2-HCDR1, A2-HCDR2 and A2-HCDR3 represented by the amino acid sequences of SEQ ID NOs: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 represented by the amino acid sequences of SEQ ID NOs: 24, 25 and 26, respectively; The liquid antibody formulation according to any one of claims 1 to 8.

10. The first antigen-binding domain comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 4; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO:4; or (iii) comprises or consists of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 4, preferably said amino acid changes do not occur in the CDR regions, and / or The light chain variable region comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 9; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO:9; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 9, and preferably said amino acid changes do not occur in the CDR regions; The liquid antibody formulation according to any one of claims 1 to 9.

11. The anti-CLDN18.2 / CD3 is an IgG type antibody (e.g., an IgG4 subtype antibody), preferably comprising a heavy chain sequence having SEQ ID NO: 9 or at least 90%, 95%, 98% or 99% identity thereto, and a light chain sequence having SEQ ID NO: 10 or at least 90%, 95%, 98% or 99% identity thereto; The liquid antibody formulation according to any one of claims 1 to 8.

12. The second antigen-binding domain comprises a heavy chain variable region and / or a light chain variable region, wherein the heavy chain variable region comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 30, 22, or 32; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32, preferably said amino acid changes do not occur in the CDR regions; and / or The light chain variable region comprises: (i) comprising or consisting of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence selected from SEQ ID NO: 27; or (ii) comprising or consisting of an amino acid sequence selected from SEQ ID NO: 27; or (iii) comprising or consisting of an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2 or 1 or less) amino acid changes (preferably amino acid substitutions, more preferably amino acid conservative substitutions) compared to an amino acid sequence selected from SEQ ID NO: 27, and preferably said amino acid changes do not occur in the CDR regions; The liquid antibody formulation according to any one of claims 1 to 11.

13. Further comprising a heavy chain constant region and / or a light chain constant region, The liquid antibody formulation according to any one of claims 1 to 11.

14. It is a bispecific antibody with an IgG-like structure. The liquid antibody formulation according to any one of claims 1 to 12.

15. the heavy chain constant region of the antibody is derived from IgG1, IgG2, IgG3 or IgG4, preferably IgG1; The liquid antibody formulation according to any one of claims 1 to 13.

16. The anti-CLDN18.2 / CD3 is recombinantly expressed in 293 cells or CHO cells; The liquid antibody formulation according to any one of claims 1 to 14.

17. The liquid antibody formulation comprises: (i) about 0.1 mg / mL to 100 mg / mL of anti-CLDN18.2 / CD3 protein; (ii) about 0.1 mg / mL to 2 mg / mL histidine; (iii) about 30 mg / mL to 70 mg / mL sorbitol; (iv) about 0.1 mg / mL to 1 mg / mL of polysorbate 80, and (v) optionally, about 0.008 mg / mL to 0.018 mg / mL of edetate disodium; wherein the liquid formulation has a pH of about 5.0 to 5.5, e.g., about 5.2; For example, the liquid antibody formulation may include (i) about 0.1 mg / mL to 10 mg / mL, for example, 0.1 mg / mL to 1 mg / mL of anti-CLDN18.2 / CD3 protein; (ii) about 1.55 mg / mL histidine; (iii) about 40 mg / mL to 60 mg / mL sorbitol or sucrose, preferably 50 mg / mL sorbitol, or 30 mg / mL to 50 mg / mL sucrose; (iv) about 0.2 mg / mL to 0.8 mg / mL, e.g., 0.2 mg / mL to 0.6 mg / mL, of polysorbate 80, and (v) about 0.008 mg / mL to 0.018 mg / mL, e.g., 0.01 mg / mL, of edetate disodium; wherein the liquid formulation has a pH of about 5.0 to 5.5, e.g., about 5.2; Alternatively, the liquid antibody formulation comprises: (i) about 1 mg / mL anti-CLDN18.2 / CD3 protein, about 1.55 mg / mL histidine, about 50.00 mg / mL sorbitol, and about 0.2 mg / mL polysorbate 80, at a pH of about 5.2; or (ii) about 50 mg / mL anti-CLDN18.2 / CD3 protein, about 1.55 mg / mL histidine, about 50.00 mg / mL sorbitol, about 0.01 mg / mL edetate disodium, and about 0.2 mg / mL polysorbate 80, at a pH of about 5.2; or (iii) about 50 mg / mL of anti-CLDN18.2 / CD3 protein, about 0.82 mg / mL of sodium acetate, about 50.00 mg / mL of sorbitol, and about 0.2 mg / mL of polysorbate 80, at pH 5.2; A liquid antibody formulation according to any one of claims 1 to 16.

18. The formulation is stable after storage, e.g., at least 2 months at 25° C. or 1 month at 40° C.±2° C., and preferably (i) the main peak has a variation of less than 1% when measured by SEC-HPLC method, and / or the preparation has a purity of greater than 96%, preferably greater than 97%, 98%; (ii) the main peak has a change of less than 2% when measured by non-reducing CE-SDS method, and / or the preparation has a purity of more than 96%, preferably more than 97%, more than 98%; (iii) When measured by CEX-HPLC, the total change in each component (main component, acidic component and basic component) of the anti-CLDN18.2 / CD3 protein in the formulation is 40% or less, and / or the change in the main component is 20% or less, for example, after storage at 40 ° C ± 2 ° C for 1 month, the total change is about 40% or less (e.g., 30% or less), or the change in the main component is 20% or less (e.g., 15% or less), or, for example, after storage at 25 ° C for 2 months, the total change is about 20% or less (e.g., 15% or less), or the change in the main component is 15% or less (e.g., 10% or less), and (iv) The relative binding activity of the anti-CLDN18.2 / CD3 protein in the formulation is 70% to 130%, for example 90% to 110%, relative to the initial value on day 0 of storage, as measured by ELISA; More preferably, the formulation is stable under shaking and / or repeated freeze-thaw cycles. The liquid antibody formulation according to any one of claims 1 to 13.

19. The liquid formulation is an injection, preferably for subcutaneous or intravenous injection, or an infusion, e.g. for intravenous infusion; A liquid antibody formulation according to any one of claims 1 to 15.

20. 20. The liquid antibody formulation according to claim 1, which is obtained by hardening the liquid antibody formulation according to claim 1, for example in the form of a lyophilized powder for injection. Solid antibody formulations.

21. A liquid antibody formulation according to any one of claims 1 to 19, or a solid antibody formulation according to claim 15. Delivery device.

22. 20. A liquid antibody formulation according to any one of claims 1 to 19 or a solid antibody formulation according to claim 15 for intravenous or intramuscular injection. Prefilled syringes.

23. Use of the liquid antibody formulation according to any one of claims 1 to 20 or the solid antibody formulation according to claim 15 in the manufacture of a delivery device, a pre-filled syringe or a medicament for treating or preventing a tumor or a pathogen infection in a subject. use.

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