Preparation containing anti-Claudin18.2 and CD3 bispecific antibody as well as preparation method and application thereof

Abstract

The present invention relates to formulations comprising anti-CLDN18.2 / CD3, in particular to pharmaceutical formulations comprising anti-CLDN18.2 / CD3, a buffering agent, a stabilizing agent and a surfactant. In addition, the invention also relates to the use of these formulations for the treatment or prevention of diseases.

Description

Preparation containing anti-Claudin18.2 and CD3 bispecific antibodies and preparation method and use thereof 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 the pharmaceutical formulations, and the therapeutic and / or prophylactic uses of the pharmaceutical formulations. Background Art

[0002] Drug stability is one of the most important indicators for ensuring drug efficacy and safety. Obtaining a good formulation is a key condition for ensuring that a drug maintains its efficacy and safety throughout its shelf life. However, due to the complexity of bispecific antibodies themselves and their degradation pathways, it is currently impossible to predict the formulation conditions required to optimize antibody stability. This is especially true considering that different antibodies typically have very different structures and CDR sequences, and these sequence differences can lead to different stability properties in solution. Therefore, given the strict safety and efficacy requirements for human antibodies, it is necessary to optimize the optimal formulation formula for each antibody individually.

[0003] Claudin-18 isoform 2 (Claudin 18.2 or CLDN18.2) is a highly selective cell lineage marker. Its expression in normal tissues is strictly restricted to differentiated epithelial cells of the gastric mucosa, but is absent in the gastric stem cell compartment. CLDN18.2 is expressed in a significant proportion of primary gastric cancers, and its expression levels are retained in gastric metastatic cancer tissues. In addition to gastric cancer, CLDN18.2 expression has also been found in pancreatic cancer, making it an ideal target for the treatment of these cancers (Singh, P., Toom, S. & Huang, Y. Anti-CLDN18.2 antibody as a 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 expressed on T cells that binds to the T cell receptor complex (TCR) and is required for T cell activation. Functional CD3 is formed by the dimerization of two of four different chains: ε, ζ, δ, and γ. CD3 dimer arrangements include γ / ε, δ / ε, and ζ / ζ. Antibodies targeting CD3 have been shown to aggregate CD3 on T cells, leading to T cell activation in a manner similar to engagement of the TCR by peptide-loaded MHC molecules. Therefore, anti-CD3 antibodies have been proposed for therapeutic purposes involving T cell activation. Furthermore, bispecific antibodies that bind to CD3 and target tumor surface antigens have been proposed to bridge tumor cells and T cells, directly activating T cells and releasing granzymes, perforins, and cytokines to kill tumors, thereby achieving the therapeutic goal of tumor suppression. CLDN18.2 is highly expressed in gastric cancer, pancreatic cancer, and gastroesophageal junction cancer, so the development of bispecific antibodies that simultaneously bind to both CD3 and Claudin18.2 could achieve therapeutic goals.

[0005] Although some bispecific antibody formulations have been proposed, there is still a need in the art for new pharmaceutical formulations that are sufficiently stable and suitable for administration to human subjects. In addition, for such bispecific antibody formulations, it is also advantageous to find simplicity and ease of formulation.

[0006] SUMMARY OF THE INVENTION

[0007] The present invention satisfies the above needs by providing a pharmaceutical formulation comprising an anti-Claudin18.2 and CD3 bispecific antibody. The antibody formulation of the present invention exhibits excellent stability against various stability-influencing factors (such as temperature and pH).

[0008] In one aspect, the present invention 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, the formulation further comprises a chelating agent.

[0009] In some aspects, the present invention provides bispecific antibodies, wherein the antibody comprises two binding domains, wherein the first binding domain specifically binds CLDN18.2 and the second binding domain specifically binds CD3.

[0010] In a preferred embodiment, the first binding domain of the present invention that specifically binds to CLDN18.2 is fully human, and / or the second binding domain is humanized.

[0011] In some aspects, the bispecific antibodies of the present invention are bispecific antibodies with an IgG-like structure.

[0012] In one aspect, the present invention relates to the following embodiments:

[0013] 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 the three complementarity determining regions A1-HCDR1, A1-HCDR2, and A1-HCDR3 contained in A1-VH as shown in SEQ ID NO: 4, and the three complementarity determining regions A1-LCDR1, A1-LCDR2, and A1-LCDR3 contained in VL as 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: 30, 22, 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.

[0014] 2. The bispecific antibody of embodiment 1, wherein

[0015] The first antigen binding domain comprises A1-HCDR1, A1-HCDR2, A1-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 1, 2, and 3, respectively, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 6, 7, and 8, respectively; and

[0016] The second antigen binding domain comprises

[0017] (i) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25 and 26, respectively; or

[0018] (ii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25 and 26, respectively; or

[0019] (iii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 31, and 21, respectively, and LCDR1, LCDR2, and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25, and 26, respectively.

[0020] 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

[0021] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 4; or

[0022] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 4; or

[0023] (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 4, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions; and / or

[0024] Light chain variable region

[0025] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 9; or

[0026] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 9; or

[0027] (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 9, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0028] 4. The antibody of any one of embodiments 1-3, 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

[0029] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or

[0030] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or

[0031] (iii) an amino acid sequence comprising one or more (preferably no more than 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: 30, 22, or 32, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions;

[0032] and / or

[0033] Light chain variable region

[0034] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 27; or

[0035] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 27; or

[0036] (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 27, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0037] 5. The antibody according to any one of embodiments 1-4, further comprising a heavy chain constant region and / or a light chain constant region.

[0038] 6. The antibody according to any one of embodiments 1-5, which is a bispecific antibody with an IgG-like structure.

[0039] 7. The antibody according to any one of embodiments 1-6, wherein the heavy chain constant region of the antibody is derived from IgG1, IgG2, IgG3, or IgG4, preferably IgG1.

[0040] 8. The antibody according to any one of embodiments 1-6, comprising two heavy chain constant regions, wherein one heavy chain constant region A1-HC is connected to the heavy chain variable region A1-VH of the first antigen domain to constitute the heavy chain that binds to CDLN18.2, and the other heavy chain constant region A2-HC is connected to the heavy chain variable region A2-VH of the second antigen binding domain to constitute the heavy chain that binds to CD3, and comprising two light chain constant regions, wherein one light chain constant region A1-LC is connected to the light chain variable region A1-VL of the first antigen domain to constitute the light chain that binds to CLDN18.8, and the other light chain constant region A2-LC is connected to the light chain variable region A2-VL of the second antigen binding domain to constitute the light chain that binds to CD3.

[0041] 9. The antibody of embodiment 8, wherein A1-HC may be the same as or different from A2-HC, and / or A1-LC may be the same as or different from A2-LC.

[0042] 10. The antibody of embodiment 8, wherein the CLDN18.2-binding portion A1-VH and A1-VL are fully human, and the CD3-binding portion A2-VH and A2-VL are humanized.

[0043] 11. The antibody of embodiment 8, wherein the heavy chain of the portion that binds to CLDN18.2

[0044] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 37;

[0045] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 37; or

[0046] (iii) comprising or consisting 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

[0047] Light chain that binds to CLDN18.2

[0048] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 38;

[0049] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38; or

[0050] (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 the amino acid sequence selected from SEQ ID NO: 38.

[0051] 12. The antibody of embodiment 8 or 11, wherein

[0052] Heavy chain that binds to CD3

[0053] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 41, 39 or 42;

[0054] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 41, 39 or 42; or

[0055] (iii) comprising or consisting 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, 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

[0056] CD3-binding light chain

[0057] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 40;

[0058] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 40; or

[0059] (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 the amino acid sequence selected from SEQ ID NO: 40.

[0060] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the present invention bind to CLDN18.2 (e.g., human CLDN18.2) with high affinity. In some embodiments, the antibodies of the present invention specifically bind to CLDN18.2 (e.g., human CLDN18.2) without binding to CLDN18.1 (e.g., human CLDN18.1). In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention have a higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, such as Zolbetuximab (abbreviated as Zmab). In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the present invention bind to CD3 (e.g., human CD3 or cynomolgus monkey CD3) with a desired affinity (e.g., lower). In some embodiments, the antibodies of the present invention are capable of binding to both human CD3 and cynomolgus monkey CD3. In some embodiments, the affinity of the antibodies is determined by thin-layer interferometry or surface plasmon resonance.

[0061] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the present invention are capable of binding to CLDN18.2 on the surface of tumor cells and CD3 on the surface of effector cells. In some embodiments, the binding is detected by flow cytometry.

[0062] In one embodiment, the concentration of the anti-CLDN18.2 / CD3 protein in the liquid antibody formulation of the present invention is about 0.1-100 mg / ml, preferably about 0.1-10 mg / ml, more preferably about 0.1-1 mg / ml. In other embodiments, the concentration of the anti-CLDN18.2 / CD3 in the liquid antibody formulation of the present invention is about 0.5, 1, 2, 3, 4, or 5 mg / ml.

[0063] In some embodiments, the buffer used in the formulations of the present invention is a histidine buffer, in particular a buffer system consisting of histidine and histidine hydrochloride. In some embodiments, the concentration of histidine in the histidine buffer of the present invention is about 0.01-10 mg / ml, in particular about 0.1-1 mg / ml, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 1 mg / ml. In one embodiment, the formulation of the present invention comprises about 0.18 mg / ml histidine. In another embodiment, the histidine buffer used in the formulation of the present invention consists of about 0.18 mg / ml histidine and about 1.84 mg / ml histidine hydrochloride.

[0064] In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 10-100 mg / ml. In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 20-60 mg / ml, for example, about 20, 30, 40, 50, 60 mg / ml.

[0065] In one embodiment, the stabilizer is selected from a polyol (e.g., sorbitol, mannitol, or a combination thereof), a sugar (e.g., sucrose, trehalose, maltose, or a combination thereof), an amino acid (e.g., arginine hydrochloride, methionine, glycine, proline, and a combination thereof, or a salt thereof), and any combination thereof. In one embodiment, the stabilizer comprises about 20-80 mg / ml of sorbitol, such as 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80 mg / ml of sorbitol. In another embodiment, the stabilizer comprises about 20-60 mg / ml of sucrose, such as 20, 30, 40, 50, 60 mg / ml of sucrose. In a preferred embodiment, the stabilizer further comprises arginine, preferably arginine hydrochloride, at a concentration of about 25-200 mM, preferably about 50-150 mM, more preferably about 50-120 mM, and most preferably about 60-100 mM, for example, about 60, 65, 70, 75, 80, 85, 90 mM.

[0066] In a preferred embodiment, the stabilizer comprises a combination of sorbitol and arginine, or a combination of sucrose and arginine. For example, a combination of about 20-40 mg / ml of sorbitol and about 50-100 mM of arginine, or about 30-60 mg / ml of sucrose and about 50-100 mM of arginine can be used.

[0067] In a preferred embodiment, the liquid antibody formulation of the invention comprises about 30-60 mg / ml (such as about 50 mg / ml) of sorbitol, or about 20-30 mg / ml (such as about 25 mg / ml) of sorbitol and 80-90 mM (about 85 mM) of arginine.

[0068] In one embodiment, the concentration of the surfactant in the liquid antibody formulation of the present invention is about 0.1-1 mg / ml. In one embodiment, the concentration of the surfactant in the liquid antibody formulation of the present invention is about 0.2-0.8 mg / ml, for example, about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg / ml.

[0069] In one embodiment, the surfactant is a nonionic surfactant. In one embodiment, the surfactant is selected from polysorbate surfactants, poloxamers, and polyethylene glycol. In one embodiment, the surfactant is selected from polysorbate surfactants. In a specific embodiment, the surfactant in the liquid antibody formulation of the present invention is polysorbate-80.

[0070] Chelating agent concentration and type:

[0071] In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the present invention is about 0.005-0.05 mg / ml. In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the present invention is about 0.008-0.018 mg / ml, for example, about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg / ml.

[0072] In one embodiment, the chelating agent is a carboxylic acid chelating agent. In one embodiment, the chelating agent is selected from disodium edetate, aminotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, dihydroxyethylglycine. In one embodiment, the chelating agent is selected from disodium edetate.

[0073] In one embodiment, the pH of the liquid formulation is about 5.0-6.0. In some embodiments, the pH of the liquid formulation is about any value between 5.0-6.0, for example, about 5.0, 5.2, 5.4, 5.6, 5.8, or 6.0. Preferably, the pH of the formulation is 5.2 ± 0.2 or 5.5 ± 0.2, preferably a pH of 5.2.

[0074] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0075] (i) about 0.1-100 mg / ml of anti-CLDN18.2 / CD3 protein;

[0076] (ii) about 0.1-2 mg / ml histidine;

[0077] (iii) about 30-70 mg / ml of sorbitol;

[0078] (iv) about 0.1-1 mg / ml polysorbate 80; and

[0079] (v) optionally, about 0.008-0.018 mg / ml of edetate disodium,

[0080] wherein the pH of the liquid formulation is about 5.0-5.5, for example, about 5.2;

[0081] For example, the liquid antibody formulation comprises

[0082] (i) about 0.1-10 mg / ml, for example 0.1-1 mg / ml, of an anti-CLDN18.2 / CD3 protein;

[0083] (ii) about 1.55 mg / ml histidine;

[0084] (iii) about 40-60 mg / ml sorbitol or sucrose, preferably 50 mg / ml sorbitol or 30-50 mg / ml sucrose;

[0085] (iv) about 0.2-0.8 mg / ml, e.g., 0.2-0.6 mg / ml polysorbate 80; and

[0086] (v) optionally, about 0.008-0.018 mg / ml, e.g., 0.01 mg / ml, of edetate disodium,

[0087] wherein the pH of the liquid formulation is about 5.0-5.5, for example, about 5.2;

[0088] Alternatively, the liquid antibody formulation comprises

[0089] (i) about 1 mg / ml anti-CLDN18.2 / CD3 protein, about 1.55 mg / ml histidine, about 50.00 mg / ml sorbitol, about 0.2 mg / ml polysorbate 80, pH about 5.2; or

[0090] (ii) about 50 mg / ml of anti-CLDN18.2 / CD3 protein, about 1.55 mg / ml of histidine, about 50.00 mg / ml of sorbitol, about 0.01 mg / ml of edetate disodium, about 0.2 mg / ml of polysorbate 80, pH about 5.2; or

[0091] (iii) about 50 mg / ml anti-CLDN18.2 / CD3 protein, about 0.82 mg / ml sodium acetate, about 50.00 mg / ml sorbitol, about 0.2 mg / ml polysorbate 80, pH 5.2.

[0092] Liquid preparation of the present invention can be stored stably for a long time, for example, at least 24 months or longer.In one embodiment, liquid preparation of the present invention can be at about -80 ℃ to about 45 ℃, for example, about -80 ℃, about -30 ℃, about -20 ℃, about 0 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃ conditions, store 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 longer, and be stable.

[0093] In one embodiment, the liquid formulation of the present invention can be stored stably for at least 24 months. In another embodiment, the liquid formulation of the present invention is stable at at least 40°C. In another embodiment, the liquid formulation of the present invention remains stable at about 2°C-8°C for at least 3 months, preferably at least 12 months, more preferably at least 24 months. In one embodiment, the liquid formulation of the present invention remains stable at room temperature or about 25°C for at least 2 months, preferably at least 3 months, more preferably at least 6 months. In another embodiment, the liquid formulation of the present invention remains stable at about 40°C for at least 2 weeks, preferably at least 1 month.

[0094] In one embodiment, the stability of the preparation can be indicated by the change of the outward appearance, visible foreign matter, protein content, turbidity, purity, and / or charge variant of the detection preparation. In one embodiment, in the forced experiment of high temperature stress, for example, after storing at least 1 week, 2 weeks or preferably 1 month at 40 ℃ ± 2 ℃, or in an accelerated experiment, for example, after storing at least 1 month or 2 months at 25 ℃ ± 2 ℃, or in a long-term experiment, for example, after storing at least 2 months or 3 months at 5 ℃ ± 3 ℃, or in an oscillation experiment (for example, at room temperature, 650r / min conditions of lucifuge, oscillate 5 days), and / or in a freeze-thaw experiment (for example, at -30 ℃ / room temperature repeated freeze-thaw 6 times), the stability of the liquid preparation of the present invention is detected. In one embodiment, relative to initial value, for example, the initial value of the 0th day or the initial value before the oscillation or freeze-thaw experiment is stored, the stability of the liquid preparation of the present invention is detected.

[0095] In one embodiment, after storage, or after an oscillation test, or after a freeze-thaw test, the stability of the liquid preparation of the present invention is checked by visual inspection, wherein the liquid preparation of the present invention remains clear to slightly opalescent in appearance, is a colorless to light yellow liquid, and is free of foreign matter. In one embodiment, visual inspection under a clarity detector shows that no visible foreign matter is present in the preparation. In one embodiment, after storage, or after an oscillation test, or after a freeze-thaw test, the stability of the liquid preparation of the present invention is checked by measuring the change in protein content, wherein, for example, by ultraviolet spectrophotometry (UV), relative to the initial value, the rate of change in protein content is no more than 20%, preferably no more than 10%, for example, 7-8%, more preferably no more than 5%, 2% or 1%. In one embodiment, after storage, or after an oscillation test, or after a freeze-thaw test, the stability of the liquid preparation of the present invention is checked by measuring the change in turbidity of the liquid preparation of the present invention, wherein, for example, by OD 350mmMethod detection, relative to the initial value, the change value does not exceed 0.06, preferably does not exceed 0.05, more preferably does not exceed 0.04, does not exceed 0.02. In one embodiment, after storage, or after an oscillation test, or after a freeze-thaw test, the stability of the liquid preparation of the present invention is checked by measuring the purity change of the liquid preparation of the present invention, wherein by size exclusion high performance liquid chromatography (SEC-HPLC), relative to the initial value, the change value (or main peak change value) of the monomer purity does not exceed 10%, for example, does not exceed 5%, 4%, 3%, for example, the change value does not exceed 2%, preferably does not exceed 1%. In one embodiment, after storage, or after an oscillation test, or after a freeze-thaw test, the stability of the liquid preparation of the present invention is checked by measuring the purity change of the liquid preparation of the present invention, wherein by non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS) method, relative to the initial value, the change value (or main peak change value) of the monomer purity does not decrease by more than 10%, for example, does not exceed 5%, 4%, 3%, 2% or 1%. In one embodiment, after storage, or after an oscillation experiment, or after a freeze-thaw experiment, the stability of the liquid formulation of the present invention is tested by cation exchange high performance liquid chromatography (CEX-HPLC), wherein the sum of the change values ​​of the charge variants (main component, acidic component and basic component) of the antibody relative to the initial value does not exceed 50%, for example, does not exceed 40%, 30%, 20%, 10%, 5%, and / or the change value of the main component does not exceed 20%, 15%, 10%, 8%, 5%. In one embodiment, after storage, or after an oscillation experiment, or after a freeze-thaw experiment, the stability of the liquid formulation of the present invention is tested by direct ELISA, wherein the relative binding activity of the antibody relative to the initial value is 70-130%, for example, 70%, 80%, 90%, 93%, 95%, 98%, 100%, 103%, 105%, 108%, 110%, 115%, 120%, 125%, 130%, preferably 90-110%.

[0096] In one embodiment, the liquid formulation of the present invention is stable after storage, for example after storage at 25°C for at least 2 months, or after storage at 40°C ± 2°C for 1 month, and preferably has one or more of the following characteristics: relative to the initial value on storage day 0,

[0097] (i) the main peak variation value is less than 1% as measured by SEC-HPLC, and / or the preparation has a purity greater than 96%, preferably greater than 97% or 98%;

[0098] (ii) the main peak variation value is less than 2% as measured by non-reduced CE-SDS method, and / or the preparation has a purity greater than 96%, preferably greater than 97% or 98%;

[0099] (iii) the sum of the changes in the components of the anti-CLDN18.2 / CD3 protein (main component, acidic component, and basic component) in the formulation does not exceed 40% and / or the change in the main component does not exceed 20% as measured by CEX-HPLC,

[0100] For example, after storage at 40°C ± 2°C for 1 month, the sum of the changes does not exceed about 40% (e.g., does not exceed 35%, 30%, 25%, 20%, 15%, 10%) or the change in the main component does not exceed 20% (e.g., does not exceed 15%, 12%, 10%, 8%), or

[0101] For example, after storage at 25°C for 2 months, the sum of the changes does not exceed about 20% (e.g., not more than 15%, 14%, 13%, 12%) or the main component changes do not exceed about 15% (e.g., not more than 10%, 8%, 7%, 6%, 5%);

[0102] (iv) the relative binding activity of the formulation against the CLDN18.2 / CD3 protein as measured by ELISA is 70%-130%, e.g., 90, 93, 95, 98, 100, 103, 105, 108, 110, 115, 120%, e.g., 90%-110%;

[0103] In preferred embodiments, the liquid formulations of the present invention are stable under shaking and / or repeated freeze-thaw cycles.

[0104] Preferably, the formulation is stable under shaking or repeated freezing and thawing, for example, after shaking at 650 rpm for 5 days at room temperature in the dark or after repeated freezing and thawing at -30°C / room temperature for 6 times, it has one or more of the following characteristics:

[0105] (i) the main peak variation value is less than 1% as measured by SEC-HPLC, and / or the preparation has a purity greater than 96%, preferably greater than 97%, 98%, or 99% purity;

[0106] (ii) the main peak variation value is less than 2% as measured by non-reduced CE-SDS method, and / or the preparation has a purity greater than 96%, preferably greater than 97% or 98%;

[0107] (iii) the sum of the changes in the components of the anti-CLDN18.2 / CD3 protein in the formulation (main component, acidic component, and basic component) does not exceed 2% as measured by CEX-HPLC;

[0108] (iv) the relative binding activity of the anti-CLDN18.2 / CD3 protein in the preparation as measured by ELISA is 70%-130%, for example, 90%-110%;

[0109] In one aspect, the liquid preparation of the present invention is a pharmaceutical preparation, preferably an injection, more preferably a subcutaneous injection or an intravenous injection. In one embodiment, the liquid preparation is an intravenous infusion.

[0110] On the other hand, the present invention provides a solid antibody formulation obtained by subjecting the liquid antibody formulation of the present invention to a solidification treatment. The solidification treatment is implemented by, for example, crystallization, spray drying, or freeze drying. In a preferred embodiment, the solid antibody formulation is, for example, in the form of a lyophilized powder injection. The solid antibody formulation can be reconstituted in an appropriate 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 appropriate solvent is selected from water for injection, an organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.

[0111] In one aspect, the present invention provides a delivery device comprising a liquid antibody formulation or a solid antibody formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a prefilled syringe comprising a liquid antibody formulation or a solid antibody formulation of the present invention, e.g., for intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous infusion.

[0112] In another aspect, the present invention provides a method for delivering an anti-CLDN18.2 / CD3 protein to a subject, such as a mammal, comprising administering to the subject a liquid antibody formulation or a solid antibody formulation of the present invention, for example, by using a delivery device such as a pre-filled syringe.

[0113] In another aspect, the present invention provides a use of the liquid antibody formulation or solid antibody formulation of the present invention for preparing a method for preventing or treating a tumor (e.g., cancer) in a subject, comprising administering to the subject an effective amount of the anti-CLDN18.2 x CD3 bispecific antibody or fragment thereof (preferably an antigen-binding fragment), pharmaceutical composition, pharmaceutical combination, or kit of the present invention.

[0114] The present invention also provides a method of administering the liquid antibody formulation or solid antibody formulation of the present invention, or a delivery device (eg, a prefilled syringe) or a drug comprising the liquid antibody formulation or solid antibody formulation to a subject.

[0115] Other embodiments of the present invention will become apparent by reference to the detailed description which follows.

[0116] BRIEF DESCRIPTION OF THE DRAWINGS

[0117] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0118] FIG1 shows a schematic diagram of the structure of the bispecific antibody used in the Examples.

[0119] FIG2 shows that the bispecific antibody of the present invention specifically kills CLDN18.2-positive gastric cancer cells NUGC-4.

[0120] FIG3 shows that the bispecific antibody specifically kills CLDN18.2-positive pancreatic cancer cells DAN-GCLDN18.2.

[0121] FIG4 shows that the bispecific antibody does not non-specifically kill cells negative for CLDN18.2 expression.

[0122] FIG5 shows the in vivo efficacy results of the bispecific antibody in the NUGC-4 gastric cancer humanized model.

[0123] FIG6 shows the in vivo efficacy results of the bispecific antibody in the DAN-G-CLDN18.2 pancreatic cancer humanized model.

[0124] FIG7 shows the PK of bispecific antibodies in mice.

[0125] Figure 8 shows the effect of changes in the pH value in the formulation, (A) protein content (UV method) change trend graph; (B) purity (SEC-HPLC method) change trend graph; (C) purity (nrCE-SDS method) change trend graph; (D) charge variant-acidic component (iCIEF method) change trend graph; (E) charge variant-main component (iCIEF method) change trend graph; (F) charge variant-basic component (iCIEF method) change trend graph.

[0126] Figure 9 shows the effects of different formulation compositions, (A) protein content (UV method) change trend graph; (B) purity (SEC-HPLC method) change trend graph; (C) purity (nrCE-SDS method) change trend graph; (D) charge variant-acidic component (iCIEF method) change trend graph; (E) charge variant-main component (iCIEF method) change trend graph; (F) polysorbate 80 content (HPLC-FLD method) change trend graph.

[0127] Detailed Description of the Invention

[0128] definition

[0129] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0130] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0131] The term "and / or" when used to link two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0132] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0133] The term "claudin" or "cldn" as used herein refers to the most important scaffold protein that determines the structure of tight junctions between cells. It participates 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 their distribution is mainly on the lateral surface of epithelial cells and on the basal cell plasma membrane. Different claudin proteins have their own specific expression in different tissues. Among them, the claudin18 (cldn18) gene is located at 3q22.3, has a molecular weight of 24kDa, contains 261 amino acid residues, and is a member of the claudins superfamily. Its protein structure includes two extracellular loops and four transmembrane regions. The two isoforms of human CLDN18, or Claudin18, are Claudin18.1 (UniProt ID: P56856-1) and Claudin18.2 (UniProt ID: P56856-2). Their primary sequences differ only in the amino acid residues at certain positions from the N-terminal signal peptide to the extracellular loop 1 (Loop 1). Specifically, in the extracellular loop 1, CLDN18.1 and CLDN18.2 differ by only eight amino acids. The two CLDN18 isoforms also share very high sequence homology across species. The extracellular loop 1 of CLDN18.2 is completely identical in sequence across species, including humans, mice, and macaques, and the homology between human and mouse CLDN18.2 proteins reaches 84%, indicating extreme sequence conservation of the CLDN18.2 protein (O. Tureci et al., Gene 481:83-92, 2011). CLDN18.2 or any variants and isoforms thereof can be isolated from cells or tissues naturally expressing them, or recombinantly produced using techniques well known in the art and / or those described herein. In one embodiment, the CLDN18.2 described herein is human CLDN18.2.

[0134] As used herein, the terms "anti-CLDN18.2 antibody," "anti-CLDN18.2," "CLDN18.2 antibody," or "antibody that binds to CLDN18.2" refer to antibodies that bind to (human) CLDN18.2 with sufficient affinity to allow the antibody to 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), thin-layer biofilm interferometry (BLI), MSD assay, surface plasmon resonance (SPR), or flow cytometry.

[0135] As used herein, the term "CD3" refers to an antigen expressed on T cells as part of a multimolecular T cell receptor (TCR) and composed of a homodimer or heterodimer formed by two of the following four receptor chains: CD3-ε, CD3-δ, CD3-ζ, and CD3-γ. Human CD3-εn (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, the CD3 described herein refers to CD3 from humans or cynomolgus monkeys.

[0136] As used herein, the term "antibody that binds to CD3" or "anti-CD3 antibody" includes antibodies and antigen-binding fragments thereof that specifically recognize or bind to a single CD3 subunit (e.g., epsilon, delta, gamma, or zeta), as well as antibodies and antigen-binding fragments thereof that specifically recognize or bind to a dimeric complex of two CD3 subunits (e.g., gamma / epsilon, delta / epsilon, and zeta / zeta CD3 dimers) and bind to them. The antibodies and antigen-binding fragments of the present invention can bind to soluble CD3, bound CD3, and / or cell-surface expressed CD3. Soluble CD3 includes native CD3 protein and recombinant CD3 protein variants, for example, monomeric and dimeric CD3 structures that lack a transmembrane region or are otherwise not bound to a cell membrane. The present invention provides antibodies that bind to human and cynomolgus monkey CD3 with low or undetectable binding affinity, thereby being able to activate human and cynomolgus monkey T cells. In some embodiments, the binding is measured, for example, by radioimmunoassay (RIA), biofilm thin layer interferometry (BLI), MSD assay, or surface plasmon resonance (SPR) or flow cytometry.

[0137] The term "cell surface expressed CD3" refers to one or more CD3 proteins that are expressed on the surface of a cell, either 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 accessible to the antigen-binding portion of an antibody. "Cell surface expressed CD3" encompasses CD3 proteins that are included within the context of a functional T cell receptor in the cell membrane. The term "cell surface expressed CD3" encompasses CD3 proteins that are expressed as part of a homodimer or heterodimer (e.g., δ / ε, γ / ε, and ζ / ζ CD3 dimers) on the cell surface.

[0138] 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 can also include natural killer cells, macrophages, granulocytes, plasma cells or B cells (lymphocytes).

[0139] As used herein, the term "antibody" is used in the broadest sense to refer to a protein that contains an antigen binding site, encompassing natural antibodies and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies.

[0140] A "bispecific" antibody is an antibody having two antigen-binding sites, each of which binds to a different epitope of the same antigen or to different epitopes of different antigens. In one embodiment, a bispecific antibody has binding specificities for a first antigen and a second antigen.

[0141] Bispecific antibody formats include IgG-like and non-IgG-like antibodies (Fan et al. (2015) Journal of Hematology & Oncology. 8:130). The most common IgG-like antibody type contains two Fab regions and an Fc region, with the heavy and light chains of each Fab region derived from separate monoclonal antibodies. Non-IgG-like bispecific antibodies lack an 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 together by peptide linkers, chemical coupling, non-covalent bonds, or other means. These formats include bispecific T-cell engagers (BiTEs).

[0142] Any bispecific antibody format or technology can be used to prepare the bispecific antibodies of the present invention. For example, an antibody or fragment thereof having a first antigen binding specificity can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) 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, but are not limited to, scFv-based or double antibody bispecific formats, IgG-scFv fusion, dual variable domains (DVD)-Ig, quadroma, knobs-into-holes, common light chains (e.g., common light chains with knobs into holes, etc.), CrossMab, CrossFab, (SEED) body, Duobody, IgG1 / IgG2, dual-action Fab (DAF)-IgG, and Mab 2 Bispecific formats.

[0143] The term "linker" as used herein refers to any molecule that enables direct connection of the different parts of a bispecific antibody. Examples of linkers establishing covalent linkages between the different antibody moieties include peptide linkers and non-protein polymers, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol.

[0144] The term "peptide linker" according to the present invention refers to an amino acid sequence, wherein the sequence connects the amino acid sequence of the first part of the antibody to the second part of the antibody. For example, a peptide linker can connect the first (variable and / or binding) domain of an antibody to a second (variable and / or binding) domain. For example, a peptide linker can also connect a part of an antibody to another part of an antibody, such as connecting an antigen-binding domain to an Fc domain or a fragment thereof. Preferably, the peptide linker has a length that is sufficient to connect two entities in a manner such that they maintain their conformations relative to each other so as not to hinder the desired activity.

[0145] Peptide linkers may or may not consist essentially of the following 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) n G, wherein n is an integer of at least 1 (and preferably 2, 3, 4, 5, 6, 7, 8, 9, 10). Useful linkers also include glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.

[0146] The term "valent" according to the present invention denotes the presence of a specified number of binding sites in an antibody molecule. Thus, the terms bivalent, trivalent, and tetravalent denote the presence of two, three, or four binding sites, respectively, in an antibody construct. The bispecific antibodies according to the present invention are at least bivalent and can be multivalent, e.g., bivalent, trivalent, tetravalent, or hexavalent.

[0147] As used herein, the term "binding domain" refers to any portion of a bispecific antibody that binds to a specific target or antigen. A binding domain is an antigen-binding site. A binding domain can be, for example, an antibody or 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.

[0148] In certain exemplary embodiments of the present invention, each antigen-binding domain of a bispecific antibody includes 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 represented by the prefix "A1," and the VH, VL, or CDR of the second antigen-binding domain may be represented by 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 (HCDRs) 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.

[0149] The term "antibody fragment" includes a portion of an intact antibody. In a preferred embodiment, the antibody fragment is an antigen-binding fragment.

[0150] An "antigen-binding fragment" refers to a molecule, other than an intact antibody, that comprises a portion of an intact antibody and binds to the antigen to which 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 antibodies; single-chain antibodies (e.g., scFv); single-domain antibodies such as VHH; diabodies or fragments thereof; or camelid antibodies.

[0151] The term "antigen" refers to a molecule that triggers an immune response. This immune response may involve the production of antibodies or the activation of specific immune cells, or both. The skilled artisan will understand that any macromolecule, including essentially all proteins or peptides, can be used as an antigen. In addition, antigens can 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.

[0152] An "antibody that binds to the same or overlapping epitope as a reference antibody" is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay, whereas conversely, the reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay.

[0153] An antibody that competes with a reference antibody for binding to its antigen is an antibody that blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the reference antibody to its antigen in a competition assay. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90% or 95% or more of the binding of the antibody to its antigen in a competition assay. Numerous types of competitive binding assays can be used to determine whether one antibody competes with another, such as solid phase direct or indirect radioimmunoassays (RIAs), solid phase direct or indirect enzyme immunoassays (EIAs), and sandwich competition assays.

[0154] 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 determining affinity are known in the art.

[0155] An antibody that exhibits the same or similar binding affinity and / or specificity as a reference antibody is an antibody that has at least 50%, 60%, 70%, 80%, 90% or more than 95% of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0156] "Complementarity determining region" or "CDR region" or "CDR" is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of a number of well-known antibody CDR assignment systems, including, for example, Chothia based on the three-dimensional structure of antibodies 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)), Kabat based on antibody sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Edition, US Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), International ImMunoGeneTics database (IMGT) (on the World Wide Web at imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures.

[0157] For example, according to different CDR definition schemes, the residues of each CDR are as follows.

[0158] A CDR can also be identified based on having the same Kabat numbering position as a reference CDR sequence (eg, any of the exemplary CDRs of the invention).

[0159] Unless otherwise indicated, in the present invention, the term "CDR" or "CDR sequence" encompasses CDR sequences determined in any of the above-mentioned ways.

[0160] Unless otherwise indicated, in the present invention, when referring to residue positions in the variable region of an antibody (including heavy chain variable region residues and light chain variable region residues), the numbering refers to the position according to 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)).

[0161] In one embodiment, the heavy chain variable region CDRs of the antibodies of the present invention are determined according to the following rules:

[0162] VH CDR1 was determined according to the AbM rule; and VH CDR2 and 3 were both determined according to the Kabat rule.

[0163] In one embodiment, the light chain variable region CDRs of an antibody of the invention are determined according to the Kabat rules.

[0164] In one embodiment, the heavy chain variable region CDRs of the antibodies of the invention are determined according to the following rules: VH CDR1 is determined according to the AbM rule; and VH CDR2 and 3 are both determined according to the Kabat rule; and the light chain variable region CDRs are determined according to the Kabat rule.

[0165] It should be noted that the boundaries of the CDRs of the variable regions of the same antibody obtained based on different assignment systems may be different. That is, the CDR sequences of the variable regions of the same antibody defined under different assignment systems may be different. Therefore, when referring to antibodies defined by specific CDR sequences defined in the present invention, the scope of the antibodies also covers antibodies whose variable region sequences contain the specific CDR sequences, but whose claimed CDR boundaries are different from the specific CDR boundaries defined in the present invention due to the application of different schemes (e.g., different assignment system rules or combinations).

[0166] Antibodies with different specificities (i.e., different binding sites for different antigens) have different CDRs (under the same assignment system). However, although CDRs are different between antibodies, only a limited number of amino acid positions in the CDRs are directly involved in antigen binding. Using at least two of the Kabat, Chothia, AbM, Contact and North methods, the minimum overlapping region can be determined, thereby providing a "minimum binding unit" for antigen binding. The minimum binding unit can be a sub-portion of a CDR. As will be appreciated by those skilled in the art, the residues of the remainder of the CDR sequence can be determined by the structure and protein folding of the antibody. Therefore, the present invention also contemplates variants of any CDR provided herein. For example, in a variant of a CDR, the amino acid residues of the minimum binding unit can remain unchanged, while the remaining CDR residues defined according to Kabat or Chothia can be replaced by conservative amino acid residues.

[0167] The term "Fc region" is used herein to define the constant regions of CH2 and CH3 of an immunoglobulin heavy chain, and the term includes native sequence Fc regions and variant Fc regions. The native Fc region can bind to different Fc receptors on the surface of immune cells, thereby enabling CDC\ADCC\ADCP effector functions. Such effector functions generally require that the Fc region be combined with a binding domain (e.g., an antibody variable domain). In some embodiments, the Fc region is mutated to enhance its CDC\ADCC\ADCP effector functions. In some embodiments, the Fc region is mutated to weaken or delete its CDC\ADCC\ADCP effector functions.

[0168] An "IgG-type antibody" refers to an antibody whose heavy chain constant region belongs to the IgG type. All antibodies of the same type have the same heavy chain constant region, while antibodies of different types differ in their heavy chain constant regions. For example, an antibody of the IgG4 type refers to an antibody whose heavy chain constant region is derived from IgG4, and an antibody of the IgG1 type refers to an antibody whose heavy chain constant region is derived from IgG1.

[0169] "Humanized" antibody refers to an antibody comprising amino acid residues from non-human CDRs and amino acid residues from people's FRs. In some embodiments, a humanized antibody will comprise substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all of the FRs correspond to those of people's antibodies. Humanized antibodies optionally can comprise at least a portion of an antibody constant region derived from a people's antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to a humanized antibody.

[0170] "Human antibody" or "fully human antibody" or "fully human antibody" are used interchangeably to refer to an antibody having an amino acid sequence that corresponds to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human source using a human antibody library or other human antibody encoding sequence. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0171] As used herein, the term "binding" or "specific binding" means that the binding effect 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 determined by enzyme-linked immunosorbent assay (ELISA) or conventional binding assays known in the art, such as by radioimmunoassay (RIA) or thin-layer interferometry or MSD assays or surface plasmon resonance (SPR).

[0172] The term "therapeutic agent" as used herein encompasses any substance effective in preventing or treating tumors, such as cancer, including chemotherapeutic agents, cytokines, angiogenesis inhibitors, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0173] 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.

[0174] "Chemotherapeutic agents" include chemical compounds useful in treating cancer or immune system disorders.

[0175] The term "small molecule drug" refers to low molecular weight organic compounds that are capable of regulating biological processes. "Small molecules" are defined as molecules with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 10 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 mimics. As therapeutic agents, small molecules can be more cell-permeable, less susceptible to degradation, and less prone to eliciting an immune response than macromolecules.

[0176] As used herein, the term "immunomodulator" refers to a natural or synthetic agent or drug that inhibits or modulates an immune response. The immune response can be a humoral response or a cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0177] The term "effective amount" refers to that amount or dosage of an antibody or fragment or composition or combination of the present invention which, after single or multiple doses, produces the desired effect in a patient in need of treatment or prevention.

[0178] A "therapeutically effective amount" is an amount effective to achieve the desired therapeutic outcome at the desired dosage and for the desired period of time. A therapeutically effective amount is also an amount in which any toxic or deleterious effects of the antibody or antibody fragment or composition or combination are outweighed by the therapeutically beneficial effects. A "therapeutically effective amount" preferably inhibits a measurable parameter (e.g., tumor volume) by at least about 40%, even more preferably by at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% relative to an untreated subject.

[0179] A "prophylactically effective amount" refers to an amount effective to achieve the desired preventive result, at the required dosage and for the required period of time. Typically, a prophylactic amount will be less than a therapeutically effective amount because a prophylactic dose is used in a subject prior to or at an earlier stage of disease.

[0180] Calculation of sequence identity between sequences is performed as follows.

[0181] To determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment or non-homologous sequences can be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, then the molecules are identical at that position.

[0182] Mathematical algorithms can be used to compare sequences and calculate percent identity between two sequences. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http: / / www.gcg.com), which has been integrated into the GAP program in the GCG software package. The Blossum 62 matrix or the PAM250 matrix is ​​used, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 is used, and a length weight of 1, 2, 3, 4, 5, or 6 is used. In another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http: / / www.gcg.com). A particularly preferred parameter set (and one that should be used unless otherwise specified) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or nucleotide sequences can also be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, a gap penalty of 4). Additionally or alternatively, the nucleic acid sequences and protein sequences described herein can be further used as "query sequences" to perform searches against public databases to, for example, identify other family member sequences or related sequences.

[0183] As used herein, the term "hybridizes under stringent conditions (e.g., low stringency, medium stringency, high stringency, or very high stringency conditions)" describes hybridization and washing conditions. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6, incorporated by reference. Aqueous and non-aqueous methods are described in the reference and either method can be used. Preferred hybridization conditions mentioned herein are as follows: 1) low stringency hybridization conditions are 6X sodium chloride / sodium citrate (SSC) at about 45°C, followed by at least two washes in 0.2X SSC, 0.1% SDS at 50°C (the temperature of the washes can be increased to 55°C for low stringency conditions); 2) moderate stringency hybridization conditions are 6X SSC at about 45°C, followed by one or more washes in 0.2X SSC, 0.1% SDS at 60°C; 3) high stringency hybridization conditions are 6X SSC at about 45°C, followed by one or more washes in 0.2X 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 in 0.2X SSC, 0.1% SDS at 65°C. Very high stringency conditions (4) are the preferred conditions and the one that should be used unless otherwise specified.

[0184] The term "anti-tumor effect" refers to a biological effect that can be demonstrated by various means, including but not limited to, for example, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival.

[0185] The terms "tumor" and "cancer" are used interchangeably herein to encompass both solid tumors and hematological tumors.

[0186] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. In certain embodiments, cancers suitable for treatment by the antibodies of the invention include gastric cancer, pancreatic cancer, or gastroesophageal junction cancer, including metastatic forms of those cancers.

[0187] The term "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues. The terms "cancer," "cancerous," and "tumor" are not mutually exclusive when referred to herein.

[0188] The term "pharmaceutical excipient" refers to a diluent, adjuvant (eg, Freund's adjuvant (complete and incomplete)), excipient, carrier, stabilizer, or the like, which is administered together with the active substance.

[0189] The term "antibody preparation" refers to a preparation that is in a form that allows the biological activity of the antibody as an active ingredient to be effectively exerted and does not contain other components that are unacceptably toxic to the subject to whom the preparation is to be administered. Such antibody preparations are generally sterile. Typically, a pharmaceutically acceptable excipient is included in the antibody preparation. A "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a subject mammal so that an effective dose of the active ingredient used in the preparation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.

[0190] The term "anti-CLDN18.2 / CD3 formulation," also referred to herein as the "antibody formulation of the present invention," refers to a preparation comprising an anti-CLDN18.2 / CD3 protein as an active ingredient and a pharmaceutically acceptable excipient. After combining the anti-CLDN18.2 / CD3 protein with the pharmaceutically acceptable excipient, the anti-CLDN18.2 / CD3 protein as the active ingredient is suitable for therapeutic or prophylactic administration to humans or non-human animals. The antibody formulation of the present invention can be prepared, for example, as an aqueous liquid formulation, e.g., ready for use in a prefilled syringe, or as a lyophilized formulation that is reconstituted (i.e., reconstituted) by dissolving and / or suspending in a physiologically acceptable solution immediately prior to use. In some embodiments, the anti-CLDN18.2 / CD3 protein formulation is in the form of a liquid formulation.

[0191] A "stable" antibody formulation means that the antibody in the formulation maintains an acceptable degree of physical and / or chemical stability after storage under specific conditions, after shaking, or after repeated freeze-thaw cycles. Although the antibody contained in the antibody formulation may not maintain 100% of its chemical structure after storage, shaking, or repeated freeze-thaw cycles, the antibody formulation is generally considered "stable" if 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 formulations of the present invention exhibit low to undetectable antibody aggregation, degradation, or chemical modification during manufacturing, preparation, transportation, and long-term storage, thereby exhibiting minimal or even no loss of biological activity of the anti-CLDN18.2 / CD3 protein, and exhibit high stability. In some embodiments, the anti-CLDN18.2 / CD3 protein formulations of the present invention substantially retain their physical and chemical stability after storage, shaking, and / or repeated freeze-thaw cycles. Preferably, the liquid formulation of the present invention is stable at room temperature or at 40°C for at least 2 weeks, and / or at 25°C for at least 2 months, and / or at 2-8°C for at least 24 months. Preferably, the liquid formulation of the present invention is stable after shaking at 650 rpm for 5 days at room temperature in the dark and / or after 1-6 freeze-thaw cycles at -30°C / room temperature.

[0192] A variety of analytical techniques are known in the art for determining 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. Alternatively, accelerated stability testing can be used. In some embodiments, stability testing is performed by subjecting the antibody formulation to various stress tests. These tests can represent extreme conditions that the formulated antibody formulation may encounter during manufacturing, storage, or transportation, or conditions that may accelerate the instability of the antibody in the antibody formulation during non-manufacturing, storage, or transportation. For example, the formulated anti-CLDN18.2 / CD3 protein formulation can be filled into glass vials to test the antibody stability under high temperature stress. For another example, the formulated anti-CLDN18.2 / CD3 protein formulation can be filled into a glass vial and shaken at 650 rpm for 5 days at room temperature in the dark, and then the stability of the antibody can be tested. For another example, the formulated anti-CLDN18.2 / CD3 protein formulation can be filled into a glass vial and repeatedly frozen and thawed at -30°C / room temperature for 1-6 times, and then the stability of the antibody can be tested. In one embodiment, freezing below -30°C for 1 day followed by thawing at room temperature constitutes one freeze-thaw cycle.

[0193] After a period of storage, or after a period of shaking, or after repeated freezing and thawing, the formulation does not show aggregation, precipitation, turbidity and / or denaturation; or shows very little aggregation, precipitation, turbidity and / or denaturation, then the antibody can be considered to "maintain its physical stability" in the formulation. Since the aggregation of antibodies in the formulation can potentially lead to an increased immune response in the patient, thereby causing safety issues. Therefore, it is necessary to minimize or prevent the aggregation of antibodies in the formulation. Light scattering can be used to measure visible aggregates in the formulation. SEC-HPLC can be used to measure soluble aggregates in the formulation. In addition, the stability of the formulation can be indicated by visual inspection of the appearance, color and / or clarity of the formulation, or by detecting the turbidity of the formulation by the OD350nm method, or by measuring the purity of the formulation by non-reduced CE-SDS method. In one embodiment, the stability of the formulation is measured by measuring the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, or after shaking, or after repeated freezing and thawing, wherein the higher the percentage of antibody monomers in the formulation, the higher the stability of the formulation.

[0194] An "acceptable level" of physical stability can mean that at least about 90% of anti-CLDN18.2 / CD3 protein monomers are detected in the formulation after storage at a specific temperature for a specific time, after shaking, or after repeated freeze-thaw cycles. In some embodiments, an acceptable level of physical stability means at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of anti-CLDN18.2 / CD3 protein monomers after storage at a specific 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 evaluating physical stability, the specific temperature at which the pharmaceutical formulation is stored can be any temperature between about -80°C and about 45°C, for example, 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, if at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of anti-CLDN18.2 / CD3 protein monomers are detected after storage at about 40°C ± 2°C for one month or four weeks, the pharmaceutical formulation is considered stable. If at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of anti-CLDN18.2 / CD3 protein monomers are detected after 2 months of storage at about 25° C., the pharmaceutical formulation is considered stable. If at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of anti-CLDN18.2 / CD3 protein monomers are detected after 9 months of storage at about 5° C., the pharmaceutical formulation is considered stable.

[0195] After a period of storage, or after a period of vibration or after repeated freezing and thawing, if the antibody in the formulation does not show significant chemical changes, the antibody can be considered to "maintain its chemical stability" in the formulation. Most chemical instabilities arise from the formation of covalently modified forms of the antibody (e.g., charge variants of the antibody). For example, basic variants can be formed by aspartic acid isomerization, N and C-terminal modification; acidic variants can be produced by deamidation, sialylation and saccharification. Chemical stability can be assessed by detecting and / or quantifying chemically altered forms of the antibody. For example, the 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 determining the charge variant percentage change value of the antibody in the formulation after storage at a specific temperature for a specific time, or after vibration or repeated freezing and thawing, wherein the smaller the change value, the higher the stability of the formulation.

[0196] An "acceptable level" of chemical stability can mean that after storage at a specific temperature for a specific period of time, or after shaking for a period of time, or after repeated freezing and thawing, the percentage change of the charge variant (e.g., the main component or the acidic component or the basic component) in the formulation does not exceed 40%, for example, not more than 30%, not more than 20%; or the sum of the percentage change of the charge variant (main component, acidic component and basic component) does not exceed 60%, for example, not more than 50%, not more than 30%. In some embodiments, an acceptable level of chemical stability can be demonstrated by a percentage change of no more than about 50%, 40%, 30%, 20%, or 15% for the principal component charge variant, or a percentage change of no more than about 60%, 50%, or 30% for the sum of the charge variants after storage at a specific 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 evaluating chemical stability, the pharmaceutical formulation can be stored at any temperature 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 to 8°C, about 5°C, about 25°C, or about 45°C. For example, a pharmaceutical formulation can be considered stable if the percent change in the principal 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 storage at 5°C for 24 months. A pharmaceutical formulation can also be considered stable if the percent change in the principal 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 storage at 25°C for 2 months. The pharmaceutical formulation may also be considered stable if the percent change in the principal component 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.

[0197] The term "lyophilized preparation" refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0198] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (eg, a lyophilized formulation) in a physiologically acceptable solution.

[0199] The term "room temperature" as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.

[0200] "Stress conditions" refers to an environment that is chemically and / or physically unfavorable for the antibody protein, which may lead to unacceptable instability of the antibody protein, for example, high temperature, shaking, freeze-thaw. "High temperature stress" refers to storing the antibody formulation at room temperature or even higher temperature (e.g., 40°C ± 2°C) for a period of time. The stability of the antibody formulation can be checked by accelerated high temperature stress testing.

[0201] As used herein, the term "parenteral administration" refers to administration other than enteral and topical administration, typically by injection or infusion, and includes, but is 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 present invention is administered to a subject parenterally. In one embodiment, the anti-CLDN18.2 / CD3 protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

[0202] I. Antibody Preparation

[0203] 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, optionally comprising a chelating agent. The pH of the antibody formulation is about 5.0-6.0. In a preferred embodiment, the liquid antibody formulation of the present invention is in the form of an injectable formulation.

[0204] (i) Anti-CLDN18.2 / CD3 Antibodies

[0205] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the present invention bind to CLDN18.2 (e.g., human CLDN18.2) with high affinity. In some embodiments, the antibodies of the present 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 thereof of the present invention have a higher binding affinity to human CLDN18.2 than known CLDN18.2 antibodies, such as Zolbetuximab (abbreviated as Zmab). In some embodiments, the anti-CLDN18.2 x CD3 bispecific antibodies of the present invention bind to CD3 (e.g., human CD3 or cynomolgus monkey CD3) with a desired affinity (e.g., lower affinity). In some embodiments, the antibodies of the present invention are capable of binding to both human CD3 and cynomolgus monkey CD3. In some embodiments, the affinity of the antibodies is determined by thin-layer interferometry or surface plasmon resonance.

[0206] In some embodiments, the anti-CLDN18.2 / CD3 bispecific antibodies of the present invention are capable of binding to CLDN18.2 on the surface of tumor cells and CD3 on the surface of effector cells. In some embodiments, the binding is detected by flow cytometry.

[0207] In some embodiments, the bispecific antibody of the present 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.

[0208] In a preferred embodiment of the present invention, the first antigen binding domain comprises three complementarity determining regions (A1-HCDRs) from the heavy chain variable region, namely A1-HCDR1, A1-HCDR2 and A1-HCDR3.

[0209] In a preferred embodiment of the present invention, the first antigen binding domain comprises three complementarity determining regions (A1-LCDR) from the light chain variable region, A1-LCDR1, A1-LCDR2 and A1-LCDR3.

[0210] In some embodiments, the first antigen binding domain comprises three complementarity determining regions from a heavy chain variable region (A1-HCDR) and three complementarity determining regions from a light chain variable region (A1-LCDR).

[0211] In some aspects, the first antigen-binding domain comprises a heavy chain variable region (A1-VH). In some aspects, the first antigen-binding domain comprises a light chain variable region (A1-VL). In some aspects, 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 complementary determining regions (A1-HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (A1-LCDR) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0212] In some embodiments, A1-VH

[0213] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 4; or

[0214] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 4; or

[0215] (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 4, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0216] In some embodiments, A1-VL

[0217] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 9; or

[0218] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 9; or

[0219] (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 9, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0220] In a preferred embodiment of the present invention, the second antigen binding domain comprises three complementarity determining regions (A2-HCDRs) from the heavy chain variable region, namely A2-HCDR1, A2-HCDR2 and A2-HCDR3.

[0221] In a preferred embodiment of the present invention, the second antigen binding domain comprises three complementarity determining regions (A2-LCDR) from the light chain variable region, A2-LCDR1, A2-LCDR2 and A2-LCDR3.

[0222] In some embodiments, the second antigen binding domain comprises three complementarity determining regions from a heavy chain variable region (A2-HCDR) and three complementarity determining regions from a light chain variable region (A2-LCDR).

[0223] In some aspects, the second antigen-binding domain comprises a heavy chain variable region (A2-VH). In some aspects, the second antigen-binding domain comprises a light chain variable region (A2-VL). In some aspects, 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 complementary determining regions (A2-HCDR) from the heavy chain variable region, HCDR1, HCDR2, and HCDR3. In some embodiments, the light chain variable region comprises three complementary determining regions (A2-LCDR) from the light chain variable region, LCDR1, LCDR2, and LCDR3.

[0224] In some embodiments, A2-VH

[0225] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 22, 30 or 32; or

[0226] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 22, 30 or 32; or

[0227] (iii) an amino acid sequence comprising one or more (preferably no more than 10, more preferably no more than 5, 4, 3, 2, 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, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0228] In some embodiments, A2-VL

[0229] (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 27; or

[0230] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 27; or

[0231] (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 27, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions.

[0232] In some embodiments, the three complementarity determining regions (A2-HCDRs) of the invention, A2-HCDR1, A2-HCDR2, and A2-HCDR3, from A2-VH are

[0233] (i) three complementarity determining regions A2-HCDR1, A2-HCDR2 and A2-HCDR3 contained in the VH as shown in SEQ ID NO: 22, 30 or 32, or

[0234] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three A2-HCDR regions relative to the sequence of (i).

[0235] In some embodiments, the three complementarity determining regions (A2-LCDRs) of the invention, A2-LCDR1, A2-LCDR2, and A2-LCDR3, from A2-VL are

[0236] (i) three complementarity determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as shown in SEQ ID NO: 27,

[0237] (ii) a sequence comprising at least one and no more than 5, 4, 3, 2 or 1 amino acid changes (preferably amino acid substitutions, preferably conservative substitutions) in the three A2-LCDR regions relative to the sequence of (i).

[0238] 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.

[0239] 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 alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 20 or 31.

[0240] 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 alterations (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence of SEQ ID NO: 21 or 29.

[0241] In some embodiments, A2-LCDR1 comprises the amino acid sequence of SEQ ID NO: 24, or consists of the amino acid sequence, 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.

[0242] In some embodiments, A2-LCDR2 comprises the amino acid sequence of SEQ ID NO: 25, or consists of said amino acid sequence, 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.

[0243] In some embodiments, A2-LCDR3 comprises the amino acid sequence of SEQ ID NO: 26, or consists of the amino acid sequence, 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.

[0244] In some embodiments, the bispecific antibodies of the present invention further comprise a heavy chain constant region. In some embodiments, the bispecific antibodies of the present invention further comprise a light chain constant region. In some embodiments, the bispecific antibodies of the present invention further comprise a heavy chain constant region and a light chain constant region. In some embodiments, the bispecific antibodies of the present invention comprise two heavy chain constant regions, one of which, A1-HC, is linked to the heavy chain variable region A1-VH of the first antigen-binding domain to form the heavy chain that binds to CDLN18.2, and the other, A2-HC, is linked to the heavy chain variable region A2-VH of the second antigen-binding domain to form the heavy chain that binds to CD3. In some embodiments, the bispecific antibodies of the present invention comprise two light chain constant regions, one of which, A1-LC, is linked to the light chain variable region A1-VL of the first antigen-binding domain to form the light chain that binds to CDLN18.2, and the other, A2-LC, is linked to the light chain variable region A2-VL of the second antigen-binding domain to form the light chain that binds to CD3. 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 the same as or different from A2-HC. In some embodiments, A1-LC may be the same as or different from A2-LC. In some embodiments, A1-HC is different from A2-HC, and A1-LC is different from A2-LC.

[0245] In some embodiments, the heavy chain constant region HC of the present invention is a heavy chain constant region of IgG1, IgG2, IgG3 or IgG4, preferably a heavy chain constant region of IgG1, such as a wild-type IgG1 heavy chain constant region or a mutant IgG1 heavy chain constant region (e.g., IgG1LALA). In some embodiments, the light chain constant region LC of the antibody of the present invention is a lambda or kappa light chain constant region.

[0246] In some preferred embodiments, the heavy chain constant region HC of the present invention is

[0247] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 5 or 23;

[0248] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 5 or 23; or

[0249] (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: 5 or 23.

[0250] In some preferred embodiments, the heavy chain constant region HC of the present invention is

[0251] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 33 or 34;

[0252] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 33 or 34; or

[0253] (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: 33 or 34.

[0254] In some embodiments, the antibody light chain constant region LC of the present invention is

[0255] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 10 or 28;

[0256] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 or 28; or

[0257] (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: 10 or 28.

[0258] In some embodiments of the present invention, the bispecific antibody of the present invention comprises a first antigen-binding domain and a second antigen-binding domain, wherein

[0259] 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 the three complementarity-determining regions A1-HCDR1, A1-HCDR2, and A1-HCDR3 contained in A1-VH as shown in SEQ ID NO:4, and the three complementarity-determining regions A1-LCDR1, A1-LCDR2, and A1-LCDR3 contained in VL as shown in SEQ ID NO:9;

[0260] The second antigen-binding domain comprises 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 three complementarity determining regions A2-LCDR1, A2-LCDR2 and A2-LCDR3 contained in A2-VL as shown in SEQ ID NO: 27.

[0261] In some specific embodiments of the present invention, the bispecific antibody of the present 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

[0262] The first antigen binding domain comprises A1-HCDR1, A1-HCDR2, A1-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 1, 2, and 3, respectively, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 6, 7, and 8, respectively; and

[0263] The second antigen binding domain comprises

[0264] (i) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25 and 26, respectively; or

[0265] (ii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25 and 26, respectively; or

[0266] (iii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively.

[0267] In some specific embodiments of the present invention, the bispecific antibody of the present 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

[0268] The first antigen-binding domain comprises A1-VH comprising, or consisting of, the amino acid sequence of SEQ ID NO:4, or an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical thereto, and A1-VL comprising, or consisting of, the amino acid sequence of SEQ ID NO:9, or an amino acid sequence that is at least 90% identical thereto;

[0269] The second antigen-binding domain comprises A2-VH having an amino acid sequence as shown in SEQ ID NO: 22, 30 or 32, or an amino acid sequence having at least 90% identity thereto, or consisting of said amino acid sequence, and A2-VL having an amino acid sequence as shown in SEQ ID NO: 27 and at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto, or consisting of said amino acid sequence.

[0270] In some specific embodiments of the present invention, the bispecific antibodies of the present invention are bispecific antibodies with an IgG-like structure, i.e., they comprise a heavy chain and a light chain that bind to CLDN18.2, as well as a heavy chain and a light chain that bind to CD3. 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), thereby forming a knob-into-hole structure. In some embodiments, the structure of the bispecific antibodies of the present invention is shown in Figure 1.

[0271] In some embodiments, the heavy chain of the CLDN18.2 binding moiety

[0272] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 37;

[0273] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 37; or

[0274] (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 the amino acid sequence selected from SEQ ID NO: 37.

[0275] In some embodiments, the light chain of the CLDN18.2 binding moiety

[0276] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 38;

[0277] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 38; or

[0278] (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 the amino acid sequence selected from SEQ ID NO: 38.

[0279] In some embodiments, the heavy chain of the CD3 binding moiety

[0280] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 39, 41 or 42;

[0281] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 39, 41 or 42; or

[0282] (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: 39, 41, or 42.

[0283] In some embodiments, the light chain of the CD3 binding moiety

[0284] (i) comprising or consisting of an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 40;

[0285] (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 40; or

[0286] (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 the amino acid sequence selected from SEQ ID NO: 40.

[0287] 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.

[0288] In preferred embodiments, the amino acid changes described herein occur in regions outside of the CDRs (e.g., in the FRs). More preferably, the amino acid changes described herein occur in regions outside of the heavy chain variable region and / or outside of the light chain variable region. In some embodiments, the amino acid changes described herein occur in the Fc region of the heavy chain constant region of an antibody. In preferred embodiments, the amino acid changes in the Fc region weaken or eliminate the ADCC and / or CDC effects of the antibody.

[0289] In some embodiments, the substitution is a conservative substitution. A conservative substitution refers to the substitution of one amino acid with another amino acid within the same class, for example, the substitution of an acidic amino acid with another acidic amino acid, the substitution of a basic amino acid with another basic amino acid, or the substitution of a neutral amino acid with another neutral amino acid. Exemplary substitutions are shown in the following table:

[0290] In certain embodiments, the substitution occurs in the CDR region of the antibody. Typically, the variant obtained has modifications (e.g., improvements) in certain biological properties (e.g., increased affinity) relative to the parent antibody and / or will have substantially the same

[0291] Certain biological properties are retained. An exemplary substitutional variant is an affinity matured antibody.

[0292] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, complement-dependent cellular cytotoxicity, Fc receptor binding, and / or antibody-dependent cellular cytotoxicity. The Fc region variant can include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid change (e.g., substitution) at one or more amino acid positions.

[0293] In certain embodiments, it may be desirable to generate cysteine ​​engineered antibodies, eg, "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues.

[0294] In certain embodiments, the antibodies provided herein may be further modified to contain other non-proteinaceous moieties known in the art and readily available. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0295] In some embodiments, in the bispecific antibodies of the present invention, the first antigen-binding domain, or the heavy and light chain combination, that specifically binds to CLDN18.2 is fully human, and the second antigen-binding domain, or the heavy and light chain combination, that binds to CD3 is humanized.

[0296] In a preferred embodiment, the anti-CLDN18.2 / CD3 bispecific antibody in the antibody preparation of the present invention is the anti-CLDN18.2 / CD3 bispecific antibody disclosed in PCT application number PCT / CN2021 / 121286 (international application date: September 28, 2021).

[0297] The amount of the antibody or antigen-binding fragment thereof contained in the antibody formulation of the present invention may vary depending on the specific intended properties of the formulation, the specific environment, and the specific purpose for which the formulation is used. In some embodiments, the antibody formulation is a liquid formulation that may contain about 0.1-100 mg / ml, preferably about 0.1-10 mg / ml, for example, about 0.5, 1, 2, 3, 4, or 5 mg / ml of the anti-CLDN18.2 / CD3 bispecific antibody.

[0298] (ii) Buffer

[0299] Buffers are agents that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffer used in the formulations of the present invention can control the pH of the formulations of the present invention to a pH range of about 5.0-6.0, for example, a pH of about 5.0-5.5. In some specific embodiments, the antibody formulations of the present invention have 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 formulations of the present invention have a pH of 5.2 ± 0.2 or 5.5 ± 0.2, preferably a pH of 5.2.

[0300] In some embodiments, the formulation of the present invention comprises a buffer system selected from the group consisting of: histidine-histidine hydrochloride buffer system, citric acid-sodium citrate buffer system, acetic acid-sodium acetate buffer system, phosphate buffer system, preferably histidine-histidine hydrochloride buffer system.

[0301] In some embodiments, the buffer used in the formulations of the present invention is a histidine buffer, in particular a buffer system consisting of histidine and histidine hydrochloride. In some embodiments, the concentration of histidine in the histidine buffer of the present invention is about 0.01-10 mg / ml mM, in particular about 0.1-1 mg / ml, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 1 mg / ml. In one embodiment, the formulation of the present invention comprises 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.

[0302] (iii) Stabilizer

[0303] Suitable stabilizers for use in the present invention can be selected from sugars, polyols, amino acids, and combinations thereof. Sugars used as stabilizers include, but are not limited to, sucrose, trehalose, maltose, and combinations thereof. Polyols used as stabilizers include, but are not limited to, sorbitol, mannitol, or combinations thereof. Amino acids used as stabilizers include, but are not limited to, arginine, arginine hydrochloride, methionine, glycine, proline, and combinations thereof.

[0304] For example, in some embodiments, the stabilizer comprises one or more selected from the group consisting of:

[0305] - a polyol selected from sorbitol, mannitol, or a combination thereof, about 10-100 mg / ml, preferably 20-40 mg / ml, such as 25 mg / ml; or 40-60 mg / ml, such as 50 mg / ml;

[0306] - a sugar selected from sucrose, trehalose, maltose, or a combination thereof, e.g., about 10-100 mg / ml, preferably 30-60 mg / ml, e.g., 40 mg / ml;

[0307] - an amino acid selected from arginine hydrochloride, methionine, glycine, proline and combinations thereof, e.g., 20-200 mM, such as about 50-110 mM, e.g., 70-100 mM, in particular about 80-90 mM.

[0308] In one embodiment, the liquid formulation of the present invention comprises sucrose as a stabilizer. The amount of sucrose in the liquid formulation of the present invention may be about 10-100 mg / ml, preferably 30-60 mg / ml, for example 40 mg / ml.

[0309] 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 can be about 10-100 mg / ml, preferably 20-40 mg / ml, such as 25 mg / ml; or 40-60 mg / ml, such as 50 mg / ml.

[0310] 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 can be about 50-110 mM, for example, 70-100 mM, in particular about 80-90 mM, for example about 17.91 mg / ml arginine hydrochloride.

[0311] In one embodiment, liquid preparation of the present invention comprises a combination of sorbitol and arginine as a stabilizer. In this combination, sorbitol can be with about 10-60mg / ml, preferably 15-40mg / ml, for example, the amount of 20-35mg / ml exists, for example, can be about 20,21,22,23,24,25,26,27,28,29,30mg / ml. In this combination, arginine can be with about 70-100mM, especially the amount of about 85mM exists. Preferably, liquid preparation of the present invention comprises the sorbitol of about 20-30mg / ml and the arginine hydrochloride of about 15-20mg / ml. More preferably, liquid preparation of the present invention comprises the sorbitol of about 25mg / ml and the arginine hydrochloride of about 17.91mg / ml.

[0312] In one embodiment, liquid preparation of the present invention comprises a combination of sucrose and arginine as a stabilizer. In this combination, sucrose can be with about 10-60mg / ml, preferably 20-50mg / ml, for example, 30-40mg / ml amount exists, for example, can be about 30,32,34,36,38,40,42,44,46,48,50mg / ml. In this combination, arginine can be with about 70-100mM, especially about 85mM amount exists. Preferably, liquid preparation of the present invention comprises the sucrose of about 30-50mg / ml and the arginine hydrochloride of about 15-20mg / ml. More preferably, liquid preparation of the present invention comprises the sucrose of about 40mg / ml and the arginine hydrochloride of about 17.91mg / ml.

[0313] In one embodiment, the liquid formulation of the present invention comprises sorbitol as the sole stabilizing agent. In this embodiment, the amount of sorbitol in the liquid formulation of the present invention can be about 30-70 mg / ml, for example, 40-60 mg / ml. For example, sorbitol can be present in an amount of about 30, 35, 40, 45, 50, 55, 60, 65 or 70 mg / ml, preferably in an amount of about 50 mg / ml.

[0314] (iv) Surfactants

[0315] As used herein, the term "surfactant" refers to organic substances with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually a hydrocarbon chain for oil solubility and an ionic group for water solubility.

[0316] In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, for example, an alkyl poly(ethylene oxide). Specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbates, such as polysorbate 20, polysorbate 80, polysorbate 60, or polysorbate 40; poloxamers, and the like. In a preferred embodiment, polysorbate 80 is included as a surfactant in the liquid formulation of the present invention.

[0317] In some embodiments, surfactants that can be used in the liquid formulations of the present invention include, but are not limited to, polysorbate surfactants (eg, polysorbate 80, polysorbate 20), poloxamers, and polyethylene glycols.

[0318] The amount of surfactant contained in the antibody formulation of the present invention may vary depending on the specific purpose of the formulation, the specific environment, and the specific purpose of the formulation. In preferred embodiments, the formulation may contain about 0.01-5 mg / ml, preferably about 0.1-1 mg / ml, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / ml of a surfactant, particularly polysorbate 80, preferably about 0.2 mg / ml of polysorbate 80.

[0319] (v) Chelating agents

[0320] As used herein, the term "chelating agent" refers to a compound that can form a chelate with a central atom, and the stability of the complex is greatly increased due to the formation of the chelate.

[0321] In one embodiment, the chelate in the liquid formulation of the present invention is a carboxylic acid-type chelating agent. In one embodiment, the chelating agent is selected from disodium edetate, aminotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, and dihydroxyethylglycine. In one embodiment, the chelating agent is selected from disodium edetate.

[0322] In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the present invention is about 0.005-0.05 mg / ml. In one embodiment, the concentration of the chelating agent in the liquid antibody formulation of the present invention is about 0.008-0.018 mg / ml, for example, about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg / ml, preferably 0.01 mg / ml.

[0323] (vi) Other excipients

[0324] The antibody liquid formulations of the present invention may optionally contain other excipients. Such other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, and the like. These and other known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are well known in the art and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., ed., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro ed., Lippincott Williams & Wilkins (2005)".

[0325] II. Preparation of Formulations

[0326] The present invention provides stable formulations comprising anti-CLDN18.2 / CD3 antibodies. The anti-CLDN18.2 / CD3 antibodies used in the formulations of the present invention can be prepared using techniques known in the art for producing antibodies. For example, the antibodies can be recombinantly produced. In a preferred embodiment, the antibodies of the present invention are recombinantly produced in 293 cells or CHO cells.

[0327] The application of antibodies as active ingredients of drugs is now very extensive. The technology for purifying therapeutic antibodies to pharmaceutical grade is well 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 using ion exchange chromatography (anion IEX and / or cation CEX chromatography) after the 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, wherein a weak partitioning anion exchange resin is used after protein A affinity chromatography.

[0328] Generally, the antibody produced by recombinant production can be purified using conventional purification methods to provide a drug substance with enough repeatability and appropriate purity for the preparation of antibody preparations. For example, after the antibody is secreted into the culture medium from the recombinant expression cell, a commercially available protein concentration filter such as the ultrafiltration device of Amicon can be used to concentrate the supernatant from the expression system. Afterwards, the purification of the antibody can be carried out using modes such as chromatography, dialysis and affinity purification. Protein A is adapted to be used as an affinity ligand for purifying IgG1, IgG2 and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. After obtaining the antibody of sufficient purity, the preparation comprising the antibody can be prepared according to methods known in the art.

[0329] For example, the preparation can be carried out by the following steps: (1) after the fermentation is completed, the fermentation broth is centrifuged to clarify and remove impurities such as cells to obtain a supernatant; (2) using affinity chromatography (e.g., a protein A column with specific affinity for IgG1, IgG2, and IgG4 antibodies) to capture antibodies; (3) performing virus inactivation; (4) refining and purification (generally, CEX cation exchange chromatography can be used) to remove impurities in the protein; (5) virus filtration (to reduce the virus titer by, for example, 4 log10 or more); (6) ultrafiltration / diafiltration (which can be used to replace the protein with a formulation buffer that is conducive to its stability and concentrate it to a suitable concentration for injection). See, for example, B. Minow, P. Rogge, K. Thompson, BioProcess International, Vol. 10, No. 6, 2012, pp. 48–57.

[0330] III. Analytical Methods for Preparations

[0331] Stability studies for biological products generally include real-time stability studies (long-term stability studies) under actual storage conditions, accelerated stability studies, and forced condition testing. Stability studies should explore and optimize research conditions based on the study objectives and the product's characteristics. Stability study plans should include long-term, accelerated, and / or forced condition testing to address various influencing factors (such as temperature, repeated freeze-thaw cycles, and vibration). Accelerated and forced condition testing facilitates understanding of product stability under short-term deviations from storage conditions and extreme conditions, and provides supporting data for determining shelf life and storage conditions.

[0332] During storage, oscillation, or repeated freeze-thaw cycles, antibodies may aggregate, degrade, or be chemically modified, leading to antibody heterogeneity (including size and charge heterogeneity), aggregates, and fragments, thus affecting the quality of the antibody preparation. Therefore, it is necessary to monitor the stability of the antibody preparation.

[0333] There are many methods known in the art that can be used to detect the stability of antibody preparations. For example, the purity of antibody preparations and the aggregation level of antibodies can be analyzed by methods such as reduced CE-SDS, non-reduced CE-SDS and SEC-HPLC; charge variants in antibody preparations can be analyzed by ion exchange chromatography (IEX) and the like. In addition, the stability of the preparation can be quickly judged by visual inspection of the appearance of the preparation. OD can also be used. 350nm The turbidity of the preparation can be monitored by spectrophotometry, which can provide information about the amount of soluble and insoluble aggregates. In addition, changes in the protein content of the preparation can be monitored using ultraviolet spectrophotometry (UV).

[0334] Non-reducing CE-SDS is a method for measuring antibody purity using a capillary separation channel. In CE-SDS, protein migration is driven by the surface charge induced by SDS binding, which is proportional to the protein's molecular weight. Because all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved within the capillary's molecular sieving gel matrix. This method has been widely used to monitor the purity of denatured intact antibodies. Typically, in non-reducing CE-SDS, the test sample is mixed with SDS sample buffer and iodoacetamide. The mixture is then incubated at 68-72°C for approximately 10-15 minutes, cooled to room temperature, and centrifuged, with the supernatant analyzed. Protein migration is monitored using a UV detector, and the electropherogram is obtained. The purity of the antibody preparation can be calculated as the percentage of the main IgG peak area 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.

[0335] Size exclusion high performance liquid chromatography, or SEC-HPLC, is another important method for antibody standards and quality control. This method mainly separates molecules based on the size or hydrodynamic radius of the molecules. Through SEC-HPLC, antibodies can be separated into three main forms: high molecular weight form (HMMS), main peak (mainly antibody monomers), and low molecular weight form (LMMS). Antibody purity can be calculated as the percentage of the main peak area on the chromatogram to the sum of all peak areas. Through SEC-HPLC, the percentage of antibody monomers in the formulation can be measured, and information on the content of soluble aggregates and shear products can be given. For further description of the SEC-HPLC method, see, for example, J. Pharm. Sci., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986). In addition, see, 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.

[0336] Accelerated stability studies can be used to examine the stability properties of a product and facilitate the screening of stable pharmaceutical formulations. For example, a formulation sample can be placed at an elevated temperature, such as approximately 40°C ± 2°C or 25°C ± 2°C, for accelerated stability studies. In addition, an oscillation test or a repeated freeze-thaw test can be performed to test the stability properties of the product. For example, an oscillation test is performed by shaking at 650 rpm for 1-5 days at room temperature in the dark. For example, a product can be frozen below -30°C for one day and then thawed at room temperature as a freeze-thaw cycle, and a repeated freeze-thaw test can be performed, wherein 1-6 repeated freeze-thaw cycles can be implemented. Product stability test indicators may 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 the antibody can be tested. For example, the binding ability of the antibody in the formulation to its antigen molecule (TIGIT) can be tested. A variety of methods are known to those skilled in the art for quantifying the specific binding of antibodies to antigens, such as immunoassays, ELISA, etc.

[0337] Abbreviation Description Example

[0338] The anti-CLDN18.2 / CD3 bispecific antibody is an antibody independently developed by Innovent Biologics (Suzhou) Co., Ltd. and disclosed in PCT application number PCT / CN2021 / 121286.

[0339] In order to develop a simple and easy-to-use injection formulation suitable for long-term storage of this fully human antibody, forced stability tests at 40°C and accelerated stability tests at 25°C were conducted to investigate the effects of different pH values, stabilizer and surfactant contents on the quality of the antibody protein. Ultimately, a formulation that is conducive to its stability was selected. The materials and methods used throughout the research process are as follows:

[0340] Materials and methods

[0341] 1.1. Materials used in the formulation studies of the present invention

[0342] Note: N / A means not applicable.

[0343] 1.2. Instruments and Equipment Used in the Preparation Studies of the Present Invention

[0344] 1.3. Test items and methods for preparation stability

[0345] The main testing items during the entire study process include: (1) detection of appearance and presence of visible foreign matter; (2) determination of protein content in the preparation by ultraviolet method (UV method); (3) detection of turbidity of the preparation by OD350nm method; (4) determination of purity of the antibody preparation by size exclusion high performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of ​​the main peak to the sum of the areas of all peaks; (5) determination of purity of the antibody preparation by non-reduced sodium dodecyl sulfate capillary electrophoresis (non-reduced CE-SDS), expressed as the percentage of the area of ​​the main peak to the sum of the areas of all peaks; (6) determination of charge variants in the antibody preparation by CEX-HPLC, expressed as the percentage of the main component, acidic component and basic component; (7) determination of relative binding activity of anti-CLDN18.2 / CD3 to antigen in the antibody preparation by direct ELISA method; (8) determination of polysorbate 80 content in the antibody preparation by HPLC-FLD.

[0346] Visible foreign matter detection

[0347] According to the method described in the Pharmacopoeia of the People's Republic of China (2015 edition, General Chapter 0904 of Part Four "Inspection Method for Visible Foreign Matter" of the State Pharmacopoeia Committee, Beijing: China Medical Science and Technology Press, 2015), a clarity detector (produced by Tianjin Tianda Tianfa, model YB-2) and an insoluble particle detector (produced by Tianjin Tianda Tianfa, model GWJ-8) were used to inspect visible foreign matter in the samples.

[0348] Protein content determination

[0349] The protein content in the sample was determined using an ultraviolet spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800) or a multi-channel microspectrophotometer (manufactured by Thermo, USA, model Nanodrop 8000).

[0350] Turbidity measurement

[0351] An ultraviolet spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800) was used to measure the absorbance of the sample at 350 nm to determine the turbidity of the sample.

[0352] Purity (SEC-HPLC method)

[0353] Use size exclusion chromatography column for separation, the mobile phase is phosphate buffer (weigh 3.12g sodium dihydrogen phosphate dihydrate, 8.77g sodium chloride and 34.84g arginine, dissolve in ultrapure water, adjust the pH to 6.8 with hydrochloric acid and make up to 1000ml), the column protection liquid is 0.05% (w / v) NaN3, the injection volume is 50μl, the flow rate is 0.5ml / min, the acquisition time is 30 minutes, the column temperature is 25℃, and the detection wavelength is 280nm. Take the sample to be tested and dilute it to 2mg / ml with ultrapure water as the test solution. Take the preparation buffer solution and dilute it with the same treatment method as above as a blank solution. Take 50μl of the blank solution and the test solution and inject them into the liquid chromatograph to start detection.

[0354] Purity (non-reduced CE-SDS method)

[0355] Capillary gel electrophoresis was used for detection. The capillary was uncoated, 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 flushed 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 tested was diluted to 2.0 mg / ml with an appropriate amount of ultrapure water. 50 μl of the diluted sample was taken into a 1.5 ml centrifuge tube, and 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 and dissolved in 45 ml of ultrapure water, and the volume was adjusted to 50 ml to prepare citric acid-phosphate buffer. 200 μl of this buffer was accurately measured, and 80 μl of 10% (w / v) sodium dodecyl sulfate solution was added. The volume was made up to 1 ml with water and mixed to obtain the obtained product), 1 μl of internal standard (10 kDa protein, 5 mg / mL) (Beckman Coulter, Cat. No. 390953), and 5 μl of 250 mmol / L Prepare a NEM solution (weigh 62 mg of N-ethylmaleimide and dissolve it in 2 ml of ultrapure water). Mix thoroughly, heat at 70 ± 2°C for 10 ± 2 minutes, cool to room temperature, and transfer to a sample vial as the test solution. Prepare a blank solution by adding the same volume of formulation buffer as the test sample and following the same procedure as above. Sample injection conditions: -5 kV for 20 seconds; separation voltage: -15 kV for 35 minutes. The capillary column temperature was maintained at 25°C, and the detection wavelength was 220 nm.

[0356] Charge variants (CEX-HPLC method)

[0357] Cation exchange chromatography (CEX-HPLC) was used for detection. Separation was performed using a MabPac SCX-10 strong cation exchange column. Mobile phase A consisted of 10 mmol / L phosphate buffer (0.51 g NaH2PO4·2H2O and 2.40 g Na2HPO4·12H2O dissolved in 800 ml of ultrapure water, the volume was adjusted to 1000 ml, and the solution was filtered through a 0.22 μm filter). Mobile phase B consisted of 10 mmol / L phosphate plus 200 mmol / L sodium chloride buffer (0.51 g NaH2PO4·2H2O, 2.40 g Na2HPO4·12H2O, and 11.69 g NaCl dissolved in 800 ml of ultrapure water, the volume was adjusted to 1000 ml, and the solution was filtered through a 0.22 μm filter). The sample was diluted to 2.0 mg / ml with ultrapure water to serve as the test solution. Dilute the formulation buffer using the same treatment as above and use as a blank solution. Inject 50 μl of each blank solution and test solution into a liquid chromatograph using a mobile phase flow rate of 1.0 ml / min, a collection time of 35 minutes, a column temperature of 35°C, a detection wavelength of 280 nm, and a sample tray temperature of 10°C. Analyze the sample and calculate the contents of the principal component, acidic components, and basic components using the area normalization method.

[0358] Polysorbate 80 content (HPLC-FLD method)

[0359] Fluorescence detection chromatography (HPLC-FLD) was used. 0.15 mol / L sodium chloride, 0.05 mol / L tris (hydroxymethylaminomethane), pH 8.0, 5% acetonitrile, 5.0 μmol / L N-phenyl-1-naphthylamine, 15 ppm 2,3-polyoxyethylene fatty alcohol ether were used for isocratic elution. The flow rate was 1.5 ml / min. The acquisition time was 3 minutes. The upper pressure limit was 100 bar. The injection volume was 10 μl. The column temperature was 30°C. The detection wavelengths were 350 nm for excitation and 420 nm for emission. The polysorbate 80 content was calculated using the standard curve method.

[0360] Example 1. Construction of a stable expression cell line

[0361] Preparation of human CLDN18.2 overexpressing cell lines

[0362] According to the manufacturer's instructions, use A cell line stably expressing human Claudin18.2 (abbreviated as CLDN18.2, hereinafter referred to as "CLDN18.2") was constructed using a kit (Invitrogen, A1369601). The full-length gene of human CLDN18.2 (UniProt ID: P56856-2) was first constructed into the pCHO1.0 vector to form a plasmid. The constructed plasmid was then transformed into CHO-S cells (Invitrogen, A1369601) and HEK293 cells (Invitrogen, A14527) using chemical transfection and electrofection, respectively. The transfected cells underwent two rounds of pressure screening to obtain cell pools expressing CLDN18.2. Then, cells highly expressing CLDN18.2 were sorted using a flow cytometer (MoFlo XDP, Beckman Coulter), and monoclonal cell lines CHO-hCLDN18.2 and HEK293-hCLDN18.2 stably expressing CLDN18.2 were obtained by dilution method.

[0363] Preparation of human CLDN18.1 overexpressing cell lines

[0364] According to the manufacturer's instructions, use A cell line stably expressing human Claudin18.1 (abbreviated as CLDN18.1, hereinafter referred to as "CLDN18.1") was constructed using a kit (Invitrogen, A1369601). The full-length human CLDN18.1 gene (UniProt ID: P56856-1) was first constructed into the pCHO1.0 vector (Invitrogen, A1369601) to generate a plasmid. This plasmid was then chemically transfected into CHO-S cells (Invitrogen, A1369601). The transfected cells underwent two rounds of pressure selection to obtain pools of cells expressing CLDN18.1. Cells expressing high levels of CLDN18.1 were then isolated using a flow cytometer (MoFlo XDP, Beckman Coulter), and a monoclonal cell line, CHO-hCLDN18.1, stably expressing CLDN18.1, was obtained by dilution.

[0365] Construction of tumor cell lines overexpressing CLDN18.2

[0366] The full-length human CLDN18.2 (UniProt ID: P56856-2) gene was constructed into the pWPT-GFP vector (Addgene, 12255), replacing the GFP sequence. The vector was then co-transfected with the lentiviral packaging vectors psPAX2 (Addgene, 12260) and pMD2.G (Addgene, 12259) into HEK293T (ATCC, CRL-3216) cells for viral packaging. The culture supernatants were collected after 48 and 72 hours of culture, respectively, and the lentivirus was concentrated using PEG8000. The concentrated lentivirus was used to transfect pancreatic cancer DAN-G cells and gastric cancer KATO III cells. CLDN18.2-expressing cells were then sorted using a flow cytometer (MoFlo XDP, Beckman Coulter) to generate the stably transfected CLDN18.2 tumor cell lines DAN-G-hCLDN18.2 and KATO III-hCLDN18.2.

[0367] Example 2: Generation of CLDN18.2 Monoclonal Clones

[0368] The present invention uses hybridoma technology to immunize mice with H2L2 fully human antibody transgenic mice (purchased from Harbour BioMed) using the cells (CHO-hCLDN18.2) obtained in Example 1. Splenocytes from the mice are then electrofused with myeloma cells. The supernatant is then collected and screened using flow cytometry (FACS) to identify hybridoma cells that specifically express anti-CLDN18.2 antibodies and secrete antibodies that do not bind to CLDN18.1. The cells to be tested (HEK293-hCLDN18.2) obtained in Example 1 are counted and diluted to 1×10 6 Cells were plated at 100 μl / well in a U-bottom 96-well plate. The culture supernatant from the hybridoma 96-well plate was centrifuged at 500 g for 5 minutes, and the cell culture medium was removed. The supernatant from the hybridoma 96-well plate was added to the U-bottom plate and the cells were resuspended. 100 μl was added to each well and the cells were incubated on ice for 30 minutes. The supernatant was removed at 500 g for 5 minutes, and the cells were washed once with PBS. 100 μl of FITC-conjugated secondary antibody against mouse Fab (1:500 dilution in PBS) was added to each well. For the positive control antibody, 100 μl of FITC-conjugated secondary antibody against human Fab was added to each well. The cells were incubated on ice for 30 minutes in the dark. The supernatant was removed at 500 g for 5 minutes, and the cells were washed once with PBS. The cells were resuspended in 50 μl of 1× PBS and analyzed on a FACS machine. Positive clones were rescreened against CHO-hCLDN18.1 using the same method as above. After two rounds of screening, the fully human antibody clone HB37A6 was obtained.

[0369] Example 3. Preparation of recombinant CLDN18.2 monoclonal antibody

[0370] 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) as full-length monoclonal antibodies.

[0371] First, expression vectors were constructed, with the heavy and light chain variable regions of HB37A6 and a control antibody (see sequence information) placed at the N-terminus of the human IgG1 heavy chain constant region (SEQ ID NO: 5) and light chain kappa constant region (SEQ ID NO: 10), respectively. These vectors were then incorporated into a pcDNA3.1 expression vector with an N-terminal signal peptide to generate the heavy and light chain expression vectors. The resulting light and heavy chain expression vectors were co-transfected into HEK293 cells using PEI (Polysciences Inc, 23966). After 7 days of culture, the supernatant was collected. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), then ultrafiltered and exchanged into PBS (Gibco, 70011-044). The concentration was determined using the A280 method and the purity was determined by SEC-HPLC. An antibody solution with a purity greater than 95% was obtained, yielding the recombinant CLDN18.2 monoclonal antibody HB37A6.

[0372] The specific transfection and purification process is as follows:

[0373] Expi293 cells (Invitrogen, A14527) were passaged according to the required transfection volume, and the cell density was adjusted to 1.5×10 6 cells / ml. The cell density on the day of transfection was approximately 3×10 6 cells / ml. Take 1 / 10 of the final volume of Opti-MEM medium (Gibco, 31985-070) as the transfection buffer, add the appropriate plasmid to the transfected cells at 1.0 μg / ml, and mix well. Add the appropriate polyethyleneimine (PEI) (Polysciences, 23966) to the plasmid (the ratio of plasmid to PEI in 293F cells is 1:3), mix well, and incubate at room temperature for 20 minutes to obtain a DNA / PEI mixture. Slowly add the DNA / PEI mixture to the cells, gently shaking the shaker while adding, and then place it in a 36.5°C, 8% CO2 incubator for culture. After seven days, obtain the cell feed solution and collect the cell supernatant for purification.

[0374] The Protein A column used for purification (Hitrap Mabselect Sure, GE, 11-0034-95) was treated with 0.1M NaOH for 2 hours. The glass bottles and other components were washed with distilled water and then dry-baked at 180°C for 4 hours. Prior to purification, the collected cell suspension was centrifuged at 4500 rpm for 30 minutes, and the supernatant was filtered through a 0.22 μm filter. The Protein A column was equilibrated with 10 column volumes of binding buffer (20 mM sodium phosphate, 150 mM NaCl, pH 7.0). The filtered supernatant was applied to the purification column and equilibrated with 10 column volumes of binding buffer. 5 ml of elution buffer (0.1 M citric acid + sodium citrate, pH 3.5) was added, and the eluate was collected. 80 μl of 2 M Tris-HCl was added to every 1 ml of eluate. The collected antibody was concentrated by ultrafiltration and exchanged into PBS (Gibco, 70011-044), and the concentration was determined.

[0375] Example 4 Preparation of anti-CD3 monoclonal antibodies

[0376] The murine CD3 antibody sp34 (US Pat. No. 8,236,308; J. Immunol. Methods., 1994, 178:195) and humanized and affinity-optimized anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, and HzSP34.97 were expressed in HEK293 cells (Invitrogen, A14527) as full-length monoclonal antibodies.

[0377] The heavy and light chain variable regions of HzSP34.24, HzSP34.87, HzSP34.97, and sp34 (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). These regions were then incorporated into the pcDNA3.1 expression vector with an N-terminal signal peptide to generate the heavy and light chain expression vectors. The resulting light and heavy chain expression vectors were co-transfected into HEK293 cells using PEI (Polysciences Inc, 23966), and the culture supernatant was collected after 7 days of culture. The supernatant was purified using a Protein A column (Hitrap Mabselect Sure, GE 11-0034-95), followed by ultrafiltration and exchange into PBS (Gibco, 70011-044). The concentration was determined by the A280 method, and the purity was determined by SEC-HPLC. An antibody solution with a purity greater than 95% was obtained, yielding recombinant anti-CD3 monoclonal antibodies HzSP34.24, HzSP34.87, and HzSP34.97, and the murine CD3 antibody sp34. For detailed transfection and purification procedures, see Example 3.

[0378] Example 5. Construction and preparation of bispecific antibodies

[0379] According to Table 2, the anti-CLDN18.2 monoclonal antibody HB37A6 was combined with 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, respectively, targeting CLDN18.2 × CD3 in a "1+1" format. See Figure 1 for schematic diagrams of their antibody structures. Specifically, the heavy chain variable region sequence of the antigen-binding domain specifically targeting CLDN18.2 is SEQ ID NO:4, and the light chain variable region sequence is SEQ ID NO:9; the heavy chain variable region sequence of the antigen-binding domain specifically targeting CD3 is SEQ ID NO:22, SEQ ID NO:30, and SEQ ID NO:32, and the light chain variable region sequence is SEQ ID NO:27. The Fc region of the antibody was selected from the IgG1 LALA sequence with a knob-in-hole structure (A. Margaret Merchant et al., Nature Biotechnology, 1998). Therefore, the heavy chain of the bispecific antibody CLDN18.2 end portion is SEQ ID NO: 37, and the light chain is SEQ ID NO: 38. The heavy chains of the bispecific antibody CD3 end portion are SEQ ID NOs: 39, 41, and 42, respectively, and the light chain is SEQ ID NO: 40.

[0380] The plasmid construction process for the bispecific antibody was as follows: the heavy chain sequence of CLDN18.2 (SEQ ID NO: 37), the light chain sequence of CLDN18.2 (SEQ ID NO: 38), the heavy chain sequence of CD3 (SEQ ID NO: 39, 41, and 42), and the light chain sequence of CD3 (SEQ ID NO: 40) were inserted into the vector pcDNA3.1 (Invitrogen, V790-20), generating the heavy chain plasmid and light chain plasmid for CLDN18.2 and the heavy chain plasmid and light chain plasmid for CD3, respectively. The heavy chain plasmid and light chain plasmid for CLDN18.2 and the heavy chain plasmid and light chain plasmid for CD3 were then transiently transfected into Expi293 cells (Invitrogen, A14527) using PEI (Polysciences, 23966), expressing the three antibody molecules for CLDN18.2 and CD3, respectively. After 7 days, the cell fermentation broth was obtained, filtered and clarified, and captured using a Hitrap Mabselect Sure Protein A column (GE Healthcare, 11-0034-95) to obtain antibodies targeting the CLDN18.2 and CD3 ends. After concentration determination using the A280 method, the antibodies were mixed at a 1:1 molar ratio. An appropriate amount of the reducing agent, GSH, was added and the reaction was allowed to proceed overnight at room temperature. The reducing agent was removed by ultrafiltration, and the reaction was terminated. The protein was then finely purified using MonoS cation exchange chromatography (GE Healthcare, 17-5168-01). Solution A consisted of 20 mM sodium phosphate buffer (pH 6.6) and Solution B consisted of 20 mM sodium phosphate buffer (pH 6.6) containing 1 M sodium chloride. The elution gradient was 0-50% (30 column volumes). The eluted protein solution was ultrafiltered and exchanged into PBS (Gibco, 70011-044). Molecular weight was determined by mass spectrometry, and purity was determined by SEC-HPLC. The obtained 030, 032 and 033 bispecific antibodies were used in the following examples.

[0381] Table 2. List of CD3 / CLDN18.2 bispecific antibodies

[0382] Example 6. Bispecific Antibody Affinity Determination

[0383] The equilibrium dissociation constant (KD) of the bispecific antibody of the present invention binding to human CD3 protein was determined using thin-layer interferometry (BLI). BLI affinity determination was performed according to existing methods (Estep, P et al., High throughput solution-based measurement of antibody-antigen affinity and epitope binning. MAbs, 2013. 5(2): p. 270-8).

[0384] Half an hour before the start of the experiment, an appropriate number of AHC (18-5060, Fortebio) sensors were soaked in SD buffer (1x PBS, 0.1% BSA, 0.05% Tween-20) based on the number of samples. 100 μl of SD buffer, each bispecific antibody, and human CD3 protein (CT026H0323H, Sino Biological, Beijing) were added to a 96-well black polystyrene half-well microplate (Greiner, 675076). Detection was performed using a Fortebio Octet Red96, with the sensor positions selected based on the sample positions. Instrument parameters were set as follows: Run steps: Baseline, Loading ~1 nm, Baseline, Association, and Dissociation; the run time for each step depended on the sample binding and dissociation rates, the rotation speed was 1000 rpm, and the temperature was 30°C. KD values ​​were analyzed using ForteBio Octet analysis software.

[0385] The affinities of the bispecific antibodies are shown in Table 3. The CD3-terminal affinity of the 030 molecule was the highest, at 7.4 nM. The CD3-terminal affinities of the 032 and 033 molecules decreased in order, at 89 nM and 440 nM, respectively.

[0386] Table 3. Bispecific antibody CD3-terminal affinity

[0387] The equilibrium dissociation constant (KD) for binding to human CLDN18.2 was determined using surface plasmon resonance (SPR). The antigen human claudin18.2 (GeneScrip, P50251802) was coupled to the surface of a CM5 chip (GE Healthcare, 29-1496-03) using an amino coupling kit (GE Healthcare, BR-1006-33) according to the manufacturer's instructions. After coupling, 1 M ethanolamine was injected to block the remaining active sites. Affinity and kinetic constants were determined by Biacore (GE Healthcare, T200) analysis of the binding and dissociation between the antigen on the chip surface and each bispecific antibody in the mobile phase according to the manufacturer's instructions. A gradient of diluted antibodies (0-100 nM, 2-fold dilution) was applied over the chip surface in increasing concentrations, with an association time of 180 s and a dissociation time of 600 s. Finally, the chip was regenerated using 10 mM Glycine pH 1.5 (GE Healthcare, BR-1003-54). Data were analyzed using Biacore T200 analysis software, using a 1:1 binding model for kinetic analysis. As shown in Table 4, the 030, 032, and 033 bispecific antibodies all utilize the same clone, HB37A6, at the CLDN18.2 end, exhibiting consistent, very strong affinity of 0.57 nM.

[0388] Table 4. Bispecific antibody CLDN18.2 end affinity

[0389] Example 7. In vitro T cell killing experiment

[0390] The 030, 032, and 033 bispecific antibodies obtained in Example 5 were subjected to an in vitro T cell killing experiment. Human peripheral blood mononuclear cells (PBMC, Allcells or Saily) were resuspended in complete medium RPMI-1640 (Hyclone, SH30809.01) + 10% fetal bovine serum (FBS, Hyclone, SH30084.03) and the PBMC was adjusted to 2×10 6 pcs / ml.

[0391] NUGC-4 or DAN-G tumor target cells overexpressing Claudin18.2, DAN-G-hCLDN18.2, and non-target L363 (DSMZ, ACC49) cells were labeled with Far-Red (Invitrogen) for 10 min, washed twice, and resuspended in complete culture medium. The cell concentration was adjusted to 2 × 10 5pcs / ml.

[0392] PBMCs were mixed with bispecific antibodies 030, 032, and 033 (the initial concentrations of 030 and 032 were 1 nM, and the initial concentration of 033 was 400 nM. All antibodies were diluted 5-fold, with a total of 10 concentration points), incubated at 37°C for 30 min, and then 50 μl of tumor target cells (1×10 4 100 μl of PBMC effector cells were added to the tube. The cells were incubated at 37°C for 24 hours, centrifuged, and resuspended in propidium iodide (PI, Invitrogen) at a final concentration of 10 μg / ml. Far-Red and PI-positive cells were detected using flow cytometry (BD, FACSCELESTA). The percentage of tumor target cell killing was calculated using FACSDiva software (BD, Celesta).

[0393] The results in Figure 2 show that the 030 and 032 molecules have very strong killing activity on gastric cancer cells NUGC-4, with EC50 values ​​of less than 1pM. The results in Figure 3 show that on pancreatic cancer cells DAN-G-hCLDN18.2, which highly express CLDN18.2, the EC50 values ​​of both are even less than 0.1pM. The killing activity of the 033 molecule on both tumor cell lines is weaker, about 1000-fold lower than that of 030 and 032, but it still achieves maximum killing (nearly 100% cell lysis). However, in the case of negative CLDN18.2 expression, the 030, 032, and 033 molecules did not show nonspecific killing (Figure 4). This shows that the 030, 032, and 033 molecules all demonstrate tumor cell-specific killing that depends on CLDN18.2 expression. Moreover, the killing effect of the bispecific antibody of the present invention is related to the abundance of CLDN18.2 on the cell surface. Within a certain expression abundance range, the higher the expression level of CLDN18.2 on the cell surface, the better the killing effect.

[0394] Example 8. In vivo efficacy experiment - gastric cancer model

[0395] This study investigated the anti-tumor effect of bispecific antibodies against NUGC-4 tumors in NOG female mice. 49 NOG female mice (Beijing Weitonglihua Laboratory Animal Technology Co., Ltd.) were selected.

[0396] PBMC cells (All cells) were revived, centrifuged, and dispersed with PBS (1×) to obtain a cell density of 2×10 7 / ml cell suspension. Take 200μl cell suspension and inject PBMC cells into the mouse's orbital vein, 4×10 6 / Only.

[0397] NUGC-4 cells were routinely recovered and subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and dispersed with PBS (1×) at a cell density of 6×10 7 The cell concentration was 3 × 10 cells / ml and mixed with matrigel gel at a ratio of 1:1. 7 On day 3, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOG humanized mice to establish a NUCG-4 tumor-bearing mouse model.

[0398] On the 7th day after cell inoculation, the maximum width and maximum length of the mouse tumor were measured with a vernier caliper to calculate the tumor volume. 3 ~168.07mm 3 Mice were divided into groups (6 mice per group) based on tumor volume. Each mouse was intravenously injected with the bispecific antibodies 030, 032, and 033 of the present invention, as well as a negative control h-IgG (Equitech-Bio, lot number 160308-02) at doses of 0.3 mg / kg and 1 mg / kg, once weekly for a total of four doses. Tumor volume was measured twice weekly. Tumor inhibition rate (TGI%) was calculated using the following formula: TGI% = 100% * (control group tumor volume – treatment group tumor volume) / (control group tumor volume – control group pre-dose tumor volume).

[0399] As shown in Figure 5, in the NUCG-4 human gastric cancer mouse model, 030 and 032 achieved a 100% TGI at a low dose of 0.3 mg / kg, and a 50% complete remission (CR) at a high dose of 1 mg / kg (3 out of 6 mice achieved complete tumor disappearance). However, 033 showed little efficacy at low doses, achieving a TGI of 20% at a dose of 1 mg / kg. Throughout the experiment, mice in both the experimental and control groups maintained their body weight.

[0400] Example 9. In vivo efficacy experiment - pancreatic cancer model

[0401] This study investigated the antitumor effect of bispecific antibodies against DAN-G-Claudin18.2 tumors in NOG female mice. PBMC cells were injected into the orbital vein of 49 NOG mice (Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd.) at 4×10 6 / mouse, inoculated with 200ul / mouse (as in Example 18). This time is recorded as day 0.

[0402] The human pancreatic cancer cell line DAN-G-CLDN18.2 constructed in Example 1 was routinely subcultured for subsequent in vivo experiments. The cells were collected by centrifugation and DAN-G-CLDN18.2 was dispersed in PBS (1×) to obtain a cell density of 10×10 6 The cell suspension was mixed with matrigel gel in a ratio of 1:1 to prepare a cell concentration of 5×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of NOD-SCID mice to establish a DNA-G pancreatic cancer humanized model with overexpression of CLDN18.2.

[0403] Seven days after tumor cell inoculation, the maximum width and maximum length of the mouse tumor were measured with a vernier caliper to calculate the tumor volume. The mice were divided into snake groups according to the tumor volume. 3 ~120.64mm 3 The mice within the range were divided into groups according to the size of the tumor (6 mice per group).

[0404] Each mouse was intravenously injected with the bispecific antibodies 030, 032, and 033 of the present invention, as well as a negative control h-IgG (Equitech-Bio, batch number 160308-02) at intraperitoneal doses of 0.3 mg / kg and 1 mg / kg once weekly for a total of four doses. Tumor volume was measured twice weekly. Tumor inhibition rate (TGI%) was calculated using the following formula:

[0405] TGI% = 100% * (control group tumor volume – treatment group tumor volume) / (control group tumor volume – control group pre-dose tumor volume). As shown in Figure 6, in the DNA-G humanized pancreatic cancer model overexpressing CLDN18.2, both 030 and 032 molecules achieved a TGI of 100% at both doses. The 033 molecule also achieved TGIs of 42% (0.3 mg / kg) and 76% (1 mg / kg), respectively. This may be related to the high expression of CLDN18.2 in the DNA-G-CLDN18.2 pancreatic cancer cells. Throughout the experiment, mice in both the experimental and control groups did not lose weight.

[0406] Example 10. Mouse PK experiment

[0407] In this study, female Balb / C mice (Vitamin B) were injected with 10 mg / kg of 030, 032, and 033 via the tail vein to investigate their pharmacokinetic properties. Blood was collected from the eyeballs at 0.086 hours, 0.5 hours, 2 hours, 6 hours, 24 hours, 48 ​​hours, 4 days, 7 days, 14 days, and 21 days after administration. Serum was collected by centrifugation at 3000 rpm for 10 minutes at 4°C. Serum antibody levels were determined by ELISA, and the in vivo half-lives of 030, 032, and 033 were calculated.

[0408] The experimental results are shown in Figure 7. The half-lives of 030, 032, and 033 in mice all achieved PK similar to that of normal monoclonal antibodies, indicating that the bispecific antibodies constructed in the present invention did not affect the half-life of the antibody.

[0409] Example 11. pH screening experiment

[0410] 1. Experimental steps

[0411] Prepare a buffer containing 1.55 mg / ml histidine and 50.00 mg / ml sorbitol. Adjust the pH to 5.0, 5.5, 6.0, 6.5, and 7.0 with hydrochloric acid. Ultrafiltration is then performed into the buffers at different pH values ​​to adjust the protein concentration to approximately 1 mg / ml. Add polysorbate 80 to a final concentration of 0.2 mg / ml. Filter and aliquot into vials, stopper, and cap. Stability testing of these samples was performed at 40°C ± 2°C. See the table for the specific protocol.

[0412] Table 5. Experimental plan

[0413] Note: (1)√ indicates sampling at that time. (2) After sampling at the above time points, all samples were first frozen at -70℃ and thawed as needed for testing.

[0414] 2. Judgment criteria

[0415] Based on the understanding of the product and the precision of the instruments and methods, the judgment standard that the quality of the sample test value has not changed compared with the initial value is set.

[0416] Table 6 Criteria for judging if quality has not changed

[0417] The results (as shown in Figure 8) show that after 4 weeks at 40°C ± 2°C, the appearance and visible foreign matter of samples at all pH conditions met the standards. Protein content and pH did not change significantly. The purity (nrCE-SDS method) of the samples did not change significantly at all pH conditions. The purity (SEC-HPLC method) increased for all samples except the pH 5.0 sample, which showed no significant change. The charge variant-acidic component increased significantly in all samples, with the acidic component increasing more rapidly with increasing pH. The charge variant-main component decreased significantly in all samples, with the main component decreasing more rapidly with increasing pH. The charge variant-basic component decreased significantly in all samples except the pH 5.0 sample, which showed no significant change. Anti-Claudin 18.2 biological activity met the standards. Anti-CD3 terminal biological activity exceeded the criteria for all conditions except the pH 5.0 sample, which met the standards.

[0418] Combining the results of the biological activity of the charge variants and the anti-CD3 end, and considering that the pH may fluctuate during actual production, the pH was finally determined to be 5.2 for the formulation determination experiment.

[0419] Example 12. Prescription determination experiment

[0420] This example mainly examines the effects of the buffer system (histidine system and sodium acetate system) and excipients (sorbitol, arginine hydrochloride and disodium edetate) on the stability of the bispecific antibody protein. For detailed prescription information, see 7.

[0421] Prepare the buffer for each formulation according to Table 7, and ultrafiltration exchange the bispecific protein into the respective formulation solution. After exchange, adjust the protein content of each formulation to 1 mg / ml. Add polysorbate 80 to a final concentration of 0.2 mg / ml. Filter and dispense into vials, stopper, and cap. Stability testing of these samples was performed at 40°C ± 2°C.

[0422] Table 7 Prescription Information

[0423] Note: Use hydrochloric acid to adjust pH

[0424] Table 8. Stability study plan

[0425] Note: (1)√ indicates sampling at that time. (2) After sampling at the above time points, all samples were first placed in a -70℃ freezer for testing and thawed and sent for testing as needed.

[0426] The evaluation criteria are shown in Table 6. The results (as shown in Figure 9) indicate that after 4 weeks at 40°C ± 2°C, all formulations met the criteria for appearance and visible foreign matter. Protein content and pH remained unchanged. The purity (SEC-HPLC) of formulations 2 and 5 remained unchanged, while the changes in the purity of the other formulations exceeded the evaluation criteria. The purity (nrCE-SDS) of formulations 3 and 4 exceeded the evaluation criteria. The decreasing trends of formulations 1 and 2 were consistent, and the decline was slower than that of formulation 5. Charge variant changes in all formulations showed a significant increase in the acidic component and a significant decrease in the main component. The anti-Claudin 18.2 end biological activity of all formulations met the criteria. The anti-CD3 end biological activity of formulations 2 and 5 also met the criteria, while all other formulations exceeded the evaluation criteria. With the exception of formulation 2, where the polysorbate 80 content remained unchanged, the other formulations showed varying degrees of decrease.

[0427] Based on the results of Examples 11 and 12, Formulation 2 was selected as the final formulation for the anti-CLDN18.2 / CD3 bispecific antibody. To avoid the use of hydrochloric acid to adjust the pH during production, the buffer system was adjusted to histidine and histidine hydrochloride. Specifically, the formulation for the anti-CLDN18.2 / CD3 bispecific antibody was: 1.0 mg / ml recombinant anti-cluster of differentiation 3 (CD3) and anti-claudin 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 disodium edetate, and 0.2 mg / ml polysorbate 80, pH 5.2.

[0428] While the exemplary embodiments of the present invention have been described above, it should be understood by those skilled in the art that these disclosures are merely exemplary and that various other substitutions, adaptations, and modifications may be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein.

[0429] Sequence information

Claims

1. A liquid antibody preparation comprising (i) anti-CLDN18.2 / CD3 protein; (ii) a buffer, (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 comprises the three complementarity determining regions A1-HCDR1, A1-HCDR2, and A1-HCDR3 contained in A1-VH as shown in SEQ ID NO: 4, and the three complementarity determining regions A1-LCDR1, A1-LCDR2, and A1-LCDR3 contained in VL as 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: 30, 22, 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. Preferably, the pH of the liquid antibody formulation is about 5.0-6.0, for example, pH 5.2±0.2 or 5.5±0.2, preferably pH 5.

2.

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

3. The liquid antibody formulation according to claim 1 or 2, characterized in that the liquid antibody formulation comprises a buffer selected from the group consisting of 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 the group consisting of histidine, histidine hydrochloride, and a combination thereof; preferably, the concentration of the buffer is about 0.01-10 mg / ml, preferably about 0.1-1 mg / ml, for example, about 0.1, 0.2, 0.3, 0.4, 0.5, or 1 mg / ml.

4. The liquid antibody formulation according to any one of claims 1 to 2, characterized in that 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 comprises one or more selected from the following: - a polyol selected from sorbitol, mannitol, or a combination thereof; - a sugar selected from sucrose, trehalose, maltose, or a combination thereof; - an amino acid selected from the group consisting of arginine hydrochloride, methionine, glycine, proline, and combinations thereof.

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

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

7. The liquid antibody formulation according to any one of claims 1 to 5, characterized in that the concentration of the surfactant is about 0.01-5 mg / ml, preferably about 0.1-1 mg / ml, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / ml, more preferably about 0.2 mg / ml.

8. The liquid antibody formulation according to any one of claim 7, further comprising a chelating agent selected from the group consisting of disodium edetate, aminotriacetic acid, diethylenetriaminepentaacetic acid, citric acid, tartaric acid, gluconic acid, hydroxyethylethylenediaminetriacetic acid, and dihydroxyethylglycine. Preferably, the chelating agent is selected from disodium edetate; Preferably, the chelating agent is about 0.008-0.018 mg / ml, such as about 0.008, 0.009, 0.010, 0.012, 0.014, 0.018 mg / ml, preferably 0.01 mg / ml.

9. The liquid antibody preparation according to any one of claims 1 to 8, wherein the anti-CLDN18.2 / CD3 protein The first antigen binding domain comprises A1-HCDR1, A1-HCDR2, A1-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 1, 2, and 3, respectively, and A1-LCDR1, A1-LCDR2, and A1-LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 6, 7, and 8, respectively; and The second antigen binding domain comprises (i) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 20 and 29, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25 and 26, respectively; or (ii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 19, 20 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NOs: 24, 25 and 26, respectively; or (iii) A2-HCDR1, A2-HCDR2, A2-HCDR3 as shown in the following amino acid sequences: SEQ ID NO: 19, 31 and 21, respectively, and LCDR1, LCDR2 and LCDR3 as shown in the following amino acid sequences: SEQ ID NO: 24, 25 and 26, respectively.

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

11. The liquid antibody preparation according to any one of claims 1 to 8, characterized in that the antibody CLDN18.2 / CD3 is an IgG-type antibody (e.g., an IgG4 subclass antibody), preferably comprising SEQ ID NO: 9, or a heavy chain sequence that is at least 90%, 95%, 98% or 99% identical thereto, and SEQ ID NO: 10, or a light chain sequence that is at least 90%, 95%, 98% or 99% identical thereto.

12. The liquid antibody preparation according to any one of claims 1 to 11, 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 (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 30, 22 or 32; or (iii) an amino acid sequence comprising one or more (preferably no more than 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: 30, 22, or 32, consisting of said amino acid sequence, preferably, said amino acid changes do not occur in the CDR regions; and / or Light chain variable region (i) comprises or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence selected from SEQ ID NO: 27; or (ii) comprises or consists of an amino acid sequence selected from SEQ ID NO: 27; or (iii) an amino acid sequence comprising one or more (preferably no more than 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 the amino acid sequence selected from SEQ ID NO: 27, consisting of said amino acid sequence. Preferably, said amino acid changes do not occur in the CDR regions. 13 . The liquid antibody formulation according to claim 1 , further comprising a heavy chain constant region and / or a light chain constant region. The liquid antibody preparation according to any one of claims 1 to 12, which is a bispecific antibody with an IgG-like structure.

15. The liquid antibody formulation according to any one of claims 1 to 13, wherein the heavy chain constant region of the antibody is derived from IgG1, IgG2, IgG3 or IgG4, preferably IgG1. 16 . The liquid antibody formulation according to claim 1 , wherein the anti-CLDN18.2 / CD3 is recombinantly expressed in 293 cells or CHO cells.

17. The liquid antibody formulation according to any one of claims 1 to 16, comprising: (i) about 0.1-100 mg / ml of anti-CLDN18.2 / CD3 protein; (ii) about 0.1-2 mg / ml histidine; (iii) about 30-70 mg / ml of sorbitol; (iv) about 0.1-1 mg / ml polysorbate 80; and (v) optionally, about 0.008-0.018 mg / ml of edetate disodium, wherein the pH of the liquid formulation is about 5.0-5.5, for example, about 5.2; For example, the liquid antibody formulation comprises (i) about 0.1-10 mg / ml, for example 0.1-1 mg / ml, of an anti-CLDN18.2 / CD3 protein; (ii) about 1.55 mg / ml histidine; (iii) about 40-60 mg / ml sorbitol or sucrose, preferably 50 mg / ml sorbitol, or 30-50 mg / ml sucrose; (iv) about 0.2-0.8 mg / ml, e.g., 0.2-0.6 mg / ml polysorbate 80; and (v) about 0.008-0.018 mg / ml, for example 0.01 mg / ml, of edetate disodium, wherein the pH of the liquid formulation is about 5.0-5.5, for example, 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, about 0.2 mg / ml polysorbate 80, pH about 5.2; or (ii) about 50 mg / ml of anti-CLDN18.2 / CD3 protein, about 1.55 mg / ml of histidine, about 50.00 mg / ml of sorbitol, about 0.01 mg / ml of edetate disodium, about 0.2 mg / ml of polysorbate 80, pH 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, about 0.2 mg / ml polysorbate 80, pH 5.

2.

18. The liquid antibody preparation according to any one of claims 1 to 13, characterized in that The formulation is stable after storage, for example after storage at 25°C for at least 2 months, or after storage at 40°C ± 2°C for 1 month, and preferably has one or more of the following characteristics: (i) the main peak variation value is less than 1% as measured by SEC-HPLC, and / or the preparation has a purity greater than 96%, preferably greater than 97% or 98%; (ii) the main peak variation value is less than 2% as measured by non-reduced CE-SDS method, and / or the preparation has a purity greater than 96%, preferably greater than 97% or 98%; (iii) the sum of the changes in the components of the anti-CLDN18.2 / CD3 protein (main component, acidic component, and basic component) in the formulation relative to the initial value on storage day 0, as measured by CEX-HPLC, does not exceed 40% and / or the change in the main component does not exceed 20%, for example, after storage at 40°C ± 2°C for 1 month, the sum of the changes does not exceed about 40% (for example, does not exceed 30%) or the change in the main component does not exceed about 20% (for example, does not exceed 15%), or after storage at 25°C for 2 months, the sum of the changes does not exceed about 20% (for example, about 15%) or the change in the main component does not exceed about 15% (for example, does not exceed about 10%); (iv) the relative binding activity of the anti-CLDN18.2 / CD3 protein in the formulation, as measured by ELISA, is 70%-130%, e.g., 90%-110%, relative to the initial value on storage day 0; More preferably, the formulation is stable under shaking and / or repeated freeze-thaw cycles.

19. The liquid antibody formulation according to any one of claims 1 to 15, characterized in that the liquid formulation is an injection, preferably for subcutaneous injection or intravenous injection, or an infusion, such as for intravenous infusion.

20. A solid antibody preparation obtained by solidifying the liquid antibody preparation according to any one of claims 1 to 19, wherein the solid antibody preparation is in the form of a lyophilized powder injection.

21. A delivery device comprising the liquid antibody formulation of any one of claims 1 to 19 or the solid antibody formulation of claim 15.

22. A prefilled syringe comprising the liquid antibody formulation according to any one of claims 1 to 19 or the solid antibody formulation according to claim 15, for intravenous or intramuscular injection.

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 for preparing a delivery device or prefilled syringe or medicament for treating or preventing tumors, pathogen infections in a subject.

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