Pharmaceutical composition of anti-CD3*CD20 bispecific antibody

Abstract

The invention belongs to the field of biological medicine, and relates to a pharmaceutical composition of an anti-CD3 * CD20 bispecific antibody, in particular to a liquid preparation and a freeze-dried preparation of the bispecific antibody and medical application of the liquid preparation and the freeze-dried preparation. More specifically, the pharmaceutical composition contains an anti-CD3 * CD20 bispecific antibody, a buffer solution, a stabilizer and a surfactant, the pH value of the pharmaceutical composition is about 4.5-6.5, and the pharmaceutical composition has good stability.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to an application with CN application number 202310886445.3 and filing date July 19, 2023. The entire contents of the CN application are hereby introduced as a whole into this application. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to a pharmaceutical composition of an anti-CD3×CD20 bispecific antibody, and specifically, to a liquid preparation and a lyophilized preparation of the bispecific antibody and their medical uses. Background Art

[0004] The formulation of antibody preparations is not only required to be suitable for administration to patients, but also to maintain a certain stability during storage and subsequent use. Improper formulations can cause therapeutic antibodies in liquid solutions to be easily degraded and aggregated. The stability of antibodies in liquid preparations depends not only on the type of excipients used in the preparation, but also on the concentration and ratio of the excipients. In addition, the preparation is also required to contain an appropriate antibody concentration, maintain antibody activity, and have a good appearance.

[0005] Bispecific antibodies targeting CD3 and CD20 can bind to T cells and B cells at the same time, and promote the killing of target B cells by cytotoxic T cells by binding to CD3-positive T cells and CD20-positive B cells. It can be used to treat cancers such as follicular lymphoma and CD20-positive B-cell non-Hodgkin's lymphoma.

[0006] Bispecific antibody drugs have large molecular weight and complex structure, and are easily degraded, aggregated or undergo undesirable chemical modifications, which lead to antibody heterogeneity (including size heterogeneity and charge heterogeneity) as well as aggregates and fragments, thus affecting the quality of antibody preparations, especially under high temperature or transportation conditions. In order to make bispecific antibodies suitable for production and administration, and to maintain biological activity and stability during storage and subsequent use, it is necessary to develop stable bispecific antibody drug preparations. There are many anti-CD3×CD20 bispecific antibodies in the prior art, and these antibodies have different amino acid sequences and structures. Therefore, for a specific antibody, it is still necessary to develop a suitable preparation type and formula.

[0007] Anti-CD3×CD20 bispecific antibodies have disadvantages such as low solubility and poor sample stability. Currently, there is a need to develop an anti-CD3×CD20 bispecific antibody preparation that can ensure drug quality during long-term storage, safe and reliable preparation process, simple formula, and convenient injection. Summary of the invention

[0008] After in-depth research and creative work, the inventors have obtained a pharmaceutical composition comprising an anti-CD3×CD20 bispecific antibody. Furthermore, the inventors have obtained a liquid preparation and a lyophilized preparation comprising an anti-CD3×CD20 bispecific antibody. The inventors surprisingly found that the pharmaceutical composition has good stability, and / or the anti-CD3×CD20 bispecific antibody as an active ingredient has good solubility. The following invention is thus provided:

[0009] One aspect of the present invention relates to a pharmaceutical composition comprising the following (a), and further comprising one or more selected from the following (b)-(d):

[0010] (a) an anti-CD3×CD20 bispecific antibody or an antigen-binding fragment thereof, which comprises a first protein functional region that specifically binds to human CD20 and a second protein functional region that specifically binds to human CD3, wherein:

[0011] The first protein functional region comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein the VH1 comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; the VL1 comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6;

[0012] The second protein functional region comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein the VH2 comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; the VL2 comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14;

[0013] (b) buffer;

[0014] (c) stabilizers;

[0015] (d) Surfactant.

[0016] In some embodiments of the present invention, the pharmaceutical composition, wherein:

[0017] The first protein functional region is an anti-CD20 immunoglobulin or an antigen-binding fragment thereof, and the second protein functional region is an anti-CD3 single-chain antibody or an antigen-binding fragment thereof.

[0018] In some embodiments of the present invention, the pharmaceutical composition, wherein the first protein functional region and the second protein functional region are independently 1, 2 or more than 2;

[0019] Preferably, the number of the first protein functional regions is 2, and the number of the second protein functional region is 1;

[0020] Preferably, the number of the first protein functional region is 1, and the number of the second protein functional region is 1.

[0021] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0022] The VH1 comprises the amino acid sequence shown in SEQ ID NO:7, and the VL1 comprises the amino acid sequence shown in SEQ ID NO:8;

[0023] and / or

[0024] The VH2 comprises the amino acid sequence shown in SEQ ID NO:15, and the VL2 comprises the amino acid sequence shown in SEQ ID NO:16.

[0025] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0026] The immunoglobulin is of human IgG1 subtype, human IgG2 subtype, human IgG3 subtype or human IgG4 subtype.

[0027] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0028] The heavy chain constant region of the immunoglobulin is Ig gamma-1 chain C region or Ig gamma-4 chain C region; the light chain constant region is Ig kappa chain C region.

[0029] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0030] The Fc region of the immunoglobulin further comprises a Knob-in-hole mutation.

[0031] In some embodiments of the present invention, the pharmaceutical composition, wherein the anti-CD3×CD20 bispecific antibody comprises a polypeptide chain represented by formula (I), formula (II), formula (III) and formula (II-2):

[0032] VH1-CH1-Fc1 formula (I),

[0033] VL1-CL formula (II),

[0034] VH1-CH1-L1-VL2-L2-VH2-L3-Fc2 formula (III); and

[0035] VL1-CL formula (II-2);

[0036] in,

[0037] Formulas (I), (II), (II-2), and (III) are connected sequentially from the N-terminus to the C-terminus;

[0038] Fc1 and Fc2 are Fc segments containing CH2 and CH3;

[0039] CH1, CH2, and CH3 represent domain 1, domain 2, and domain 3 of the heavy chain constant region, respectively;

[0040] CL indicates light chain constant region;

[0041] L1, L2 and L3 represent connecting peptides, and L1, L2 and L3 are the same or different;

[0042] Optionally, there is a hinge region between CH1 and Fc1.

[0043] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0044] Formula (I) and Formula (II), as well as Formula (III) and Formula (II-2) are connected via a disulfide bond; and Formula (I) and Formula (III) are connected via a disulfide bond and / or a Knob into Hole structure of the CH3 domain;

[0045] Preferably, Fc1 in formula (I) is knob-Fc, and Fc2 in formula (III) is hole-Fc; or Fc1 in formula (I) is hole-Fc, and Fc2 in formula (III) or (III) is knob-Fc.

[0046] In some embodiments of the present invention, the pharmaceutical composition, wherein the connecting peptide comprises a polypeptide composed of n amino acid sequences GGGGS (SEQ ID NO: 20) in series, or comprises a polypeptide in which serine (S) and alanine (A) are sequentially connected to the N-terminus of a polypeptide composed of n amino acid sequences shown in SEQ ID NO: 20 in series, wherein n is independently selected from 1, 2, 3, 4, 5 and 6.

[0047] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0048] The amino acid sequence of the polypeptide chain represented by formula (I) is shown in SEQ ID NO: 19,

[0049] The amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are both as shown in SEQ ID NO: 17, and

[0050] The amino acid sequence of the polypeptide chain represented by formula (III) is shown in SEQ ID NO:18.

[0051] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0052] The buffer is a buffer containing acetic acid or citric acid or histidine;

[0053] The stabilizer is a stabilizer comprising sugar; and / or

[0054] The surfactant is a surfactant comprising polysorbate.

[0055] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0056] The buffer is selected from one or more of acetate buffer, citrate buffer and histidine hydrochloride buffer;

[0057] The stabilizer is selected from one or more of sucrose and trehalose; and / or

[0058] The surfactant is selected from one or more of polysorbate 80 and polysorbate 20.

[0059] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0060] The pH of the pharmaceutical composition is (4.5±0.5) to (6.5±0.5), preferably (4.5±0.5) to (6.0±0.5), (4.5±0.5) to (5.5±0.5) or (5.5±0.5) to (6.0±0.5).

[0061] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0062] The buffer is selected from acetate buffer,

[0063] The stabilizer is selected from sucrose or trehalose, and

[0064] The surfactant is selected from polysorbate 80.

[0065] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0066] The buffer is selected from citrate buffer,

[0067] The stabilizer is selected from sucrose or trehalose, and

[0068] The surfactant is selected from polysorbate 80.

[0069] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0070] The concentration of the buffer solution is (5±0.5) to (50±5) mM, preferably (10±1) to 50±5) mM, more preferably (10±1) to (20±2) mM, and more preferably 20±1 mM.

[0071] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0072] The concentration of the stabilizer is (100±10) to (300±30) mM, preferably (150±10) to (280±20) mM, more preferably (200±20) to (230±20) mM, and particularly preferably 230±10 mM.

[0073] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0074] In terms of w / v, the concentration of the surfactant is 0.01% to 0.1%, preferably 0.01% to 0.08%, and more preferably 0.02% to 0.05%.

[0075] In some embodiments of the present invention, the pharmaceutical composition, wherein

[0076] The concentration of the anti-CD3×CD20 bispecific antibody or its antigen-binding fragment is (0.5 mg / ml±0.05 mg / ml) to (20 mg / ml±2 mg / ml), preferably (1±0.1 mg / ml to (10 mg / ml±1 mg / ml), and more preferably (2±0.2 mg / ml) to (5 mg / ml±0.5 mg / ml).

[0077] In some embodiments of the present invention, the pharmaceutical composition comprises:

[0078] 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 20±2 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, pH 4.5-5.5; or

[0079] 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 20±2 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, pH 5.0-5.5; or

[0080] 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 20±2 mM citrate buffer, 230±10 mM sucrose or trehalose, 0.02%±0.01% (w / v) polysorbate 80, pH 5.5-6.0; or

[0081] 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 10±2 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, pH 5.5±0.5; or

[0082] 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 10±2 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, pH 5.5±0.2; or

[0083] 2 mg / L anti-CD3×CD20 bispecific antibody, 10 mM citrate buffer, 234 mM sucrose or trehalose, 0.04% (w / v) polysorbate 80, pH 5.5.

[0084] In some embodiments of the present invention, the pharmaceutical composition is a liquid preparation or a lyophilized preparation.

[0085] In some embodiments of the present invention, the pharmaceutical composition is for subcutaneous administration or intravenous administration.

[0086] The pharmaceutical composition according to any one of the present invention is used for treating or preventing cancer; preferably, the cancer is selected from one or more of acute B lymphocytic leukemia, diffuse large B cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia and Burkitt's lymphoma; preferably, the non-Hodgkin's lymphoma is CD20-positive B cell non-Hodgkin's lymphoma.

[0087] Another aspect of the present invention relates to use of any one of the pharmaceutical compositions of the present invention in the preparation of a medicament for treating or preventing cancer; preferably, the cancer is selected from one or more of acute B lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia and Burkitt's lymphoma; preferably, the non-Hodgkin's lymphoma is CD20-positive B-cell non-Hodgkin's lymphoma.

[0088] Another aspect of the present invention relates to a method for treating or preventing cancer, comprising the step of administering to a subject in need thereof an effective amount of the pharmaceutical composition described in any one of the present invention; preferably, the cancer is selected from one or more of acute B lymphocytic leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia and Burkitt's lymphoma; preferably, the non-Hodgkin's lymphoma is CD20-positive B-cell non-Hodgkin's lymphoma.

[0089] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined in this article, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention belongs. It should be understood that the present invention is not limited to specific methods, reagents, compounds, compositions or biological systems, and of course the above can be changed. It should also be understood that the terms used in the present invention are only for the purpose of describing specific embodiments and are not intended to be limiting.

[0090] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes combinations of two or more polypeptides, etc.

[0091] The term "pharmaceutical composition" or "preparation" refers to a mixture containing one or more antibodies of the present invention and other components, such as physiologically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity.

[0092] The term "liquid preparation" refers to a preparation in a liquid state, and is not intended to refer to a resuspended lyophilized preparation. The liquid preparation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state change methods, such as spray drying). The liquid preparation dosage form of the present invention is also referred to as a water injection.

[0093] The term "aqueous liquid formulation" refers to a liquid formulation using water as a solvent. In some embodiments, the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying and / or freezing to maintain stability (eg, chemical and / or physical stability and / or biological activity).

[0094] The term "lyophilized preparation" is also called lyophilized powder injection, which refers to sterile powder for injection prepared by freeze drying. The lyophilized preparation dosage form of the present invention is also called lyophilized powder injection.

[0095] The term "excipient" refers to an agent that can be added to a formulation to provide desired properties (e.g., consistency, increased stability) and / or to adjust osmotic pressure. Examples of commonly used excipients include, but are not limited to, sugars, polyols, amino acids, surfactants, and polymers.

[0096] As used herein, "about" when referring to a measurable value (e.g., an amount, a duration, etc.) is intended to encompass variations of ±20% or ±10% relative to the particular value, including ±5%, ±1%, and ±0.1%, as such variations are suitable for performing the disclosed methods.

[0097] The term "buffer pH of about 4.5-6.5" refers to an agent that, through the action of its acid / base conjugate components, renders the solution containing the agent resistant to pH changes. The buffer used in the formulations of the present invention may have a pH in the range of about 4.5 to about 6.5, or a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.0 to about 6.0.

[0098] Herein, examples of "buffers" that control the pH within this range include acetates (e.g., sodium acetate), succinates (e.g., sodium succinate), gluconic acid, histidine, histidine hydrochloride, methionine, citrate, phosphate, citrate / phosphate, imidazole, acetic acid, acetate, citrate, combinations thereof, and other organic acid buffers.

[0099] "Histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine and histidine salts, such as histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate, such as a histidine buffer containing histidine and histidine hydrochloride; the histidine buffer of the present invention also includes a histidine buffer containing histidine and acetate (such as sodium salt or potassium salt).

[0100] "Citrate buffer" is also called citrate buffer, which is a buffer including citrate ions. Examples of citrate buffer include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, etc. The preferred citrate buffer is citric acid-sodium citrate buffer.

[0101] "Acetate buffer" is also called acetate buffer, which is a buffer comprising acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, etc. The preferred acetate buffer is acetic acid-sodium acetate buffer.

[0102] The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, salts, polyols and their metabolites as defined below, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, trehalose, arginine or its salts (such as arginine hydrochloride), glycine, alanine (α-alanine, β-alanine), betaine, leucine, lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, γ-aminobutyric acid (GABA), opines, alanine, octopine, strombine) and trimethylamine N-oxide (TMAO), human serum albumin (hsa), bovine serum albumin (bsa), α-casein, globulin, α-lactalbumin, LDH, lysozyme, myoglobin, ovalbumin and RNAaseA. Some stabilizers, such as sodium chloride, calcium chloride, magnesium chloride, mannitol, sorbitol, sucrose, etc., can also play a role in controlling osmotic pressure. The stabilizer specifically used in the present invention is selected from one or more of polyols, amino acids, salts, and sugars. Preferred salts are sodium chloride, preferred sugars are sucrose and trehalose, and preferred polyols are sorbitol and mannitol. Preferred amino acids are arginine or its salt (such as arginine hydrochloride), glycine, and proline. Preferred stabilizers are sodium chloride, mannitol, sorbitol, sucrose, trehalose, arginine hydrochloride, glycine, proline, sodium chloride-sorbitol, sodium chloride-mannitol, sodium chloride-sucrose, sodium chloride-trehalose, arginine hydrochloride-mannitol, and arginine hydrochloride-sucrose.

[0103] The term "surfactant" generally includes agents that protect proteins such as antibodies from air / solution interface-induced stress, solution / surface-induced stress to reduce the aggregation of antibodies or minimize the formation of particulate matter in the formulation. Exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, polyethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene-polyoxypropylene copolymers (poloxamers, Pluronic), sodium lauryl sulfate (SDS).

[0104] The term "isotonic" means that the formulation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured using a vapor pressure or freezing point depression osmometer.

[0105] The term "stable formulation" is a formulation in which the antibody substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or upon storage. A pharmaceutical formulation may be stable even if the antibody contained therein does not retain 100% of its chemical structure or biological function after a certain period of storage. In some cases, an antibody formulation that maintains about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of its structure or function after a certain period of storage may also be considered "stable". Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee, ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991), and Jones, A. (1993) Adv. Drug Delivery Rev. 10: 29-90 (both incorporated by reference).

[0106] The stability of the formulation can be measured by determining the percentage of natural antibodies remaining therein (and other methods) after the formulation has been stored at a certain temperature for a certain period of time. Among other methods, the percentage of natural antibodies can be measured by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography [SEC-HPLC]), and "natural" means not aggregated and not degraded. In some embodiments, the stability of the protein is determined by the percentage of monomeric protein in a solution with a low percentage of degradation (e.g., fragmentation) and / or aggregated protein. In some embodiments, the formulation can be stably stored at room temperature, about 25-30°C or 40°C 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 longer, with no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% aggregated form of antibodies.

[0107] Stability can be measured by determining the percentage of antibody that migrates in the more acidic fraction of the main fraction of the antibody (the "major charged form") during ion exchange (among other methods), where stability is inversely proportional to the percentage of the antibody in the acidic form. The percentage of "acidified" antibody can be measured by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography [CEX-HPLC]), among other methods. In some embodiments, an acceptable level of stability means that after a formulation is stored at a certain temperature for a certain period of time, no more than about 49%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the antibody is detectable in the acidic form. The storage period before measuring stability can be 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 longer. When evaluating stability, the temperature at which the pharmaceutical preparation is allowed to be stored can be any temperature in the range of about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 2-8°C, about 5°C, about 25°C, or about 40°C.

[0108] An antibody "retains its physical stability" in the pharmaceutical composition if it shows substantially no signs of, for example, aggregation, precipitation, and / or denaturation when visually inspected for color and / or clarity or when measured by UV light scattering or by size exclusion chromatography. Aggregation is the process by which individual molecules or complexes associate covalently or non-covalently to form aggregates. Aggregation may proceed to the extent that a visible precipitate is formed.

[0109] The stability of the formulation, e.g., physical stability, can be assessed by methods known in the art, including measuring the apparent extinction (absorbance or optical density) of a sample. Such extinction measurements are related to the turbidity of the formulation. The turbidity of a formulation is, in part, an inherent property of the protein dissolved in solution, and is typically measured by turbidimetry and measured in nephelometric turbidity units (NTU).

[0110] The turbidity level that varies with, for example, the concentration of one or more components in the solution (e.g., protein and / or salt concentration) is also referred to as the "emulsification" or "emulsified appearance" of the formulation. The turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity. The reference standard for determining the turbidity level of a pharmaceutical composition can be based on the European Pharmacopoeia standard (European Pharmacopoeia, 4th edition, "Directorate for the Quality of Medicine of the Council of Europe" (EDQM), Strasbourg, France). According to the European Pharmacopoeia standard, a clear solution is defined as a solution having a turbidity less than or equal to the turbidity of a reference suspension having about 3 according to the European Pharmacopoeia standard. Turbidimetric turbidity measurements can detect Rayleigh scattering in the absence of association or non-ideal effects, which generally varies linearly with concentration. Other methods for evaluating physical stability are well known in the art.

[0111] An antibody "retains its chemical stability" in a pharmaceutical composition if its chemical stability at a given time point is such that the antibody is considered to still retain its biological activity as defined below. Chemical stability can be assessed, for example, by detecting or quantifying chemically altered forms of the antibody. Chemical alterations can include size changes (e.g., shortening), which can be assessed using, for example, size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time of flight mass spectrometry (MALDI / TOF MS). Other types of chemical alterations include charge changes (e.g., occurring as a result of deamidation or oxidation), which can be assessed by, for example, ion exchange chromatography.

[0112] An antibody "retains its biological activity" in a pharmaceutical composition if the antibody in the pharmaceutical composition is biologically active for its intended purpose. For example, a formulation of the invention is considered stable if, after storage at a temperature such as 5°C, 25°C, 45°C, etc. for a certain period of time (e.g., 1 to 12 months), the anti-CD20×CD3 antibody contained in the formulation binds to the CD3 antigen with an affinity that is at least 90%, 95% or more of the binding affinity of the antibody before the storage. Binding affinity can also be determined, for example, by ELISA or plasma resonance techniques.

[0113] In the context of the present invention, in a pharmacological sense, a "therapeutically effective amount" or "effective amount" of an antibody refers to an amount that is effective in preventing or treating or alleviating the symptoms of a disorder that the antibody can effectively treat. In the present invention, a "therapeutically effective amount" or "therapeutically effective dose" of a drug is any amount of the drug that protects a subject from the onset of the disease or promotes disease regression when used alone or in combination with another therapeutic agent, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of damage or disability caused by disease pain. The ability of a drug to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that predict human efficacy, or by measuring the activity of the agent in an in vitro assay. A therapeutically effective amount of a drug includes a "prophylactically effective amount," i.e., any amount of the drug that inhibits the development or recurrence of a disease when given alone or in combination with other therapeutic drugs to a subject at risk of the disease or a subject with a recurring disease.

[0114] The term "subject" or "patient" is intended to include mammalian organisms. Examples of subjects / patients include humans and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the invention, the subject is a human.

[0115] The terms "administering", "giving" and "treating" refer to introducing a composition comprising a therapeutic agent into a subject using any of a variety of methods or delivery systems known to those skilled in the art. The routes of administration of anti-CD20×CD3 antibodies include intravenous, intramuscular, subcutaneous, peritoneal, spinal or other parenteral routes of administration, such as injection or infusion. "Parenteral administration" refers to administration methods that are usually by injection other than enteral or topical administration, including but not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraframe, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, intradural and intrasternal injection and infusion and in vivo electroporation.

[0116] The term "antibody" as used herein should be understood to include complete antibody molecules and antigen-binding fragments thereof. The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment") as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to human CD20 or CD3 or an epitope thereof.

[0117] The term "full-length antibody" or "complete antibody molecule" as used herein refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (about 50-70 kDa at full length) and two light (L) chains (about 25 kDa at full length) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into highly variable complementary determining regions (CDRs) and regions separated by more conservative framework regions (FRs). Each VH or VL region consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system, such as effector cells, and the first component (Clq) of the classical complement system.

[0118] As used herein, the term "CDR" refers to the complementary determining region within the variable sequence of an antibody. There are three CDRs in each variable region of the heavy and light chains, which are named HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region. The exact boundaries of these CDRs are defined differently in different systems.

[0119] The precise amino acid sequence boundaries of the variable region CDRs of the antibodies of the invention can be determined using any of a number of well-known schemes, including Chothia (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)), based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat (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), the international ImMunoGeneTics database (IMGT) (1999 Nucleic Acids Research, 27, 209-212), and the North CDR definition based on affinity propagation clustering using a large number of crystal structures. The CDRs of the antibodies of the present invention can be defined by a person skilled in the art according to any scheme in the art (e.g., different assignment systems or combinations).

[0120] As used herein, "antigen-binding fragments" include fragments of antibodies or derivatives thereof, generally including at least one fragment of the antigen-binding region or variable region (e.g., one or more CDRs) of a parent antibody, which retains at least some of the binding specificity of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments. When the binding activity of an antibody is expressed on a molar concentration basis, the binding fragment or its derivative generally retains at least 10% of the antigen-binding activity of the parent antibody. Preferably, the binding fragment or its derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen-binding affinity of the parent antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly change its biological activity (referred to as "conservative variants" or "functional conservative variants" of antibodies).

[0121] The term "bispecific antibody" refers to an antibody molecule that can bind to two independent antigens or has binding specificity for different epitopes within the same antigen. For example, in some embodiments, one arm of the bispecific antibody molecule binds to a tumor-associated antigen and the other arm binds to an immune cell-associated antigen (e.g., a CD3 molecule), which can activate and initiate cellular immune-related mechanisms at tumor cells. The structure of bispecific antibodies is diverse.

[0122] Pharmaceutical preparations

[0123] The present invention provides a pharmaceutical composition comprising an anti-CD3×CD20 bispecific antibody and an optional buffer, a stabilizer, and a surfactant.

[0124] In some embodiments of the present invention, the provided pharmaceutical composition comprises an anti-CD3×CD20 bispecific antibody or an antigen-binding fragment thereof, wherein the anti-CD3×CD20 bispecific antibody comprises two first protein functional regions that specifically bind to human CD20 and one second protein functional region that specifically binds to human CD3.

[0125] The bispecific antibody in the pharmaceutical composition of the present invention can be a murine antibody, a chimeric antibody, or a humanized antibody, preferably a humanized antibody, and more preferably any one of the anti-CD3×CD20 bispecific antibodies derived from Chinese patent application CN2022100685060 and patent application PCT / CN2023072510. All the contents disclosed in the patent applications with application numbers CN2022100685060 and PCT / CN2023072510 are incorporated herein by reference.

[0126] In some embodiments of the present invention, the anti-CD3×CD20 bispecific antibody comprises the following polypeptide chains:

[0127] (1) A polypeptide chain represented by formula (I) and formula (II) having a CD20 binding domain:

[0128] VH1-CH1-Fc1 formula (I),

[0129] VL1-CL formula (II), and

[0130] (2) A polypeptide chain represented by formula (III) and formula (II-2) having CD20 and CD3 binding domains:

[0131] VH1-CH1-L1-VL2-L2-VH2-L3-Fc2 formula (III);

[0132] VL1-CL formula (Ⅱ-2);

[0133] Wherein VH represents the heavy chain variable region; VL represents the light chain variable region; Fc comprises CH2 and CH3; CH1, CH2 and CH3 represent domain 1, domain 2 and domain 3 of the heavy chain constant region, respectively; VL represents the light chain variable region; CL represents the light chain constant region; L1, L2 and L3 independently represent connecting peptides, each connecting peptide is the same or different; optionally, there is a hinge region between CH1 and Fc1; formula (Ⅰ), (Ⅱ), (Ⅱ-2) and (Ⅲ) are connected sequentially from N-terminus to C-terminus, respectively; the antigen binding site formed by VH1 and VL1 binds to CD20, and the antigen binding site formed by VL2 and VH2 binds to CD3.

[0134] Further, in some embodiments, the connecting peptide of the present invention comprises a polypeptide composed of n amino acid sequences GGGGS (SEQ ID NO: 20) in series, or comprises a polypeptide in which serine (S) and alanine (A) are sequentially connected to the N-terminus of a polypeptide composed of n amino acid sequences shown in SEQ ID NO: 20 in series, wherein n is independently selected from 1, 2, 3, 4, 5 or 6.

[0135] Furthermore, in some embodiments, the formula (I) and the formula (II), and the formula (III) and the formula (II-2) are connected via a disulfide bond; and the formula (I) and the formula (III) are connected via a disulfide bond and a Knob into Hole structure of the CH3 domain;

[0136] Further, in some embodiments, Fc1 in formula (I) is knob-Fc, and Fc2 in formula (III) is hole-Fc; or Fc1 in formula (I) is hole-Fc, and Fc2 in formula (III) is knob-Fc.

[0137] Furthermore, in some embodiments, the CH1-Fc1 in formula (I) and the Fc2 in formula (III) are in the form of IgG, such as IgG1, IgG2, IgG3 or IgG4, and / or the CL in formula (II) and formula (II-2) is from λ or κ chain.

[0138] In some embodiments, the bispecific antibody of the present invention, wherein:

[0139] The VH1 comprises the amino acid sequence as shown in SEQ ID NO:7, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO:7; and

[0140] The VL1 comprises the amino acid sequence as shown in SEQ ID NO:8; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:8.

[0141] The VH2 comprises the amino acid sequence as shown in SEQ ID NO: 15, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO: 15; and

[0142] The VL2 comprises the amino acid sequence as shown in SEQ ID NO:16; or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:16.

[0143] In some embodiments, the bispecific antibody of the present invention, wherein:

[0144] The polypeptide chain of formula (I) comprises the amino acid sequence as shown in SEQ ID NO:19, or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the amino acid sequence as shown in SEQ ID NO:19; and

[0145] The polypeptide chain of formula (II) and (II-2) comprises the amino acid sequence as shown in SEQ ID NO:17, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:17.

[0146] In some embodiments, the bispecific antibody of the present invention, wherein the polypeptide chain of formula (III) comprises the amino acid sequence as shown in SEQ ID NO:18, or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:18.

[0147] In some embodiments, the present invention provides an anti-CD3×CD20 bispecific antibody named TZT7, wherein TZT7 comprises the following polypeptide chains:

[0148] (1) A polypeptide chain represented by formula (I) and formula (II) having a CD20 binding domain:

[0149] VH1-CH1-Fc1 formula (I),

[0150] VL1-CL formula (II), and

[0151] (2) A polypeptide chain represented by formula (III) and formula (II-2) having CD20 and CD3 binding domains:

[0152] VH1-CH1-L1-VL2-L2-VH2-L3-Fc2 formula (III);

[0153] VL1-CL formula (Ⅱ-2);

[0154] The amino acid sequence of the polypeptide chain of formula (I) is shown as SEQ ID NO:19, the amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are shown as SEQ ID NO:17, and the amino acid sequence of the polypeptide chain of formula (III) is shown as SEQ ID NO:18.

[0155] In some embodiments, the concentration of the anti-CD3×CD20 bispecific antibody or antigen-binding fragment thereof is 0.5 mg / ml±0.05 mg / ml to 50 mg / ml±5 mg / ml, or 0.5 mg / ml±0.05 mg / ml to 40 mg / ml±4 mg / ml, or 0.5 mg / ml±0.05 mg / ml to 30 mg / ml±3 mg / ml, or 0.5 mg / ml±0.05 mg / ml to 20 mg / ml±2 mg / ml. ml, or 1±0.1mg / ml~10mg / ml±1mg / ml, or 2±0.2mg / ml~8mg / ml±0.8mg / ml, or 2±0.2mg / ml~6mg / ml±0.6mg / ml, or 2±0.2mg / ml~5mg / ml±0.5mg / ml, the concentration of non-limiting embodiments is for example: 2±0.5mg / ml, or 2±0.4mg / ml, or 2±0.3mg / ml, or 2±0.1mg / ml.

[0156] In some embodiments, in the pharmaceutical composition of the present invention, the buffer is a buffer containing acetic acid or citric acid or histidine.

[0157] In some embodiments, in the pharmaceutical composition of the present invention, the stabilizer is a stabilizer comprising sugar.

[0158] In some embodiments, in the pharmaceutical composition of the present invention, the surfactant is a surfactant comprising polysorbate.

[0159] In some embodiments, the buffer in the pharmaceutical composition of the present invention is selected from one or more of acetate buffer, citrate buffer or histidine buffer.

[0160] In some embodiments, the buffer in the pharmaceutical composition of the present invention is an acetate buffer, preferably, the acetate buffer is an acetate-sodium acetate buffer or an acetate-potassium acetate buffer, preferably an acetate-sodium acetate buffer. Preferably, the pH of the acetate buffer in the pharmaceutical composition of the present invention is about 4.5 to about 5.5.

[0161] In some embodiments, the buffer in the pharmaceutical composition of the present invention can be a histidine buffer, including a histidine-hydrochloride buffer or a histidine-acetate buffer, preferably a histidine-hydrochloride buffer. More preferably, the histidine-hydrochloride buffer is made of histidine and histidine hydrochloride, preferably L-histidine and L-histidine monohydrochloride. Preferably, the pH of the histidine buffer in the pharmaceutical composition of the present invention is about 5.0 to about 6.5.

[0162] In some embodiments, the buffer in the pharmaceutical composition of the present invention is a citrate buffer, preferably, the citrate buffer is a citric acid-sodium citrate buffer. Preferably, the pH of the citrate buffer in the pharmaceutical composition of the present invention is about 5.5 to about 6.0.

[0163] In some embodiments, the concentration of the buffer in the pharmaceutical composition of the present invention is 5±0.5~100±10mM, 10±1~100±10mM, or 10±1~80±10mM, or 10±1~60±10mM, or 10±1~50±10mM, or 10±1~40±5mM, or 10±1~30±5mM, or 15±2~30±3mM, or 10±1~20±2mM, non-limiting examples of concentrations include 8±2mM, 10±1mM, 10±2mM, 15±2mM, 20±2mM, or 20±1mM.

[0164] In some embodiments, the pH of the pharmaceutical composition of the present invention is 4.5±0.5 to 6.5±0.5, 4.5±0.5 to 5.5±0.5, 5.5±0.5 to 6.0±0.5, 5.0±0.5 to 6.5±0.5, 4.5±0.3 to 6.5±0.3, 4.5±0.3 to 5.5±0.3, 5.5±0.3 to 6.0±0. 3, 5.0±0.3~6.5±0.3, 4.5±0.2~6.5±0.2, 4.5±0.2~5.5±0.2, 5.5±0.2~6.0±0.2, or 5.0±0.2~6.5±0.2, non-limiting examples of concentrations include 4.4, 4.6, 5.4, 5.6, 5.8, 5.9, 6.1, 6.4, or 6.6.

[0165] In some embodiments, the stabilizer in the pharmaceutical composition of the present invention is selected from one or more of arginine hydrochloride, sodium chloride, mannitol, sorbitol, sucrose and trehalose. Preferably, the stabilizer comprises sugar, and more preferably, the stabilizer is selected from trehalose or sucrose.

[0166] The concentration of the stabilizer in the pharmaceutical composition of the present invention is about 10mM to 400mM, 20mM to 300mM, 30mM to 200mM, 100±10 to 300±30mM, 120±10 to 280±20mM, 140±10 to 280±20mM, 150±10 to 280±20mM, 150±10 to 250±20mM, 200 ±20~230±20mM, 200±10~230±10mM, or 220±10~230±10mM, the concentration of non-limiting examples are: 230±10mM, 225mM, 226mM, 227mM, 228mM, 229mM, 230mM, 231mM, 232mM, 233mM, 234mM, or 235mM.

[0167] In some embodiments, the surfactant in the pharmaceutical composition of the present invention comprises polysorbate, and the surfactant can be selected from polysorbate 80, polysorbate 20 and poloxamer 188. The preferred surfactant is polysorbate 80. In terms of w / v, the concentration of the surfactant in the pharmaceutical composition of the present invention is about 0.001% to 0.1%, 0.01% to 0.1%, 0.01% to 0.08%, 0.02% to 0.08%, 0.02% to 0.06%, 0.02% to 0.05%, or 0.02% to 0.04%. The concentration of non-limiting embodiments is about 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, or 0.08%, preferably 0.02%, 0.04%.

[0168] In some embodiments, the buffer in the pharmaceutical composition of the present invention is selected from acetate buffer, the stabilizer is selected from sucrose or trehalose, and the surfactant is selected from polysorbate 80.

[0169] In some embodiments, the buffer in the pharmaceutical composition of the present invention is selected from citrate buffer, the stabilizer is selected from sucrose or trehalose, and the surfactant is selected from polysorbate 80.

[0170] In some embodiments, the buffer in the pharmaceutical composition of the present invention is selected from histidine buffer, the stabilizer is selected from sucrose or trehalose, and the surfactant is selected from polysorbate 80.

[0171] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 20±2 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 4.5-5.5.

[0172] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 20±2 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.0-5.5.

[0173] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 20±2 mM citrate buffer, 230±10 mM sucrose or trehalose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.5-6.0.

[0174] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 10±2 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, and a pH of 5.5±0.2.

[0175] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 10±2 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.03%±0.01% (w / v) polysorbate 80, and a pH of 5.5±0.2.

[0176] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 4.5-5.5.

[0177] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.0-5.5.

[0178] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM citrate buffer, 230±10 mM trehalose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.5-6.0;

[0179] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM citrate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.5-6.0;

[0180] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 10±1 mM citrate buffer, 234±10 mM trehalose or sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.5-6.0.

[0181] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 10±1 mM citrate buffer, 234±5 mM trehalose or sucrose, 0.02%±0.01% (w / v) polysorbate 80, and a pH of 5.5-6.0.

[0182] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM citrate buffer, 234±10 mM sucrose, 0.04%±0.01% (w / v) polysorbate 80, and pH 5.5±0.2.

[0183] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM citrate buffer, 234±5 mM sucrose, 0.04%±0.01% (w / v) polysorbate 80, and pH 5.5±0.2.

[0184] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 10±1 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, and pH 5.5±0.5.

[0185] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 10±1 mM citrate buffer, 234±5 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, and pH 5.5±0.5.

[0186] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 10±1 mM citrate buffer, 234±10 mM sucrose, 0.04%±0.01% (w / v) polysorbate 80, and pH 5.5±0.2.

[0187] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 10±1 mM citrate buffer, 234±5 mM sucrose, 0.04%±0.01% (w / v) polysorbate 80, and pH 5.5±0.2.

[0188] In some embodiments, the pharmaceutical composition of the present invention comprises 2±0.1 mg / L of anti-CD3×CD20 bispecific antibody, 20±1 mM histidine buffer, 230±10 mM sucrose, 0.04%±0.01% (w / v) polysorbate 80, and a pH of 5.0, 6.0 or 6.5.

[0189] In another aspect, the present invention provides a liquid preparation or a lyophilized preparation comprising the above pharmaceutical composition.

[0190] In some embodiments, the liquid preparation of the pharmaceutical composition of the present invention is subjected to freezing, primary drying, and secondary drying steps to obtain the lyophilized preparation of the present invention.

[0191] Medical Uses

[0192] On the other hand, the present invention also provides the use of the pharmaceutical composition described in any embodiment of the present invention in the preparation of a medicament for treating or preventing CD20-positive mediated diseases, and a method of administering a therapeutically effective amount of the pharmaceutical composition described in any embodiment of the present invention to an individual or patient in need to treat or prevent CD20-positive mediated diseases, especially cancer.

[0193] In some embodiments, the cancer of the present invention is selected from follicular lymphoma, CD20-positive B-cell non-Hodgkin's lymphoma, acute B-cell leukemia, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, non-Hodgkin's lymphoma, chronic myeloid leukemia or Burkitt's lymphoma.

[0194] The entire contents of the following three prior applications are hereby incorporated by reference into this application:

[0195] Prior application 1: Chinese application, application number 2022100685060, application date January 20, 2022, invention name is “Anti-CD3 and anti-CD20 bispecific antibodies and their uses”;

[0196] Prior application 2: PCT application, application number PCT / CN2023 / 072510, application date January 17, 2023, invention name “Anti-CD3 and anti-CD20 bispecific antibodies and uses thereof”;

[0197] Prior application 3: Chinese application, application number 2023100718073, application date January 17, 2023, the name of the invention is “Anti-CD3 and anti-CD20 bispecific antibodies and their uses”.

[0198] Advantageous Effects of the Invention

[0199] The pharmaceutical composition of the present invention has good stability and / or safety, can effectively treat or prevent cancer, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0200] Figure 1 : Schematic diagram of the structure of the anti-CD3×CD20 bispecific antibody TZT7.

[0201] Figure 2 : Binding and dissociation curves of the antibody drug composition and the antigen human CD3ε protein.

[0202] The partial sequences involved in the present invention are shown in Table A below:

[0203] Table A

[0204]

[0205]

[0206]

[0207]

[0208] DETAILED DESCRIPTION

[0209] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0210] Preparation Example 1: Design and preparation of bispecific antibody molecule TZT7

[0211] 1. Construction of the expression vector of the bispecific antibody molecule TZT7

[0212] The expression vectors were constructed in the following order: GeneScript Biotech was commissioned to synthesize the gene encoding JSCD20 LC-2, which was digested with BSPQI and connected to the HXT2 vector to obtain the first expression vector HXT2-JS CD20 LC-2. GeneScript Biotech was commissioned to synthesize the gene encoding JSCD20 HC-2, which was digested with HindIII and NheI and connected to HXT4s-Mut-h to obtain the second expression vector HXT4s-JS CD20 HC-2h. GenScript Biotech was commissioned to synthesize the gene encoding JSCD3ScFv, which was digested with HindIII and NheI and ligated to the HX4-JSCD20HC-2-G4 FC mut b vector to obtain the third expression vector HX4-JS CD20 HC-2JSCD3ScFv NVL-G4 FC b V2.

[0213] 2. Expression of the bispecific antibody molecule TZT7

[0214] Transient expression and purification of the bispecific antibody molecule TZT7: The three plasmids constructed above were extracted using an endotoxin-controlled kit for subsequent mammalian cell expression. The CHO-K1 cells being cultured (which have been modified at the genome level to make them suitable for transient expression) were counted. When the cell density was 2*10 6 / mL-6*10 6 / mL, use CD CHO medium for subculture and amplification, and dilute the cell density to 1.5*10 one day before transfection. 6 / mL-2.0*10 6 / mL, the next day when the cell density reached about 3.5*10 6 / mL for transfection. First add one-tenth of the transfection volume of culture medium, then add 1-2μg / mL of plasmid, and finally add 3-14μg / mL of PEI, mix well and incubate at room temperature for no more than 30 minutes, and finally slowly add the transfection mixture to the pre-treated cells, mixing while adding. The transfected mixture is placed in a shaker for culture at 36.5℃, 120rmp, and 7% CO2. The culture period is 6-10 days after transfection, and the feed is replenished every two days.

[0215] 3. Purification of the bispecific antibody molecule TZT7

[0216] After the above transfection mixture was cultured, the precipitate was discarded by centrifugation at 1000g for 10min, and then the cell supernatant was collected by centrifugation at 12000g for 30min and sterile filtered. Purification was performed using an AKTA Avant purifier. The column filled with Praesto Jetted A50 affinity filler was first CIPed for 15-20min with 0.1M NaOH, and then equilibrated with PBS buffer for 3-5CV before loading. After loading, the affinity chromatography elution buffer was used for elution, and finally the target protein was eluted with acetic acid-sodium acetate buffer at pH 3.8. The sample was neutralized with 1M Tris buffer, adjusted to pH 5.5, and the sample was sent to SEC-HPLC for testing the purity of the sample. The remaining samples were purified in the next step; weak cationic filler EMD COOM filler was used for purification, the equilibrium solution was pH 5.5 50mM acetic acid-sodium acetate system, the eluent was pH 5.5 50mM acetic acid-sodium acetate + 1M NaCl buffer system, and the target protein was collected by linear elution. The final SEC-HPLC monomer purity can reach more than 95%, and the bispecific antibody molecule TZT7 is obtained. The schematic diagram of the structure of the bispecific antibody molecule TZT7 is shown in the figure Figure 1 shown.

[0217] The amino acid sequence of the bispecific antibody molecule TZT7 is as follows:

[0218] HC1 (HXT4s-JS CD20HC-2Mut h): SEQ ID NO: 19;

[0219] LC (HXT2-JS CD20 LC-2): SEQ ID NO: 17;

[0220] HC2 (HX4-JS CD20 HC-2JS CD3ScFv NVL-G4 FC b V2): SEQ ID NO: 18.

[0221] The CDR and variable region amino acid sequences of the bispecific antibody molecule TZT7 are shown in Table B.

[0222] Table B: CDR and variable region amino acid sequences of bispecific antibody molecules

[0223] (KABAT solution)

[0224]

[0225] Example 1: Stability test of a pharmaceutical composition comprising an anti-CD3×CD20 bispecific antibody

[0226] 1.1 Experimental samples

[0227] TZT7 stock samples were prepared using Millipore 3 0.11m 2 Ultrafiltration concentration (UF / DF) was performed, and then dialyzed into the corresponding buffer containing a stabilizer. The concentration of TZT7 was determined respectively, and the corresponding buffer containing a stabilizer and a surfactant were added according to the determined concentration, and the final concentration of TZT7 was adjusted to 2 mg / mL, and the contents of other components were respectively as shown in Table 2, to obtain the test drug compositions numbered FS1-1 to FS1-6, FS3-1 and FS3-4, and the corresponding dosage form was a water injection. The above-mentioned newly prepared different test drug compositions were aseptically filled into 2R cillin bottles, and then stability sampling and testing were performed according to the stability investigation conditions.

[0228] TZT7 stock samples were prepared using Millipore 3 0.11m 2 Ultrafiltration concentration (UF / DF) was performed, and then dialyzed into the corresponding buffer containing a stabilizer. The concentration of TZT7 was measured respectively, and the corresponding buffer containing a stabilizer and a surfactant were added according to the measured concentration, and the final concentration of TZT7 was adjusted to 2 mg / mL, and the contents of other components were shown in Table 2; then freeze-dried, and stability sampling was performed according to the stability test conditions. After sampling, the TZT7 concentration was re-dissolved with water for injection to 2 mg / mL, and the drug compositions to be tested numbered Lyo-1 to Lyo-2 were obtained, and the corresponding dosage forms were freeze-dried powder injections. The freeze-drying process parameters are shown in Table 1.

[0229] The components of different drug compositions to be tested are shown in Table 2, wherein: acetate buffer refers to acetic acid-sodium acetate buffer; histidine buffer refers to L-histidine-L-histidine hydrochloride buffer; citrate buffer refers to citric acid-sodium citrate buffer.

[0230] Table 1: Freeze-drying process parameters

[0231]

[0232] Table 2: Components of different tested pharmaceutical compositions

[0233]

[0234] 1.2 Experimental methods and results

[0235] Stability test conditions include:

[0236] (1) Placed in an environment at 5±3℃ for 2 weeks, 4 weeks or 3 months, hereinafter referred to as “long-term (5±3℃)”;

[0237] (2) Placed in an environment of 40±2℃ for 2W, 4W or 3M, hereinafter referred to as "high temperature (40±2℃)";

[0238] (3) Placed in an environment at 25±2℃ for 2W, 4W or 3M, hereinafter referred to as "accelerated (25±2℃)".

[0239] 1.2.1 Appearance test

[0240] Test method: Use visual inspection to test the appearance of each pharmaceutical composition (sample). During the test, ensure that the light intensity of the clarity detector is maintained between 1000 Lux and 1500 Lux. Keep the sample at the same level as the eye, and gently shake or turn it upside down to avoid bubbles; perform visual inspection in front of a black background and a white background respectively. Record the visual inspection results in terms of color, opalescence, and visible foreign matter.

[0241] Test results: All samples had no obvious visible foreign matter and no obvious opalescence at T0; samples FS3-1 and FS3-4 had a small amount of particles after being placed under high temperature conditions for four weeks; samples Lyo-1 and Lyo-2 had no obvious visible foreign matter after being placed under high temperature conditions for four weeks, as shown in Table 3. The above results show that freeze-dried powder injections are more stable than water injections.

[0242] Table 3: Appearance results

[0243]

[0244] Note: “ / ” means not tested.

[0245] 1.2.2SEC-HPLC (size exclusion high performance liquid chromatography) purity test

[0246] Test method: Using a SEC column ( BEH SEC purity was detected by HPLC (Waters instrument) with a flow rate of 7.8mm×30cm, 3.5μm. Each drug composition (sample) was diluted to 1.0mg / mL with the mobile phase, mixed and added to the inner liner tube, and directly injected for analysis; the mobile phase composition was 50mM phosphate solution, 300mM anhydrous sodium sulfate, pH 6.8±0.2. The relative percentages of the main peak, polymers and oligomers were calculated by area normalization method. The components of the reference product used in the test include: 2.65mM citric acid monohydrate, 7.34mM trisodium citrate dihydrate, 234mM sucrose, polysorbate 80 0.2mg / mL, pH5.5±0.2; the pretreatment method and test method of the reference product were the same as those of the sample. The test parameters are shown in Table 4 below.

[0247] Table 4: SEC-HPLC purity test parameters

[0248]

[0249] Test results: SEC-HPLC purity test results are shown in Table 5 and Table 6.

[0250] Table 5: Water injection SEC-HPLC purity test results (%)

[0251]

[0252]

[0253] Note: “ / ” means not tested, and “ND” means not detected.

[0254] Table 6: Lyophilized powder injection SEC-HPLC purity test results (%)

[0255]

[0256]

[0257] From the results in Table 5 above, it can be seen that the purity of all water injection samples showed a significant downward trend after being placed under high temperature conditions for 4 weeks, which was manifested by a significant increase in the proportion of fragment peaks and aggregate peaks. The acetic acid system (FS1-1, FS1-2, FS1-3) and the citric acid system (FS3-1 and FS3-4) performed relatively well; the SEC-HPLC purity of some samples showed a downward trend after being placed under accelerated conditions for 4 weeks; all samples showed no significant change in SEC-HPLC purity after being placed under long-term conditions for 4 weeks.

[0258] From the results in Table 6 above, it can be seen that all powder injection samples showed no significant changes after being placed under different conditions for 4 weeks.

[0259] 1.2.3icIEF purity test

[0260] Test method:

[0261] Take 8.00μL sample solution (1.4mg / mL) and ultrapure water (blank sample) and mix them with 92.0μL icIEF MasterMix respectively, centrifuge at 10000rpm for 5min, take 75.0μL supernatant and add it to 96-well plate, pay attention to avoid bubbles, and then centrifuge the 96-well plate at 1000g for 5min to remove bubbles. The components of the reference product used in the test include: 2.65mM citric acid monohydrate, 7.34mM trisodium citrate dihydrate, 234mM sucrose, polysorbate 80 0.2mg / mL, pH5.5±0.2; the pretreatment method and test method of the reference product are the same as the sample. The sample preparation and method parameters are shown in Tables 7 and 8 below.

[0262] Table 7: icIEF Master Mix Preparation

[0263]

[0264] Table 8: Sample run method parameters

[0265]

[0266] Test results: The icIEF purity test results are shown in Tables 9 and 10.

[0267] Table 9: Purity test results of water injection icIEF (%)

[0268]

[0269]

[0270] Note: “ / ” means not tested, and “ND” means not detected.

[0271] Table 10: Lyophilized powder injection icIEF purity test results (%)

[0272]

[0273] From the results in Table 9 above, it can be seen that after being placed under high temperature conditions for 4 weeks, the icIEF purity of all water injection samples showed a downward trend, mainly due to the increase of acidic peaks; the acetic acid system (FS1-2, FS1-3) and the citric acid system (FS3-1, FS3-4) performed relatively well, and the icIEF purity of FS1-6 decreased the fastest, and there was no significant difference between other samples; after being placed under accelerated conditions for 4 weeks, the icIEF purity of some samples showed a downward trend; after being placed for 4 weeks for a long time, the icIEF purity of all samples did not change significantly. From the results in Table 10 above, it can be seen that after all powder injection samples were placed under different temperature conditions for 4 weeks, the icIEF purity of all samples did not change significantly.

[0274] 1.2.4 rCE-SDS purity test

[0275] Test method: Use a capillary electrophoresis instrument (Maurice Instrument) for rCE-SDS purity detection. Take buffer (Maurice 1×Sample Buffer PLUS) to dilute each drug composition (sample) to 1 mg / mL, then take 95 μL of the diluted sample and mix it evenly with 5 μL of 2-mercaptoethanol, centrifuge at 3000 rpm at room temperature for 30 seconds, and then incubate at 70°C for 15 minutes. After incubation, cool to room temperature and centrifuge at 12000 rpm at room temperature for 5 minutes. Take out 75 μL of sample solution from the sample tube into a 96-well plate to avoid bubbles, centrifuge at 1000g at room temperature for 10 minutes, and detect using a capillary electrophoresis instrument (Maurice). The ingredients of the reference sample used in the test include: 2.65mM citric acid monohydrate, 7.34mM trisodium citrate dihydrate, 234mM sucrose, polysorbate 80 0.2mg / mL, pH5.5±0.2; the pretreatment method and test method of the reference sample are the same as those of the sample.

[0276] Test results: The rCE-SDS purity test results are shown in Table 11.

[0277] Table 11: rCE-SDS purity test results (%)

[0278]

[0279] Note: “ / ” means not tested.

[0280] From the results in Table 11 above, it can be seen that the rCE-SDS purity of the water injection samples FS1-4, FS1-5, FS3-1 and FS3-4 samples decreased significantly after being placed under high temperature conditions for 4 weeks, while other prescriptions did not show significant changes; some samples were placed under accelerated conditions for 4 weeks, and all prescriptions did not show significant changes; all samples were placed under long-term conditions for 4 weeks, and the rCE-SDS purity did not show significant changes.

[0281] From the results in Table 11 above, it can be seen that after all powder injection samples were placed under different temperature conditions for 4 weeks, the rCE-SDS purity of all samples did not change significantly.

[0282] 1.2.5 nrCE-SDS purity results

[0283] Test method: Use a capillary electrophoresis instrument (Maurice Instrument) for nrCE-SDS purity detection. Calculate the amount of diluent and sample required to dilute each drug composition (sample) to 1 mg / mL according to the final volume of 100 μL, and then add the diluent, 5.0 μL 0.25M NEM (ethylmaleimide), and sample to a 1.5 mL EP tube and mix well; ultrapure water is used as a blank control, and the dilution ratio is consistent with the reference product (the dilution multiple is at least 2 times). Centrifuge at 3000 rpm at room temperature for 30 seconds, and then incubate at 70°C for 5 minutes. After incubation, cool to room temperature and centrifuge at 12000 rpm at room temperature for 5 minutes. Take out 75 μL of sample solution from each sample tube to a 96-well plate to avoid bubbles, centrifuge at 1000g at room temperature for 10 minutes, and detect using a capillary electrophoresis instrument (Maurice). The ingredients of the reference product used in the test include: citric acid 0.56 mg / mL, sodium citrate 2.16 mg / mL, sucrose 78.73 mg / mL and polysorbate 80 0.2 mg / mL; the pretreatment method and test method of the reference product are the same as those of the sample.

[0284] Test results: The nrCE-SDS purity test results are shown in Table 12.

[0285] Table 12: nrCE-SDS purity test results (%)

[0286]

[0287]

[0288] Note: “ / ” means not tested.

[0289] From the results in Table 12 above, it can be seen that the nrCE-SDS purity of all water injection samples showed a downward trend after being placed under high temperature conditions for 4 weeks, especially FS1-4, FS1-5 and FS1-6, where the purity decreased at the fastest rate; some samples showed a slight downward trend in nrCE-SDS purity after being placed under accelerated conditions for 4 weeks; all samples showed no obvious changes in nrCE-SDS purity after being placed under long-term conditions for 4 weeks.

[0290] From the results in Table 12 above, it can be seen that after all powder injection samples were placed under different temperature conditions for 4 weeks, the nrCE-SDS purity of all samples did not change significantly.

[0291] 1.2.6 Binding activity test

[0292] Test method: This method uses an indirect method, using Recombinant Human CD3E (C-6His) as an antigen coated on a 96-well plate. TZT7 injection can bind to C-6His, and Mouse Anti-Human IgG4 Fc-HRP can specifically bind to TZT7 bound to the solid phase antigen (C-6His). Under the action of hydrogen peroxide, it can catalyze TMB to develop a blue color. The depth of blue is positively correlated with the amount of Mouse Anti-Human IgG4 Fc-HRP bound, and it turns yellow after termination with 2M hydrochloric acid. The absorbance (OD value) is measured at a wavelength of 450nm / 620nm using an enzyme reader, and the ability of the TZT7 antibody to bind to C-6His is examined by the effective binding activity (EC50) of the standard curve. See the table below for details:

[0293] Table 13: Binding activity test method

[0294]

[0295]

[0296] Test results: The binding activity test results are shown in Table 14.

[0297] Table 14: Binding activity test results (%)

[0298]

[0299] Note: “ / ” means not tested.

[0300] From the results in Table 14 above, it can be seen that the binding activity of all samples did not change significantly after being placed under high temperature, accelerated or long-term conditions for 4 weeks.

[0301] 1.2.7 Cell activity test

[0302] Test method: TZT7 is a bispecific antibody targeting CD20 and CD3. One end of TZT7 binds to CD20 to recruit target cells with high CD20 expression, and the other end binds to CD3 to activate T cells, thereby enhancing the killing effect of T cells on target cells. In this research system, the target cells are Raji (B lymphoma cells) cells, and the effector cells are Jurkat NFAT D9 cells that overexpress the downstream fluorescent reporter gene NFAT-luc in Jurkat cells. NFAT (nuclear factor of activated Tcells) is a key transcription factor for downstream signals of T cell activation. Adding TZT7 antibody to this system will activate the first signal of T cell activation. When T cells are activated, NFAT expression is upregulated, and the expression of the luc luciferase gene coupled to it is also upregulated. This study detected the biological activity of TZT7 injection by measuring the expression of the luc luciferase gene.

[0303] Test results: The cell activity test results are shown in Table 15.

[0304] Table 15: Cell activity test results (%)

[0305]

[0306] Note: “ / ” means not tested.

[0307] From the results in Table 15 above, it can be seen that the cell activity of all samples did not change significantly after being placed under high temperature, accelerated or long-term conditions for 4 weeks.

[0308] 1.2.8 Insoluble particle test

[0309] Test method: HIAC insoluble particle counter was used as the detection instrument, and the insoluble particles in each pharmaceutical composition (sample) were statistically determined by the light obscuration method. The operation mode used was Run counter, and the setting parameters were as shown in Table 16 below.

[0310] Table 16: Insoluble Particle Test Parameters

[0311]

[0312]

[0313] Test results: The test results of insoluble particles are shown in Table 17. Among them, ≥10 μm means that the particle size is between 10 and 25 μm, and ≥25 μm means that the particle size is greater than or equal to 25 μm.

[0314] Table 17: Insoluble particles test results (unit: particles / mL)

[0315]

[0316] Note: “ / ” means not tested.

[0317] From the results in Table 17 above, it can be seen that after all samples were placed under high temperature, accelerated and long-term conditions for 4 weeks, the insoluble particles showed no significant changes.

[0318] In summary, from the results of FS1-1 to FS1-6, FS3-1 and FS3-4, it can be seen that under high temperature conditions, there is no significant difference in the results of insoluble particles, binding activity and cell activity among all samples; from the appearance results, FS1-6, FS3-1 and FS3-4 showed an increase in particles and enhanced opalescence after 4 weeks of storage, and the appearance was relatively unstable in the water injection state; from the SEC-HPLC results, the content of aggregates and fragments peaks in all water injection prescriptions showed an upward trend, among which the acetic acid system (FS1-1, FS1-2, FS1-4) showed an upward trend. -3) and citric acid systems (FS3-1 and FS3-4) performed relatively well; from the results of rCE-SDS, the purity of FS1-4, FS1-5, FS3-1 and FS3-4 decreased significantly, and other prescriptions did not show significant changes; from the results of nrCE-SDS purity, the purity of FS1-4, FS1-5 and FS1-6 decreased the fastest; from the results of icIEF purity, the acetic acid system (FS1-2, FS1-3) and citric acid system (FS3-1, FS3-4) performed relatively well, the purity of FS1-6 icIEF decreased the fastest, and there was no significant difference between other samples. Under accelerated conditions, the purity of SEC-HPLC, icIEF, and nrCE-SDS showed a slight downward trend, and in the long-term stability experiment, the indicators of all samples did not change significantly.

[0319] From the results of Lyo-1 and Lyo-2, it can be seen that under high temperature conditions, judging from the stability results of appearance, SEC-HPLC, icIEF, rCE-SDS and nrCE-SDS, the stability of the lyophilized powder injection is higher than that of the water injection; from the perspective of various indicators, there is no obvious difference among the samples of lyophilized powder injection.

[0320] Based on the results of the water injection prescription investigation, the acetic acid buffer system (FS1-1, 2, 3) and the citric acid buffer system (FS3-1, 4) are more stable than other prescriptions. In order to obtain a more stable preparation, the product is considered to be freeze-dried, and the citric acid system is selected for subsequent freeze-dried prescription investigation. And from the stability data of the freeze-dried prescription of the citric acid buffer system (Lyo-1, 2), the stability of the freeze-dried powder injection is good under different temperature conditions. Considering the cost of raw materials and excipients, sucrose (Lyo-1) is selected as a protective agent. In order to reduce the number of insoluble particles in the sample after reconstitution, 0.04% Tween content is finally selected as the preferred prescription for freeze-dried preparations. The freeze-dried preparation prescription is selected as: pH 5.5, 234mM sucrose and 0.04% polysorbate 80 are added to the 10mM citric acid system.

[0321] Example 2: Antigen affinity determination of pharmaceutical compositions

[0322] 2.1 Experimental methods

[0323] CD3ε receptor binding experiment: This experiment uses the molecular interaction analyzer OctetRED96e from ForteBio and uses biomembrane interferometry technology to perform affinity analysis on the antibody drug composition. This experiment uses Protein G probe to capture the antibody drug composition (5μg / mL), and then injects gradient dilutions of human CD3ε protein. The gradient dilution concentrations of human CD3ε protein are 100nM and 25nM to detect the binding signal. The kinetic 1:1 model analysis of Octet analysis software Data Analysis Software is used to fit the affinity K D value.

[0324] 2.2 Experimental Results

[0325] CD3ε binding experiment: The experimental results are shown in Table 18 and Figure 2 As shown, the pharmaceutical composition has specific binding activity with human CD3ε protein. CD3ε affinity K of the pharmaceutical composition D The value is 2.03E-09M.

[0326] Table 18: Summary of affinity between antibody drug composition and human CD3E protein

[0327] Receptor Name Sample name ka(1 / Ms) kd(1 / s) <![CDATA[K D (M)]]> human CD3ε TZT7 1.10E+05 2.24E-04 2.03E-09

[0328] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A pharmaceutical composition comprising the following items (a) to (d): (a) an anti-CD3×CD20 bispecific antibody or an antigen-binding fragment thereof, comprising a first protein functional region that specifically binds to human CD20 and a second anti-protein functional region that specifically binds to human CD3, wherein the first protein functional region is an anti-CD20 immunoglobulin or an antigen-binding fragment thereof, and the second protein functional region is an anti-CD3 single-chain antibody or an antigen-binding fragment thereof, in: The first protein functional region comprises a heavy chain variable region VH1 and a light chain variable region VL1, wherein the VH1 comprises HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, and HCDR3 as shown in SEQ ID NO:3; the VL1 comprises LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, and LCDR3 as shown in SEQ ID NO:6; The second protein functional region comprises a heavy chain variable region VH2 and a light chain variable region VL2, wherein the VH2 comprises HCDR1 as shown in SEQ ID NO:9, HCDR2 as shown in SEQ ID NO:10, and HCDR3 as shown in SEQ ID NO:11; the VL2 comprises LCDR1 as shown in SEQ ID NO:12, LCDR2 as shown in SEQ ID NO:13, and LCDR3 as shown in SEQ ID NO:14; The concentration of the anti-CD3×CD20 bispecific antibody or antigen-binding fragment thereof is (0.5 mg / ml±0.05 mg / ml) to (20 mg / ml±2 mg / ml), preferably (1±0.1 mg / ml to (10 mg / ml±1 mg / ml), and more preferably (2±0.2 mg / ml) to (5 mg / ml±0.5 mg / ml); (b) a buffer selected from one or more of an acetate buffer, a citrate buffer and a histidine-hydrochloride buffer; the concentration of the buffer is (5±0.5) to (50±5) mM, preferably (10±1) to (50±5) mM, more preferably (10±1) to (20±2) mM, and more preferably 20±1 mM; (c) a stabilizer selected from one or more of sucrose and trehalose; the concentration of the stabilizer is (100±10) to (300±30) mM, preferably (150±10) to (280±20) mM, more preferably (200±20) to (230±20) mM, and particularly preferably 230±10 mM; (d) a surfactant; one or more selected from polysorbate 80 and polysorbate 20; the concentration of the surfactant is 0.01% to 0.1%, preferably 0.01% to 0.08%, and more preferably 0.02% to 0.05% on a w / v basis; The pH of the pharmaceutical composition is (4.5±0.5) to (6.5±0.5), preferably (4.5±0.5) to (6.0±0.5), (4.5±0.5) to (5.5±0.5) or (5.5±0.5) to (6.0±0.5).

2. The pharmaceutical composition according to claim 1, wherein The first protein functional region and the second protein functional region are independently 1, 2 or more than 2; Preferably, the number of the first protein functional regions is 2, and the number of the second protein functional region is 1; Preferably, the number of the first protein functional region is one, and the number of the second protein functional region is one.

3. The pharmaceutical composition according to claim 1 or 2, wherein The VH1 comprises the amino acid sequence shown in SEQ ID NO:7, and the VL1 comprises the amino acid sequence shown in SEQ ID NO:8; and / or The VH2 comprises the amino acid sequence shown in SEQ ID NO:15, and the VL2 comprises the amino acid sequence shown in SEQ ID NO:

16.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein The immunoglobulin is of human IgG1 subtype, human IgG2 subtype, human IgG3 subtype or human IgG4 subtype.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein The heavy chain constant region of the immunoglobulin is Ig gamma-1chain C region or Ig gamma-4chain C region; the light chain constant region is Ig kappa chain C region.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the anti-CD3×CD20 bispecific antibody comprises a polypeptide chain represented by formula (I), formula (II), formula (III) and formula (II-2): VH1-CH1-Fc1 formula (I), VL1-CL formula (II), VH1-CH1-L1-VL2-L2-VH2-L3-Fc2 formula (III); and VL1-CL formula (II-2); in, Formulas (I), (II), (II-2), and (III) are connected sequentially from the N-terminus to the C-terminus; Fc1 and Fc2 are Fc segments containing CH2 and CH3; CH1, CH2, and CH3 represent domain 1, domain 2, and domain 3 of the heavy chain constant region, respectively; CL indicates light chain constant region; L1, L2 and L3 represent connecting peptides, and L1, L2 and L3 are the same or different; Optionally, there is a hinge region between CH1 and Fc1.

7. The pharmaceutical composition according to claim 6, wherein Formula (I) and Formula (II), as well as Formula (III) and Formula (II-2) are connected via a disulfide bond; and Formula (I) and Formula (III) are connected via a disulfide bond and / or a Knob into Hole structure of the CH3 domain; Preferably, Fc1 in formula (I) is knob-Fc, and Fc2 in formula (III) is hole-Fc; or Fc1 in formula (I) is hole-Fc, and Fc2 in formula (III) or (III) is knob-Fc.

8. The pharmaceutical composition according to claim 6 or 7, wherein The connecting peptide comprises a polypeptide composed of n amino acid sequences shown in SEQ ID NO: 20 connected in series, or a polypeptide composed of n amino acid sequences shown in SEQ ID NO: 20 connected in series with serine and alanine connected in sequence at the N-terminus of the polypeptide, wherein n is independently selected from 1, 2, 3, 4, 5 and 6.

9. The pharmaceutical composition according to claim 6, wherein The amino acid sequence of the polypeptide chain represented by formula (I) is shown in SEQ ID NO: 19, The amino acid sequences of the polypeptide chains of formula (II) and formula (II-2) are both as shown in SEQ ID NO: 17, and The amino acid sequence of the polypeptide chain represented by formula (III) is shown in SEQ ID NO:

18.

10. The pharmaceutical composition according to any one of claims 1 to 9, comprising: 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 20±2 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, pH 4.5-5.5; or 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 20±2 mM acetate buffer, 230±10 mM sucrose, 0.02%±0.01% (w / v) polysorbate 80, pH 5.0-5.5; or 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 20±2 mM citrate buffer, 230±10 mM sucrose or trehalose, 0.02%±0.01% (w / v) polysorbate 80, pH 5.5-6.0; or 2±0.2 mg / L of anti-CD3×CD20 bispecific antibody, 10±2 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, pH 5.5±0.5; or 2±0.2 mg / L anti-CD3×CD20 bispecific antibody, 10±2 mM citrate buffer, 234±10 mM sucrose or trehalose, 0.04%±0.01% (w / v) polysorbate 80, pH 5.5±0.2; or 2 mg / L anti-CD3×CD20 bispecific antibody, 10 mM citrate buffer, 234 mM sucrose or trehalose, 0.04% (w / v) polysorbate 80, pH 5.

5.

11. The pharmaceutical composition according to any one of claims 1 to 10, which is a liquid preparation or a lyophilized preparation.

12. Use of the pharmaceutical composition according to any one of claims 1 to 11 in the preparation of a medicament for treating or preventing cancer; preferably, the cancer is selected from one or more of acute B lymphocytic leukemia, diffuse large B cell lymphoma, chronic lymphocytic leukemia, follicular lymphoma, non-Hodgkin's lymphoma, chronic myeloid leukemia and Burkitt's lymphoma; preferably, the non-Hodgkin's lymphoma is CD20-positive B-cell non-Hodgkin's lymphoma.

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