Recombinant fully human anti-TIGIT monoclonal antibody formulation, its preparation method and uses

Abstract

This invention provides formulations comprising anti-TIGIT antibodies, including anti-TIGIT antibodies, buffers, stabilizers, and surfactants. Furthermore, this invention also provides the use of these formulations in the treatment or prevention of diseases.

Description

Technical Field

[0001] This invention relates to the field of antibody formulations. More specifically, this invention relates to pharmaceutical formulations comprising recombinant fully human antibodies against T-cell immune receptors containing immunoglobulin motifs and tyrosine inhibitory motif domains of immune receptors (also known as anti-TIGIT antibodies), particularly stable liquid formulations, methods for preparing said pharmaceutical formulations, and therapeutic and / or prophylactic uses of said pharmaceutical formulations. Background Technology

[0002] Drug stability is a crucial indicator of drug efficacy and safety. Obtaining a suitable formulation is essential for maintaining the efficacy and safety of a drug throughout its shelf life. However, due to the complexity of antibodies themselves and their degradation pathways, it is currently impossible to predict the formulation conditions required for optimizing antibody stability. This is especially true considering that different antibodies often possess very different CDR sequences, and these sequence differences lead to varying stability properties in solution. Therefore, given the stringent requirements for the safety and efficacy of human antibodies, it is necessary to optimize the formulation individually for each antibody.

[0003] TIGIT (a T-cell immune receptor containing immunoglobulin motifs and immunoreceptor tyrosine inhibitory motif domains, also known as WUCAM, Vstm3, or Vsig9) was initially discovered as a member of the CD28 family using bioinformatics alignment. Antibodies binding to human TIGIT have been shown to be useful in cancer treatment. See, for example, WO 2006 / 124667. In mouse models, antibody blockade of both PD-L1 and TIGIT can lead to a synergistic enhancement of CD8+ T cell-mediated tumor rejection. Groganet et al. (2014) J. Immunol. 192(1) Suppl. 203.15; Johnston et al. (2014) Cancer Cell 26:1-15. Similar results were obtained in animal models of melanoma. Inozume et al. (2014) J. Invest. Dermatol. 134: S121-Abstract 693. Anti-TIGIT antibodies that bind with high specificity to TIGIT have been described, for example, in PCT application number PCT / CN2019 / 097665.

[0004] Although several anti-TIGIT antibody formulations have been proposed, there remains a need in the field for novel pharmaceutical formulations containing sufficiently stable anti-TIGIT antibodies suitable for administration to human subjects. Furthermore, for such antibody formulations, simplicity and ease of use in formulation formulation are also advantageous. Invention Overview

[0006] This invention addresses the aforementioned needs by providing a pharmaceutical formulation containing an antibody that specifically binds to TIGIT. The antibody formulation of this invention exhibits excellent stability against various stability-influencing factors (such as temperature, repeated freeze-thaw cycles, and agitation).

[0007] In one aspect, the present invention therefore provides a liquid antibody formulation comprising (i) an anti-TIGIT antibody protein; (ii) a buffer; (iii) a stabilizer; and (iv) a surfactant.

[0008] In one embodiment, the anti-TIGIT antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO:7 or a sequence having at least 90% identity with it, and the light chain variable region comprises the sequence of SEQ ID NO:8 or a sequence having at least 90% identity with it.

[0009]

[0010] In one embodiment, the anti-TIGIT antibody comprises:

[0011] The heavy chain VH CDR1 of -YTFTEYYMH (SEQ ID NO:1);

[0012] The heavy chain VH CDR2 of -IISPSAGSTKYAQKFQG (SEQ ID NO:2);

[0013] The heavy chain VH CDR3 of -ARDHDIRLAGRLADY (SEQ ID NO:3);

[0014] -RASQGISSWLA (SEQ ID NO:4) light chain VL CDR1;

[0015] -AASSLQS (SEQ ID NO:5) light chain VL CDR2; and

[0016] -QQAVILPIT (SEQ ID NO:6) light chain VL CDR3.

[0017] In one embodiment, the anti-TIGIT antibody is an IgG4 type antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises the sequence of SEQ ID NO:9 or a sequence having at least 90% identity with it, and wherein the light chain comprises the sequence of SEQ ID NO:10 or a sequence having at least 90% identity with it.

[0018]

[0019] Preferably, the anti-TIGIT antibody is the anti-TIGIT monoclonal antibody ADI-30278 disclosed in PCT application number PCT / CN2019 / 097665 (international application date: July 25, 2019), which consists of the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10.

[0020] In one embodiment, the anti-TIGIT antibody is an anti-TIGIT antibody recombinantly expressed in 293 cells or CHO cells.

[0021] In one embodiment, the concentration of anti-TIGIT antibody in the liquid antibody formulation of the present invention is about 1-150 mg / ml. In another embodiment, the concentration of anti-TIGIT antibody in the liquid antibody formulation of the present invention is about 10-100 mg / ml, preferably 20-60 mg / ml, and especially about 50 mg / ml. In other embodiments, the concentration of anti-TIGIT antibody in the liquid antibody formulation of the present invention is about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 mg / ml.

[0022] In one embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-50 mM. In another embodiment, the concentration of the buffer in the liquid antibody formulation of the present invention is about 5-30 mM, for example, about 5, 10, 15, 20, 25, or 30 mM. In one embodiment, the buffer is a histidine buffer, preferably, the buffer is composed of histidine and histidine hydrochloride. In a preferred embodiment, the buffer is about 5-30 mM histidine buffer, for example, 10-20 mM, such as about 10 mM histidine.

[0023] In one embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 50-500 mM. In another embodiment, the concentration of the stabilizer in the liquid antibody formulation of the present invention is about 100-400 mM, for example about 100, 150, 200, 250, 300, 350, or 400 mM.

[0024] In one embodiment, the stabilizer is selected from polyols (e.g., sorbitol), sugars (e.g., sucrose), amino acids (e.g., arginine or arginine hydrochloride), 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, or 80 mg / ml sorbitol. In yet another embodiment, the stabilizer comprises about 20-60 mg / ml of sucrose, such as 20, 30, 40, 50, or 60 mg / ml sucrose. In a preferred embodiment, the stabilizer further comprises arginine, such as about 25 mM-200 mM, for example 50-150 mM, or 50 mM-120 mM, preferably about 60-100 mM, for example about 60, 65, 70, 75, 80, 85, or 90 mM arginine. Preferably, arginine is provided as a stabilizer by arginine hydrochloride.

[0025] 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 sorbitol and about 50-100 mM arginine, or about 30-60 mg / ml sucrose and about 50-100 mM arginine, can be used.

[0026] In a preferred embodiment, the liquid antibody formulation of the present invention comprises about 30-60 mg / ml (e.g., about 50 mg / ml) of sorbitol, or about 20-30 mg / ml (e.g., about 25 mg / ml) of sorbitol and 80-90 mM (e.g., about 85 mM) of arginine.

[0027] 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 another 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, or 0.8 mg / ml.

[0028] In one embodiment, the surfactant is a nonionic surfactant. In another 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.

[0029] 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 any value in the range of about 5.0-6.0, such as 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, and more preferably, the pH is 5.2.

[0030] In one embodiment, the liquid antibody formulation of the present invention comprises:

[0031] (i) Anti-TIGIT antibody protein at approximately 10-100 mg / ml, such as 10-80 mg / ml, for example, 10, 20, 30, 40, 50, 60, 70 or 80 mg / ml antibody protein;

[0032] (ii) Approximately 5-50 mM of histidine buffer, such as 5-30 mM, for example 5, 10, 15, 20, 25, 30 mM;

[0033] (iii) about 50-300 mM of sorbitol, sucrose, or any combination thereof, for example, 50, 80, 100, 120, 140, 160, 180, 200, 250, 300 mM.

[0034] (iv) Approximately 0.1-1 mg / ml of polysorbate 80, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0 mg / ml; and

[0035] (v) Optionally, approximately 50-120 mM of arginine,

[0036] The pH of the liquid formulation is about 5.0-5.5, for example, about 5.2.

[0037] For example, the liquid antibody formulation may contain

[0038] (i) Approximately 10-60 mg / ml of anti-TIGIT antibody protein, such as 20, 30, 40, 50, 55, 60 mg / ml;

[0039] (ii) Approximately 10 mM histidine buffer;

[0040] (iii) Approximately 10-50 mg / ml of sorbitol or sucrose, such as 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / ml of sorbitol, or such as 10, 15, 20, 25, 30, 35, 40, 45, 50 mg / ml of sucrose.

[0041] (iv) Approximately 0.2–0.8 mg / ml, e.g., 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg / ml, e.g., 0.3–0.6 mg / ml polysorbate 80; and

[0042] (v) Approximately 60-100 mM, such as 60, 65, 70, 75, 80, 85, 90, 95, 100 mM, especially arginine at approximately 85 mM.

[0043] The pH of the liquid formulation is about 5.0-5.5, for example, about 5.2.

[0044] In a preferred embodiment, the liquid antibody formulation comprises:

[0045] (i) Approximately 50 mg / ml of anti-TIGIT antibody protein, approximately 0.21 mg / ml of histidine, approximately 1.81 mg / ml of histidine hydrochloride, approximately 25.00 mg / ml of sorbitol, approximately 17.91 mg / ml of arginine hydrochloride, approximately 0.50 mg / ml of polysorbate 80, pH approximately 5.2; or

[0046] (ii) Approximately 50 mg / ml of anti-TIGIT antibody protein, approximately 0.21 mg / ml of histidine, approximately 1.81 mg / ml of histidine hydrochloride, approximately 25.00 mg / ml of sorbitol, approximately 17.91 mg / ml of arginine hydrochloride, approximately 0.20 mg / ml of polysorbate 80, pH approximately 5.2; or

[0047] (iii) Approximately 0.21 mg / ml histidine, approximately 1.81 mg / ml histidine hydrochloride, approximately 40.00 mg / ml sucrose, approximately 17.91 mg / ml arginine hydrochloride, approximately 0.20 mg / ml polysorbate 80, pH 5.2.

[0048] The liquid formulation of the present invention can be stored stably for a long period of time, for example, at least 24 months or longer. In one embodiment, the liquid formulation of the present invention can be stored stably for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months, or longer at temperatures ranging from about -80°C to about 45°C, for example, at -80°C, about -30°C, about -20°C, about 0°C, about 5°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C.

[0049] In one embodiment, the liquid formulation of the present invention can be stably stored for at least 24 months. In another embodiment, the liquid formulation of the present invention is stable at at least 40°C. In yet another embodiment, the liquid formulation of the present invention 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, for example, about 25°C for at least 2 months, preferably at least 3 months, more preferably at least 6 months. In yet 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.

[0050] In one embodiment, the stability of the formulation can be indicated by detecting changes in its appearance, visible foreign matter, protein content, turbidity, purity, and / or charge variants. In one embodiment, the stability of the liquid formulation of the present invention can be detected in a forced high-temperature stress test, such as after storage at 40°C ± 2°C for at least 1 week, 2 weeks, or preferably 1 month; or in an accelerated test, such as after storage at 25°C ± 2°C for at least 1 month or 2 months; or in a long-term test, such as after storage at 5°C ± 3°C for at least 2 months or 3 months; or in a shaking test (e.g., shaking at 650 rpm for 5 days at room temperature in the dark); and / or in a freeze-thaw test (e.g., repeated freeze-thaw cycles at -30°C / room temperature 6 times). In one embodiment, the stability of the liquid formulation of the present invention is detected relative to an initial value, such as the initial value on day 0 of storage, or the initial value before the shaking or freeze-thaw test.

[0051] In one embodiment, the stability of the liquid formulation of the present invention is visually inspected after storage, or after an shaking experiment, or after a freeze-thaw experiment, wherein the liquid formulation of the present invention remains clear to slightly opalescent in appearance, is a colorless to pale yellow liquid, and is free of foreign matter. In one embodiment, visual inspection under a clarity meter shows no visible foreign matter in the formulation. In one embodiment, the stability of the liquid formulation of the present invention is checked by measuring changes in protein content after storage, or after an shaking experiment, or after a freeze-thaw experiment, wherein, for example by ultraviolet spectrophotometry (UV), the change rate of protein content relative to the initial value does not exceed 20%, preferably not more than 10%, for example 7-8%, more preferably not more than 5%, 2%, or 1%. In one embodiment, the stability of the liquid formulation of the present invention is checked by measuring changes in turbidity after storage, or after an shaking experiment, or after a freeze-thaw experiment, wherein, for example by OD... 350mmThe stability of the liquid formulation of the present invention is checked by measuring the change in purity of the liquid formulation of the present invention relative to the initial value, as determined by the method of size exclusion high performance liquid chromatography (SEC-HPLC), wherein the change in monomer purity (or change in the main peak value) relative to the initial value does not exceed 10%, for example, not more than 5%, 4%, 3%, or for example, not more than 2%, preferably not more than 1%. In one embodiment, the stability of the liquid formulation of the present invention is checked by measuring the change in purity of the liquid formulation of the present invention after storage, or after a shaking experiment, or after a freeze-thaw experiment, wherein the change in monomer purity (or change in the main peak value) relative to the initial value does not exceed 10%, for example, not more than 5%, 4%, 3%, 2%, or 1%, by non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS). In one embodiment, the stability of the liquid formulation of the present invention is detected by cation exchange high-performance liquid chromatography (CEX-HPLC) after storage, or after a shaking test, or after a freeze-thaw test, wherein the sum of the changes in the charge variants (main component, acidic component, and basic component) of the antibody relative to the initial value does not exceed 50%, for example, not exceeding 40%, 30%, 20%, 10%, or 5%, and / or the change in the main component does not exceed 20%, 15%, 10%, 8%, or 5%. In one embodiment, the stability of the liquid formulation of the present invention is detected by direct ELISA after storage, or after a shaking test, or after a freeze-thaw test, 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, or 130%, preferably 90-110%.

[0052] 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 day 0 of storage,

[0053] (i) The change in the main peak is less than 1% as measured by SEC-HPLC, and / or the formulation has a purity of more than 96%, preferably more than 97% or 98%;

[0054] (ii) The change in the main peak is less than 2% as measured by the non-reducing CE-SDS method, and / or the formulation has a purity of more than 96%, preferably more than 97% or 98%.

[0055] (iii) As measured by CEX-HPLC, the sum of the changes in the components (main component, acidic component, and basic component) of the anti-TIGIT antibody protein in the formulation does not exceed 40% and / or the change in the main component does not exceed 20%.

[0056] For example, after storage at 40°C ± 2°C for 1 month, the total change does not exceed approximately 40% (e.g., not exceeding 35%, 30%, 25%, 20%, 15%, 10%) or the change in the main component does not exceed 20% (e.g., not exceeding 15%, 12%, 10%, 8%).

[0057] For example, after storage at 25°C for 2 months, the total change value does not exceed about 20% (e.g., not more than 15%, 14%, 13%, 12%) or the change value of the main component does not exceed about 15% (e.g., not more than 10%, 8%, 7%, 6%, 5%).

[0058] (iv) The relative binding activity of the anti-TIGIT antibody protein in the formulation is 70%-130%, for example, 90, 93, 95, 98, 100, 103, 105, 108, 110, 115, 120%, for example, 90%-110%, as measured by ELISA.

[0059] In a preferred embodiment, the liquid formulation of the present invention is stable under shaking and / or repeated freeze-thaw cycles.

[0060] Preferably, the formulation is stable under shaking or repeated freeze-thaw cycles, for example, after shaking for 5 days at 650 rpm in the dark at room temperature or after repeated freeze-thaw cycles at -30°C / room temperature 6 times, and has one or more of the following characteristics:

[0061] (i) The change in the main peak is less than 1% as measured by SEC-HPLC, and / or the formulation has a purity of more than 96%, preferably more than 97%, 98%, or 99%.

[0062] (ii) The change in the main peak is less than 1% as measured by the non-reducing CE-SDS method, and / or the formulation has a purity of more than 96%, preferably more than 97% or 98%.

[0063] (iii) The total variation of each component (main component, acidic component and basic component) of the anti-TIGIT antibody protein in the formulation, as measured by CEX-HPLC, does not exceed 2%;

[0064] (iv) The relative binding activity of the anti-TIGIT antibody protein in the formulation is 70%-130%, for example, 90%-110%, as measured by ELISA.

[0065] In one aspect, the liquid formulation 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 formulation is an intravenous infusion.

[0066] On the other hand, the present invention provides a solid antibody formulation obtained by solidifying the liquid antibody formulation of the present invention. The solidification process is carried out by, for example, crystallization, spray drying, or freeze-drying. In a preferred embodiment, the solid antibody formulation is, for example, a lyophilized powder for injection. The solid antibody formulation can be reconstituted in a suitable solvent before use to form the reconstituted formulation of the present invention. The reconstituted formulation is also a liquid antibody formulation of the present invention. In one embodiment, the suitable solvent is selected from water for injection, organic solvents for injection, including but not limited to oils for injection, ethanol, propylene glycol, etc., or combinations thereof.

[0067] In one aspect, the present invention provides a delivery device comprising the liquid or solid antibody formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a pre-filled syringe comprising the liquid or solid antibody formulation of the present invention, for example for intravenous, subcutaneous, intradermal, or intramuscular injection, or intravenous infusion.

[0068] In another aspect, the present invention provides a method for delivering anti-TIGIT antibody protein to a subject, such as a mammal, comprising the step of administering the subject a liquid or solid antibody formulation of the present invention, said delivery being carried out, for example, by using a delivery device using a pre-filled syringe.

[0069] In another aspect, the present invention provides the use of the liquid or solid antibody formulation of the present invention for the preparation of a delivery device or pre-filled syringe or drug for blocking the binding of TIGIT to CD155 in a subject to reduce or eliminate the immunosuppressive effect of TIGIT, or for the preparation of a delivery device (e.g., a pre-filled syringe) or drug for the treatment or prevention of tumors or pathogen infections in a subject, said tumors being, for example, cancers, including but not limited to gastrointestinal cancers, such as colon cancer.

[0070] The present invention also provides a method for reducing or eliminating the immunosuppressive effect of TIGIT by administering to a subject the liquid antibody preparation or solid antibody preparation of the present invention, or a delivery device (e.g., a pre-filled syringe) or drug containing the liquid antibody preparation or solid antibody preparation of the present invention.

[0071] The present invention also provides a method for treating a subject’s disease, such as the aforementioned tumor or pathogen infection, by administering the liquid antibody preparation or solid antibody preparation of the present invention, or a delivery device (e.g., a pre-filled syringe) containing the liquid antibody preparation or solid antibody preparation, or a drug, to the subject.

[0072] Other embodiments of the present invention will become clear from the following detailed description. Brief description of the attached diagram

[0074] The preferred embodiments of the invention described in the following detailed description will be better understood when read in conjunction with the accompanying drawings. The drawings show presently preferred embodiments for illustrative purposes. However, it should be understood that the invention is not limited to the precise arrangement and means of the embodiments shown in the drawings.

[0075] Figure 1 shows the trend of antibody charge variants-principal components under different pH conditions, as detected by CEX-HPLC in the pH screening experiment of Example 1.

[0076] Figure 2 shows the trend of antibody charge variant - acidic component changes under different pH conditions, as detected by CEX-HPLC in the pH screening experiment of Example 1 (CEX-HPLC method).

[0077] Figure 3 shows the trend of charge variant changes detected by CEX-HPLC in the 40°C stability confirmation experiment of Example 2.

[0078] Figure 4 shows the trend of charge variant changes detected by CEX-HPLC in the 25°C stability confirmation experiment of Example 2. Invention Details

[0080] Before describing the invention in detail, it should be understood that the invention is not limited to the specific methods and experimental conditions described herein, as these methods and conditions can be modified. Furthermore, the terminology used herein is for illustrative purposes only and is not intended to be restrictive.

[0081] definition

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this invention, the following terms are defined below.

[0083] The term “about” when used in conjunction with a numeric value means to cover a range of numeric values ​​that have a lower limit of 5% less than the specified numeric value and an upper limit of 5% greater than the specified numeric value.

[0084] When the term “and / or” is used to connect two or more options, it should be understood to mean any one of the options or any two or more of the options.

[0085] As used herein, the terms “comprising” or “including” mean to include the stated 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 specified, they also cover situations consisting of the mentioned elements, integers, or steps. For example, when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of that specific sequence.

[0086] In this document, the term "TIGIT" refers to "T-cell immune receptor containing Ig and ITIM domains." This term also includes variants, isotypes, homologs, and species homologs of TIGIT. TIGIT is also known as VSIG9, VSTM3, and WUCAM. The amino acid and nucleic acid sequences of the human and mouse forms of GITR can be found in GenBank accessions NP_776160 (human amino acid sequence) and NP_001139797 (mouse amino acid sequence). TIGIT proteins may also include fragments of TIGIT, such as fragments containing extracellular domains, for example, fragments that maintain the ability to bind to any of the antibodies of this invention.

[0087] In this article, the term “antibody” is used in the broadest sense to refer to a protein that contains an antigen-binding site, encompassing natural and artificial antibodies of various structures, including but not limited to complete antibodies and antigen-binding fragments of antibodies.

[0088] The terms “full-length antibody,” “complete antibody,” and “intact antibody” are used interchangeably herein to refer to glycoproteins comprising at least two heavy chains (H) and two light chains (L) linked by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Each light chain consists of a light chain variable region (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 hypervariable regions (complementarity-determining regions (CDRs) interspersed with more conserved regions (framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions do not directly participate in antibody-antigen binding but exhibit various effector functions.

[0089] The terms "human antibody" or "fully human antibody" are used interchangeably herein to refer to antibodies that include variable regions in which both the framework region and the CDR region are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, that constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of this invention may include amino acid sequences not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or point-specific mutagenesis or in vivo somatic mutations), for example in the CDR—particularly in CDR3. However, as used herein, the term "human antibody" does not include antibodies in which the CDR sequence is derived from a germline of another mammalian species (e.g., mouse) and transplanted with a human framework sequence.

[0090] The term "antibody preparation" refers to a preparation in a form that allows the biological activity of the antibody, as the active ingredient, to be effectively exerted, and which does not contain any other components that would have unacceptable toxicity to the subject to whom the preparation is administered. Such antibody preparations are typically sterile. Generally, antibody preparations contain pharmaceutically acceptable excipients. "Pharmaceutical-acceptable" excipients are reagents that can be rationally administered to test mammals 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.

[0091] The term "anti-TIGIT antibody formulation," also referred to herein as "the antibody formulation of the present invention," means a preparation comprising an anti-TIGIT antibody protein as the active ingredient and comprising a pharmaceutically acceptable excipient. When combined with a pharmaceutically acceptable excipient, the anti-TIGIT antibody 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 a liquid formulation in an aqueous form, such as for use with a pre-filled syringe, or as a lyophilized formulation, reconstituted (i.e., rehydrated) by dissolving and / or suspending in a physiologically acceptable solution just before use. In some embodiments, the anti-TIGIT antibody protein formulation is in liquid formulation form.

[0092] A "stable" antibody formulation means that the antibodies in the formulation retain an acceptable level of physical and / or chemical stability after storage under specific conditions, or after shaking, or after repeated freeze-thaw cycles. Although the antibodies contained in an antibody formulation may not maintain 100% of their chemical structure after storage, shaking, or repeated freeze-thaw cycles, an antibody formulation is generally considered "stable" if it retains approximately 90%, 95%, 96%, 97%, 98%, or 99% of its antibody structure or function. In some specific embodiments, the anti-TIGIT antibody protein formulation of the present invention exhibits low to undetectable antibody aggregation, degradation, or chemical modification during manufacturing, preparation, transportation, and long-term storage, resulting in minimal or even no loss of biological activity of the anti-TIGIT antibody protein, demonstrating high stability. In some embodiments, the anti-TIGIT antibody protein formulation of the present invention substantially retains its 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 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 r / min for 5 days at room temperature in the dark and / or after repeated freeze-thaw cycles at -30°C / room temperature 1-6 times.

[0093] Various 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 selected temperatures and for selected storage times. For example, the storage time can be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability tests 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 periods. For example, a formulated anti-TIGIT antibody protein formulation can be filled into glass vials to test antibody stability under high-temperature stress. For example, the prepared anti-TIGIT antibody protein formulation can be filled into glass vials and shaken at 650 rpm for 5 days at room temperature in the dark, and then the antibody stability can be checked. Another example is that the prepared anti-TIGIT antibody protein formulation can be filled into glass vials and subjected to 1-6 freeze-thaw cycles at -30°C / room temperature, and then the antibody stability can be checked. In one embodiment, freezing at -30°C for 1 day followed by thawing at room temperature constitutes one freeze-thaw cycle.

[0094] If, after a period of storage, or after shaking for a period of time, or after repeated freeze-thaw cycles, 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 have "maintained its physical stability" in the formulation. Since antibody aggregation in the formulation can potentially lead to an increased immune response in patients, thus causing safety issues, it is necessary to minimize or prevent antibody aggregation in the formulation. Light scattering methods can be used to determine visible aggregates in the formulation. SEC-HPLC can be used to determine soluble aggregates in the formulation. In addition, the appearance, color, and / or clarity of the formulation can be visually inspected, or determined by OD... 350nm The stability of a formulation can be indicated by detecting its turbidity or by determining its purity using a non-reducing CE-SDS method. In one embodiment, the stability of the formulation is measured by determining the percentage of antibody monomers in the formulation after storage at a specific temperature for a specific time, after shaking, or after repeated freeze-thaw cycles, wherein a higher percentage of antibody monomers in the formulation indicates higher stability.

[0095] "Acceptable level" physical stability can be expressed as the presence of at least approximately 90% of the anti-TIGIT antibody protein monomers in the formulation after storage at a specific temperature for a specific time, followed by shaking or repeated freeze-thaw cycles. In some embodiments, acceptable level physical stability 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 longer indicates at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-TIGIT antibody protein monomers, respectively. When assessing physical stability, the specific storage temperature for a pharmaceutical preparation can be any temperature ranging from about -80°C to about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-TIGIT antibody protein monomers are detected after storage at about 40°C ± 2°C for 1 month or 4 weeks, the pharmaceutical preparation is considered stable. If at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-TIGIT antibody protein monomers are detected after storage at about 25°C for 2 months, the pharmaceutical preparation is considered stable. If, after being stored at approximately 5°C for 9 months, at least approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the anti-TIGIT antibody protein monomers are detected, the pharmaceutical preparation is considered stable.

[0096] If the antibody in the formulation does not show significant chemical changes after a period of storage, or after a period of agitation, or after repeated freeze-thaw cycles, the antibody can be considered to have “maintained its chemical stability” in the formulation. Most chemical instabilities arise from the formation of covalently modified forms of the antibody (e.g., charge variants). For example, aspartic acid isomerization, N- and C-terminal modifications can form basic variants; deamidation, sialylation, and glycosylation can produce acidic variants. Chemical stability can be assessed by detecting and / or quantifying the chemically modified forms of the antibody. For example, charge variants of the antibody in the formulation can be detected by cation exchange chromatography (CEX) or imaging capillary isoelectric focusing electrophoresis (iCIEF). In one embodiment, the stability of the formulation is measured by determining the percentage change in the charge variant of the antibody in the formulation after storage at a specific temperature for a specific time, or after agitation, or after repeated freeze-thaw cycles, wherein a smaller change indicates higher stability of the formulation.

[0097] "Acceptable" chemical stability can be expressed as the percentage change of charge variants (e.g., main component, acidic component, or basic component) in the formulation not exceeding 40%, for example, not exceeding 30% or 20%, after storage at a specific temperature for a specific time, or after shaking for a period of time, or after repeated freeze-thaw cycles; or the sum of the percentage changes of charge variants (main component, acidic component, and basic component) not exceeding 60%, for example, not exceeding 50% or 30%. In some embodiments, 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 longer, an acceptable level of chemical stability can be demonstrated by a percentage change in the charge variant of the main component not exceeding about 50%, 40%, 30%, 20%, or 15%; or a total percentage change in the charge variant not exceeding about 60%, 50%, or 30%. When assessing chemical stability, the storage temperature of the pharmaceutical preparation can be any temperature from about -80°C to about 45°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C. For example, if the percentage change in charge variants of the main component 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 2 months, the pharmaceutical preparation can be considered stable. If the percentage change in charge variants of the main component 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 preparation can also be considered stable. If, after being stored at 40°C for one month, the percentage change in charge variants of the main component is less than approximately 50%, 40%, 30%, 20%, 16%, 15%, 14%, 13%, 12%, 10%, 5%, or 4%, the pharmaceutical preparation can also be considered stable.

[0098] The term "lyophilized formulation" refers to a composition obtained or that can be obtained by freeze-drying a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0099] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

[0100] The term “room temperature” as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, and more preferably 25°C.

[0101] "Stress conditions" refer to chemically and / or physically unfavorable environments for antibody proteins, which can lead to unacceptable instability of the antibody protein, such as high temperature, agitation, or freeze-thaw cycles. "High-temperature stress" refers to storing the antibody preparation at room temperature or even higher temperatures (e.g., 40°C ± 2°C) for a period of time. Accelerated high-temperature stress tests can be used to examine the stability of antibody preparations.

[0102] As used herein, the term "parenteral administration" means administration other than enteral and local administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, tracheal, subcutaneous, subcutaneous, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, the stable anti-TIGIT antibody protein formulation of the present invention is administered parenterally to a subject. In one embodiment, the anti-TIGIT antibody protein formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

[0103] I. Antibody preparations

[0104] This invention provides a stable liquid antibody formulation comprising (i) an anti-TIGIT antibody, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, wherein the antibody formulation has a pH of about 5.0-6.0. In a preferred embodiment, the liquid antibody formulation of this invention is in injectable form.

[0105] (i) Anti-TIGIT antibody

[0106] "Anti-TIGIT antibody" refers to an antibody that can bind to TIGIT molecules with sufficient affinity, such that the antibody can be used as a therapeutic and / or preventative agent targeting TIGIT molecules.

[0107] In some embodiments, such as by bio-optical interferometry, the anti-TIGIT antibody in the antibody formulation of the present invention can achieve high affinity, for example, at 10... -7 M or smaller, preferably 10-20 x 10 -10 M of K DThis antibody specifically binds to human TIGIT, thereby mediating a highly effective blocking effect on the binding of TIGIT and its ligand CD155, reducing or eliminating inhibitory signaling caused by TIGIT binding to its ligand. In some embodiments, the anti-TIGIT antibody in the antibody formulation of the present invention inhibits the growth of tumors (e.g., gastrointestinal tumors, preferably colorectal cancer) containing infiltrating lymphocytes expressing human TIGIT, and preferably when used in combination with an anti-PD1 antibody, it achieves a significantly better antitumor effect than when the antibody is administered alone.

[0108] In some embodiments, the anti-TIGIT antibody in the antibody formulation of the present invention comprises: a heavy chain variable region (VH) of SEQ ID NO:7 or having at least 90% identity with it; and a light chain variable region (VL) of SEQ ID NO:8 or having at least 90% identity with it. A “variable region” or “variable domain” is a domain in the heavy or light chain of the antibody that participates in the binding of the antibody to its antigen. Generally, the heavy chain variable region (VH) and the light chain variable region (VL) can be further subdivided into hypervariable regions (HVR, also known as complementarity-determining regions (CDRs)) interspersed with more conserved regions (i.e., framework regions (FRs)). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0109] In some embodiments, the anti-TIGIT antibody in the antibody formulation of the present invention comprises the VH CDR1, 2, and 3 sequences in the heavy chain variable region shown in SEQ ID NO:7 and the VL CDR1, 2, and 3 sequences in the light chain variable region shown in SEQ ID NO:8. The “complementarity-determining region” or “CDR region” (which may be used interchangeably with the hypervariable region “HVR” herein) is the amino acid region in the antibody variable region that is primarily responsible for binding to the antigenic epitope. The CDRs of the heavy and light chains are generally referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. CDRs located within the antibody heavy chain variable domain are referred to as VH CDR1, VH CDR2, and VH CDR3, while CDRs located within the antibody light chain variable domain are referred to as VL CDR1, VL CDR2, and VL CDR3. Various schemes for determining the CDR sequence in a given VH or VL amino acid sequence are known in the art. For example, the Kabat complementarity-determining region (CDR) is determined based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the location of a structural loop (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The AbM HVR is a compromise between the Kabat HVR and the Chothia structural loop and is used by the AbM antibody modeling software from Oxford Molecular. "Contact" HVR is based on the analysis of available complex crystal structures. HVR can also be determined based on having the same Kabat numbering position as a reference CDR sequence (e.g., the exemplary CDR disclosed herein). In one embodiment, the anti-TIGIT antibody of the present invention has VHCDR1 of SEQ ID NO: 1, VH CDR2 of SEQ ID NO: 2, VH CDR3 of SEQ ID NO: 3; and VL CDR1 of SEQ ID NO: 4, VL CDR2 of SEQ ID NO: 5 and VL CDR3 of SEQ ID NO: 6.

[0110] In some embodiments, the anti-TIGIT antibody in the antibody formulation of the present invention may comprise a heavy chain variable region (VH) having at least 90%, 95%, 98%, or 99% or higher identity with SEQ ID NO:7; and / or a light chain variable region (VL) having at least 90%, 95%, 98%, or 99% or higher identity with SEQ ID NO:8. In this document, "sequence identity" refers to the degree of sequence similarity on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison window. The "sequence identity percentage" can be calculated as follows: Two best-aligned sequences are compared in a comparison window to determine the number of positions in the two sequences that contain the same nucleic acid bases (e.g., A, T, C, G, I) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions. The number of matching positions is divided by the total number of positions in the comparison window (i.e., the window size), and the result is multiplied by 100 to produce the sequence identity percentage. The best alignment performed to determine the sequence identity percentage can be implemented in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for aligning sequences, including any algorithms required to achieve maximum alignment across the full length of the sequence being compared or within the target sequence region.

[0111] In some embodiments, the VH sequence of the antibody of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared to SEQ ID NO: 7, preferably said different residues being conserved amino acid substitutions. In some embodiments, the VL sequence of the antibody of the present invention has no more than 10, preferably no more than 5, 4, or 3 different residues compared to SEQ ID NO: 8, preferably said different residues being conserved amino acid substitutions. "Conservative substitution" refers to an amino acid change that results in the replacement of a certain amino acid with a chemically similar amino acid. Providing a table of conserved substitutions for functionally similar amino acids is well known in the art. In any embodiment of the present invention, in a preferred aspect, the conserved substituted residues are from the following table of conserved substitutions, preferably the preferred substitution residues shown in Table A.

[0112] Table A

[0113] Original residues Exemplary replacement Preferred conserved amino acid substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp(D) Glu;Asn Glu Cys(C) Ser;Ala Ser Gln(Q) Asn; Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Leucine Leu

[0114] Leu(L) Leucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; Leucine Leu

[0115] In some embodiments, the anti-TIGIT antibody in the antibody formulation of the present invention is an antibody in the form of IgG. "An antibody in the form of IgG" refers to an antibody whose heavy chain constant region belongs to the IgG form. All antibodies of the same IgG form have the same heavy chain constant region, while antibodies of different IgG forms have different heavy chain constant regions. For example, an antibody in the form of IgG1 refers to an antibody whose heavy chain constant region Ig domain is the Ig domain of IgG1.

[0116] In a preferred embodiment, the anti-TIGIT antibody in the antibody formulation of the present invention is the anti-TIGIT monoclonal antibody ADI-30278 disclosed in PCT application number PCT / CN2019 / 097665 (international application date: July 25, 2019), which has a heavy chain of SEQ ID NO:9 and a light chain of SEQ ID NO:10. In one embodiment, the anti-TIGIT antibody is an IgG4 type antibody produced by recombinant expression in CHO cells and purified. Preferably, the antibody in the liquid formulation of the present invention exhibits significant antitumor activity. For example, in a mouse tumor model inoculated with mouse MC38 cells, administration of the antibody formulation of the present invention can lead to a significant tumor-suppressive effect, especially when used in combination with an anti-PD-1 antibody.

[0117] The amount of antibody or its antigen-binding fragment contained in the antibody formulation of the present invention may be varied depending on the specific purpose 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 1-150 mg / mL, preferably about 10-100 mg / mL, for example about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, or 100 mg / mL of anti-TIGIT antibody.

[0118] (ii) Buffer

[0119] A buffer is a reagent that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffer used in the formulation of the present invention can control the pH of the formulation of the present invention within a pH range of approximately 5.0-6.0, for example, approximately 5.0-5.5. In some specific embodiments, the antibody formulation of the present invention has a pH of approximately 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, or 5.8. For example, the antibody formulation of the present invention has a pH of 5.2 ± 0.2 or 5.5 ± 0.2, preferably a pH of 5.2.

[0120] In some embodiments, the formulation of the present invention comprises a buffer system selected from the following: histidine-histidine hydrochloride buffer system, citrate-sodium citrate buffer system, acetate-sodium acetate buffer system, phosphate buffer system, preferably histidine-histidine hydrochloride buffer system.

[0121] In some embodiments, the buffer used in the formulations of the present invention is a histidine buffer, particularly a buffer system composed of histidine and histidine hydrochloride. In some embodiments, the concentration of histidine in the histidine buffer of the present invention is about 5-50 mM, particularly about 5-30 mM, for example, about 5, 10, 15, 20, 25, or 30 mM. In one embodiment, the formulation of the present invention contains about 10 mM of histidine. In another embodiment, the histidine buffer used in the formulation of the present invention is composed, for example, about 0.21 mg / ml of histidine and about 1.81 mg / ml of histidine hydrochloride.

[0122] (iii) Stabilizers

[0123] Suitable stabilizers for use in this invention may be selected from sugars, polyols, and 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.

[0124] For example, in some embodiments, the stabilizer comprises one or more of the following:

[0125] - A polyol selected from sorbitol, mannitol, or combinations thereof, at a concentration of about 10-100 mg / ml, preferably 20-40 mg / ml, for example 25 mg / ml; or 40-60 mg / ml, for example 50 mg / ml;

[0126] - Sugars selected from sucrose, trehalose, maltose, or combinations thereof, for example, about 10-100 mg / ml, preferably 30-60 mg / ml, for example 40 mg / ml;

[0127] - Selected from arginine hydrochloride, methionine, glycine, proline and combinations thereof, for example, 20-200 mM, such as about 50-110 mM, such as 70-100 mM, especially about 80-90 mM.

[0128] 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.

[0129] In one embodiment, the liquid formulation of the present invention comprises sorbitol as a stabilizer. The amount of sorbitol in the liquid formulation of the present invention may be about 10-100 mg / ml, preferably 20-40 mg / ml, for example 25 mg / ml; or 40-60 mg / ml, for example 50 mg / ml.

[0130] In one embodiment, the liquid formulation of the present invention comprises arginine as a stabilizer. The amount of arginine in the liquid formulation of the present invention may be about 50-110 mM, for example, 70-100 mM, especially about 80-90 mM, for example, about 17.91 mg / ml arginine hydrochloride.

[0131] In one embodiment, the liquid formulation of the present invention comprises sorbitol as a single stabilizer. In this embodiment, the amount of sorbitol in the liquid formulation of the present invention may be about 30-70 mg / ml, for example, 40-60 mg / ml. For example, sorbitol may 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.

[0132] In one embodiment, the liquid formulation of the present invention comprises a combination of sorbitol and arginine as a stabilizer. In this combination, sorbitol may be present in an amount of about 10-60 mg / ml, preferably 15-40 mg / ml, for example 20-35 mg / ml, such as about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg / ml. In this combination, arginine may be present in an amount of about 70-100 mM, particularly about 85 mM. Preferably, the liquid formulation of the present invention comprises about 20-30 mg / ml of sorbitol and about 15-20 mg / ml of arginine hydrochloride. More preferably, the liquid formulation of the present invention comprises about 25 mg / ml of sorbitol and about 17.91 mg / ml of arginine hydrochloride.

[0133] In one embodiment, the liquid formulation of the present invention comprises a combination of sucrose and arginine as a stabilizer. In this combination, sucrose may be present in an amount of about 10-60 mg / ml, preferably 20-50 mg / ml, for example 30-40 mg / ml, such as about 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 mg / ml. In this combination, arginine may be present in an amount of about 70-100 mM, particularly about 85 mM. Preferably, the liquid formulation of the present invention comprises about 30-50 mg / ml of sucrose and about 15-20 mg / ml of arginine hydrochloride. More preferably, the liquid formulation of the present invention comprises about 40 mg / ml of sucrose and about 17.91 mg / ml of arginine hydrochloride.

[0134] (iv). Surfactants

[0135] As used herein, the term "surfactant" refers to an organic substance with an amphiphilic structure; that is, it consists of groups with opposite solubility tendencies, typically oil-soluble hydrocarbon chains and water-soluble ionic groups.

[0136] In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, such as an alkyl poly(ethylene oxide). Specific nonionic surfactants that may be included in the formulation of the present invention include, for example, polysorbates, such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40; poloxamer, etc. In a preferred embodiment, the liquid formulation of the present invention contains polysorbate-80 as a surfactant.

[0137] In some embodiments, surfactants that can be used in the liquid formulations of the present invention include, but are not limited to, polysorbate surfactants (e.g., polysorbate 80, polysorbate 20), poloxamer, and polyethylene glycol.

[0138] The amount of surfactant contained in the antibody formulation of the present invention can be varied depending on the specific purpose of the formulation, the specific environment, and the specific purpose for which the formulation is used. In some preferred embodiments, the formulation may contain about 0.01-5 mg / ml, preferably about 0.1-1 mg / ml, for example about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / ml of surfactant, especially polysorbate-80, preferably about 0.5 mg / ml of polysorbate-80.

[0139] (v) Other excipients

[0140] The antibody liquid formulations of the present invention optionally include other excipients. These other excipients include, for example, antimicrobial agents, antistatic agents, antioxidants, chelating agents, gelatin, etc. These and other known pharmaceutical excipients and / or additives suitable for the formulations of the present invention are well known in the art, for example, as listed in "The Handbook of Pharmaceutical Excipients, 4th Edition, Rowe et al., eds., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st Edition, Gennaro, ed., Lippincott Williams & Wilkins (2005)".

[0141] II. Preparation of the formulation

[0142] This invention provides a stable formulation comprising an anti-TIGIT antibody protein. The anti-TIGIT antibody protein used in the formulation of this invention can be prepared using techniques known in the art for antibody production. For example, the antibody can be prepared recombinantly. In a preferred embodiment, the antibody of this invention is prepared recombinantly in 293 cells or CHO cells.

[0143] Antibodies are now widely used as active pharmaceutical ingredients. Techniques for purifying therapeutic antibodies to pharmaceutical grade are well-known in the field. 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 a protein A capture step. Kelley et al. (Weak partitioning chromatography for anion exchange purification of monoclonal antibodies, Biotechnology and Bioengineering 101 (2008) 553–566) describe a two-column purification method using a weakly partitioning anion exchange resin after protein A affinity chromatography.

[0144] Generally, recombinant monoclonal antibodies can be purified using conventional purification methods to provide pharmaceutical substances with sufficient reproducibility and adequate purity for the formulation of antibody preparations. For example, after the antibody is secreted from the recombinant expression cells into the culture medium, the supernatant from the expression system can be concentrated using a commercially available protein concentrator filter, such as an Amicon ultrafiltration device. The antibody can then be purified using methods such as chromatography, dialysis, and affinity purification. Protein A is well-suited as an affinity ligand for the purification of IgG1, IgG2, and IgG4 type antibodies. Other antibody purification methods, such as ion exchange chromatography, can also be used. After obtaining antibodies of sufficient purity, formulations containing the antibodies can be prepared according to methods known in the art.

[0145] For example, the preparation can be carried out using the following steps: (1) after fermentation, centrifuge the fermentation broth to clarify and remove impurities such as cells to obtain a supernatant; (2) capture the antibody using affinity chromatography (e.g., a protein A column with specific affinity for IgG1, IgG2, and IgG4 antibodies); (3) inactivate the virus; (4) purify the protein (usually using CEX cation exchange chromatography) to remove impurities from the protein; (5) filter the virus (to reduce the virus titer by, for example, more than 4 log10); (6) ultrafiltration / difiltration (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.

[0146] III. Analytical methods for formulations

[0147] 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 studies. Stability studies should explore and optimize research conditions based on the research objectives and the product's inherent characteristics; for various influencing factors (such as temperature, repeated freeze-thaw cycles, vibration, etc.), long-term, accelerated, and / or forced-condition testing protocols should be developed. Accelerated and forced-condition testing is helpful in understanding the product's stability under short-term deviations from storage conditions and extreme conditions, and provides supporting data for determining shelf life and storage conditions.

[0148] During the storage, shaking, or repeated freeze-thaw cycles of antibody preparations, antibodies may aggregate, degrade, or undergo chemical modification, leading to antibody heterogeneity (including size and charge heterogeneity) and the formation of aggregates and fragments, thereby affecting the quality of the antibody preparation. Therefore, it is necessary to monitor the stability of antibody preparations.

[0149] Various methods are known in the art for detecting the stability of antibody formulations. For example, the purity of antibody formulations and the assessment of antibody aggregation levels can be analyzed using methods such as reduced CE-SDS, non-reduced CE-SDS, and SEC-HPLC; charge variants in antibody formulations can be analyzed using methods such as cIEF, iCIEF, and ion exchange chromatography (IEX). Furthermore, the stability of a formulation can be quickly determined by visually inspecting its appearance. OD can also be used... 350nm This method can detect changes in the turbidity of the formulation, providing information about the amount of soluble and insoluble aggregates. Additionally, ultraviolet spectrophotometry (UV method) can be used to detect changes in the protein content of the formulation.

[0150] Non-reducing CE-SDS is a capillary-based method for determining antibody purity. In CE-SDS, protein migration is driven by the surface charge induced by SDS binding, which is proportional to the protein's molecular weight. Since all SDS-protein complexes have similar mass-to-charge ratios, electrophoretic separation based on molecular size or hydrodynamic radius can be achieved in a capillary molecular sieve gel matrix. This method has been widely used to monitor the purity of denatured, intact antibodies. Generally, 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. The supernatant is used for analysis. Protein migration is detected using a UV detector to obtain an electrophoretic pattern. Antibody purity can be calculated as the percentage of the IgG main peak area relative to the sum of all peak areas. For a further description of the CE-SDS method, please refer to, for example, Richard R. et al., Application of CE SDS gel in development of biopharmaceutical antibody-based products, Electrophoresis, 2008, 29, 3612-3620.

[0151] Size exclusion high-performance liquid chromatography (SEC-HPLC) is another important method for antibody standardization and quality control. This method primarily separates molecules based on differences in molecular size or hydrodynamic radius. SEC-HPLC can separate antibodies into three main forms: high molecular weight monosodium molecule (HMMS), the main peak (primarily antibody monomer), and low molecular weight monosodium molecule (LMMS). Antibody purity can be calculated as the percentage of the main peak area relative to the sum of all peak areas on the chromatogram. SEC-HPLC can also measure the percentage of antibody monomers in a pharmaceutical product, providing information on the content of soluble aggregates and shear products. For further description of the SEC-HPLC method, see, for example, J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res., 11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm. Bio. Anal., 14:1133-1140 (1986). In addition, see, for example, R. Yang et al., High resolution separation of recombinant monoclonal antibodies by size exclusionultra-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.

[0152] Charge variants of antibodies in antibody preparations can be determined by cation exchange high-performance liquid chromatography (CEX-HPLC). In this assay, peaks eluting from the CEX-HPLC column earlier than the retention time of the main peak (or main component) are labeled as "acidic peaks" (or acidic components), while peaks eluting from the CEX-HPLC column later than the retention time of the main peak are labeled as "basic peaks" (or basic components).

[0153] Accelerated stability studies can be used to examine the stability properties of products, which is beneficial for screening stable drug formulations. For example, accelerated stability studies can be conducted by placing formulation samples at elevated temperatures, such as approximately 40°C ± 2°C or 25°C ± 2°C. Furthermore, shaking experiments or repeated freeze-thaw cycles can be performed to assess the stability properties of products. For example, a shaking experiment can be conducted by shaking at 650 rpm for 1-5 days at room temperature in the dark. A repeated freeze-thaw cycle can be performed by thawing a product frozen below -30°C for one day at room temperature, with 1-6 cycles possible. Indicators of product stability can include appearance, visible foreign matter, protein content, turbidity, purity (SEC-HPLC, non-reducing CE-SDS), and charge variants (iCIEF, CEX-HPLC). In addition, antibody efficacy or biological activity can be detected. For example, the binding ability of an antibody to its antigen molecule (TIGIT) in a formulation can be detected. Various methods known to those skilled in the art can be used to quantify the specific binding of antibodies to antigens, such as immunoassays and ELISA.

[0154] IV. Uses of the formulation

[0155] The antibody preparation of the present invention, which contains anti-TIGIT antibody protein, reduces immunosuppression and can be used to treat or prevent tumors, pathogen infections, etc.

[0156] In one embodiment, the formulation of the present invention can be used in a subject to block the binding of TIGIT to CD155 to reduce or eliminate the immunosuppressive effect of TIGIT. In another embodiment, the formulation of the present invention can be used in a subject to treat or prevent tumors or pathogen infections. The tumor is, for example, a gastrointestinal cancer, such as colon cancer.

[0157] In one embodiment, the formulation of the present invention can be administered in combination with a second therapeutic agent, such as an anti-PD-1 antibody.

[0158] This invention also provides the use of the formulations of this invention in the preparation of a medicament for delivering anti-TIGIT antibody protein to a mammal. This invention further provides a method for treating or preventing one or more of the above-described diseases and conditions using the formulations of this invention. Preferably, the mammal is a human.

[0159] The antibody preparation of the present invention can be administered to subjects or patients via a variety of routes. For example, administration can be performed by infusion or via syringe. Therefore, in one aspect, the present invention provides a delivery device (e.g., a syringe) comprising the antibody preparation of the present invention (e.g., a pre-filled syringe). The patient will receive an effective amount of anti-TIGIT antibody protein as the main active ingredient, i.e., an amount sufficient to treat, improve, or prevent a target disease or condition.

[0160] Therapeutic effects may include reduction of physiological symptoms. The optimal effective dose and concentration of an antibody for any particular subject will depend on a variety of factors, including the patient's age, weight, health status and / or sex, the nature and extent of the disease, the activity of the specific antibody, the body's clearance of it, and any other possible treatments administered in combination with the antibody preparation. For specific cases, the effective dose delivered can be determined within the judgment of the clinician.

[0161] The following embodiments are described to aid in understanding the invention. It is not intended, and should not be construed in any way, as limiting the scope of the invention.

[0162] Abbreviation Description

[0163] abbreviations Full name CE-SDS Sodium dodecyl sulfate capillary gel electrophoresis CEX-HPLC Cation exchange high performance liquid chromatography ELISA Enzyme-linked immunosorbent assay SEC-HPLC Size exclusion high performance liquid chromatography Example

[0164] The recombinant fully human anti-TIGIT monoclonal antibody ADI-30278 is an antibody independently developed by Innovent Biologics (Suzhou) Co., Ltd., and is disclosed in PCT application number PCT / CN2019 / 097665. This antibody can effectively block the binding of TIGIT to its ligand CD155, relieve the inhibitory effect of CD155 on the downstream IL2 signaling pathway, and inhibit tumor growth when administered in vivo, especially when used in combination with anti-PD-1 antibodies, where the tumor-suppressing effect is particularly significant.

[0165] To develop a simple and easy-to-use injectable formulation suitable for long-term stable storage of this fully human antibody, the effects of different pH values, stabilizer contents, and surfactant contents on the antibody protein content were investigated through forced stability tests at 40°C and accelerated stability tests at 25°C. Ultimately, a formulation favorable for its stability was selected. The materials and methods used in the entire research process are as follows:

[0166] Materials and methods

[0167] 1.1. Materials used in the formulation research of this invention

[0168]

[0169]

[0170] Note: N / A indicates not applicable.

[0171] 1.2. Instruments and equipment used in the formulation research of this invention

[0172] name Origin and Brand model serial number Electronic balance Sartorius, Germany BSA3202S PD-A1-186 Electronic balance Mettler Switzerland XPE3003S PD-A1-247 Constant temperature and humidity chamber BINDER, Germany KBF P 720 PD-A1-070 Biochemical incubator Shanghai Jinghong SHP-150 PD-A1-200 Medical refrigerator Qingdao Haier HYC-360 PD-A1-166 Medical refrigerator Qingdao Haier HYC-360 PD-A1-165 Ultra-low temperature freezer Thermo USA 907 PD-A1-175 Clarity tester Tianjin Tianda Tianfa YB-2 PD-A1-033 UV-Vis spectrophotometer Shimadzu UV-1800 AS-A1-037 pH meter Mettler Switzerland FE20 PD-A1-161 Multichannel micro spectrophotometer Thermo USA Nanodrop 8000 PD-A1-052 benchtop refrigerated centrifuge Thermo USA SL16R PD-A1-082 Clean bench Suzhou Airtech SW-CJ-2FD QC-A1-011 Medium flow manual peristaltic pump Watson Marlow, UK 520S / R2 PD-A1-235 Filling machine Watson Marlow, Denmark FP50 PD-C14-115 Insoluble particulate detector Tianjin Tianda Tianfa GWJ-8 QC-A1-094

[0173] 1.3. Test items and methods for formulation stability

[0174] The main testing items during the entire research process included: (1) detecting the appearance and presence of visible foreign matter; (2) determining the protein content in the preparation by ultraviolet (UV) method; (3) detecting the turbidity of the preparation by OD350nm method; (4) determining the 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) determining the 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) determining the charge variants in the antibody preparation by CEX-HPLC method, expressed as the percentage of the main component, acidic component and basic component; (7) determining the relative binding activity of the anti-TIGIT antibody to the TIGIT antigen in the antibody preparation by direct ELISA method.

[0175] Visible foreign object detection

[0176] According to the method described in the Pharmacopoeia of the People's Republic of China (2015 edition, Part IV, General Chapter 0904 "Visible Foreign Matter Inspection Method", Beijing: China Medical Science and Technology Press, 2015), visible foreign matter in the sample was inspected using a clarity detector (manufactured by Tianjin Tianda Tianfa, model YB-2) and an insoluble particulate matter detector (manufactured by Tianjin Tianda Tianfa, model GWJ-8).

[0177] Protein content determination

[0178] The protein content in the sample was determined using a UV spectrophotometer (Shimadzu UV-1800, Japan) or a multi-channel micro spectrophotometer (Thermo Nanodrop 8000, USA).

[0179] Turbidity measurement

[0180] The absorbance of the sample at 350 nm was measured using a UV spectrophotometer (manufactured by Shimadzu, Japan, model UV-1800) to determine the turbidity of the sample.

[0181] Purity (SEC-HPLC method)

[0182] Separation was performed using a size exclusion column. The mobile phase was phosphate buffer (3.12 g sodium dihydrogen phosphate dihydrate, 8.77 g sodium chloride, and 34.84 g arginine were dissolved in ultrapure water, and the pH was adjusted to 6.8 with hydrochloric acid and brought to a final volume of 1000 ml). The column protectant was 0.05% (w / v) NaN3. The injection volume was 50 μl, the flow rate was 0.5 ml / min, the acquisition time was 30 min, the column temperature was 25℃, and the detection wavelength was 280 nm. The test sample was diluted with ultrapure water to a concentration of 2 mg / ml to prepare the test solution. The formulation buffer was diluted using the same method to prepare the blank solution. 50 μl each of the blank solution and the test solution were injected into the liquid chromatograph, and detection was initiated.

[0183] Purity (non-reducing CE-SDS method)

[0184] Capillary gel electrophoresis was used for detection. The capillary was an uncoated capillary with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before electrophoresis, the capillary column was rinsed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi. Dilute the sample to be tested with an appropriate amount of ultrapure water to 2.0 mg / mL. Take 50 μl of the diluted sample into a 1.5 mL centrifuge tube and add 45 μl of pH 6.5 sample buffer (weigh 0.32 g of citrate monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate, dissolve in 45 mL of ultrapure water, and bring the volume to 50 mL to prepare citrate-phosphate buffer; accurately measure 200 μl of this buffer, add 80 μl of 10% (w / v) sodium dodecyl sulfate solution, add water to 1 mL, and mix well), 1 μl of internal standard (10 kDa protein, 5 mg / mL) (Beckman Coulter, catalog number: 390953), and 5 μl of 250 mmol / L [unclear - possibly a specific solution or product] to the buffer. The NEM solution (62 mg of N-ethylcis-butyrendiimide dissolved in 2 ml of ultrapure water) was thoroughly mixed and heated at 70 ± 2 °C for 10 ± 2 minutes. After cooling to room temperature, it was transferred to a sample vial as the test solution. A blank solution was prepared by taking the same volume of the formulation buffer as the test solution and performing the same procedure. Sample injection conditions: -5 kV for 20 seconds; separation voltage: -15 kV for 35 minutes. The capillary column temperature was controlled at 25 °C, and the detection wavelength was 220 nm.

[0185] Charge variants (CEX-HPLC method)

[0186] The sample was detected using cation exchange chromatography (CEX-HPLC). A MabPac SCX-10 strong cation exchange column was used for separation. Mobile phase A was 10 mmol / L phosphate buffer (0.51 g of NaH₂PO₄·2H₂O and 2.40 g of Na₂HPO₄·12H₂O were dissolved in 800 mL of ultrapure water and brought to a final volume of 1000 mL, then filtered through a 0.22 μm filter). Mobile phase B was 10 mmol / L phosphate + 200 mmol / L sodium chloride buffer (0.51 g of NaH₂PO₄·2H₂O, 2.40 g of Na₂HPO₄·12H₂O and 11.69 g of NaCl were dissolved in 800 mL of ultrapure water and brought to a final volume of 1000 mL, then filtered through a 0.22 μm filter). The sample was diluted with ultrapure water to a concentration of 2.0 mg / mL to prepare the test solution. Dilute the formulation buffer solution using the same method described above to prepare a blank solution. Inject 50 μl each of the blank solution and the test solution into the liquid chromatograph. Set the mobile phase flow rate to 1.0 mL / min, acquisition time to 35 minutes, column temperature to 35°C, detection wavelength to 280 nm, and sample pan temperature to 10°C. Perform injection analysis and calculate the contents of the principal component, acidic component, and basic component using the area normalization method.

[0187] Relative binding activity (direct ELISA method)

[0188] Human TIGIT antigen (purchased from Acrobiosystems, TIT-H52H3) was used to coat 96-well microplates at 0.5 μg / ml, 100 μl / well, overnight at 4°C. After washing, blocking buffer (2% BSA-PBST, 300 μl / well) was added and the plates were blocked at 37°C for 2 h. Serially diluted test samples were added at 100 μl / well to microplates after discarding the blocking buffer. A negative control was set up with only 100 μl of dilution buffer (2% BSA-PBST) per well. The plates were incubated at 37°C for 60 min. After washing, HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, catalog number A80-104P, USA) diluted with 2% BSA-PBST was added as a secondary antibody (100,000-fold dilution, 100 μl / well), and the reaction was carried out at 37°C for 30 min. After washing the plate, add 100 μl of TMB chromogenic solution to each well. After developing for 10 min, add 100 μl of 1 mol / L H₂SO₄ to each well to terminate the reaction. Measure the OD value at 450 nm using 620 nm as the reference wavelength. Plot the concentration values ​​of each concentration gradient sample on the x-axis and the OD450 nm - OD620 nm values ​​of each gradient sample on the y-axis. Calculate the EC using Prism four-parameter fitting. 50 Reflects the binding activity of the antibody to each antigen. Relative binding activity (%) = (EC50% of the test sample) / (C60% of the antigen). 50 EC of the reference product 50*100%, wherein the reference is a stable anti-TIGIT antibody that has not been subjected to any stress treatment.

[0189] Example 1. Preparation and purification of anti-TIGIT antibody

[0190] According to PCT application number PCT / CN2019 / 097665, an anti-TIGIT antibody ADI-30278 specifically binding to TIGIT was obtained. This antibody has the heavy chain sequence of SEQ ID NO: 9 and the light chain sequence of SEQ ID NO: 10, and is a fully human antibody. PCT application number PCT / CN2019 / 097665 is hereby incorporated by reference in its entirety.

[0191] In short, the antibody was recombinantly expressed in CHO cells and purified through processes such as filtration, chromatography, virus inactivation, and re-filtration. Samples used in the following pH screening tests were products purified by affinity chromatography with a protein content of 17.0 mg / ml. Samples used in the following formulation determination tests were products purified by nanofiltration with a protein content of 11.5 mg / ml.

[0192] Example 2. pH Screening Test

[0193] 2.1 Experimental Procedure

[0194] This example investigates the effect of histidine buffer systems at pH 5.0, 5.5, 6.0, 6.5, and 7.0 on the stability of the purified anti-TIGIT antibody from Example 1 in order to obtain a better pH range.

[0195] Prepare 10 mM histidine, 5% (w / v) sorbitol buffer solutions. Adjust the pH to 5.0, 5.5, 6.0, 6.5, and 7.0 with hydrochloric acid. Ultrafilter the purified anti-TIGIT antibody from Example 1 into these buffer solutions at different pH values, adjusting the protein concentration to approximately 20 mg / ml. Add polysorbate 80 to 0.20 mg / ml. Filter, dispense into vials, stopper, and crimp cap. The stability of the samples was investigated at 40℃±2℃; the specific experimental protocol is shown in Table 1.

[0196] Table 1. Experimental Scheme

[0197]

[0198] Note: (1) X indicates that the sample was taken at that point. (2) All samples taken at the above time points were first placed in an ultra-low temperature freezer for storage and testing.

[0199] 2.2 Judgment Criteria

[0200] Based on our understanding of the product and the precision of the instruments and methods, we established a criterion for determining whether the sample quality had changed compared to the initial value. This criterion is used to determine whether the sample has changed. The criterion is shown in Table 2.

[0201] Table 2. Criteria for determining that quality has not changed

[0202] Testing items Criteria for determining no change Appearance (observation method) Clear to slightly milky, colorless to pale yellow liquid, free of foreign matter. Visible foreign objects (visible foreign object inspection method) If it conforms to the Pharmacopoeia of the People's Republic of China (2015 Edition, Part IV), then 0904 Protein content (UV method) Change rate ≤ 10% <![CDATA[Turbidity (OD 350nm method)]]> Change value ≤ 0.02 Purity (SEC-HPLC method) The change in the main peak is ≤1%. Purity (non-reducing CE-SDS method) The change in the main peak is ≤2%. Charge variants (CEX-HPLC method) The changes in the main components and acid-base components are ≤2%. Relative binding activity (direct ELISA method) It should be 70%–130%.

[0203] 2.3 Experimental Results

[0204] (1) Appearance and visible foreign objects

[0205] After concentration and solution replacement, the pH 6.5 and pH 7.0 samples showed turbidity and precipitation, therefore accelerated stability testing was not conducted. The pH 5.0, pH 5.5, and pH 6.0 samples, after being stored at 40℃±2℃ for 2 weeks, showed acceptable appearance and no visible foreign matter.

[0206] (2) Protein content

[0207] The protein content detection results are shown in Table 3. The results indicate that after being placed at 40℃±2℃ for 2 weeks, the protein content of samples at pH 5.0, pH 5.5, and pH 6.0 did not change significantly.

[0208] Table 3. Protein content results after pH screening (UV method, mg / ml)

[0209]

[0210] (3) Turbidity

[0211] The turbidity results are shown in Table 4. The results indicate that after being placed at 40℃±2℃ for 2 weeks, the turbidity of samples at pH 5.0 and pH 5.5 did not change; however, the turbidity of sample at pH 6.0 showed a certain upward trend.

[0212] Table 4. Turbidity results of pH screening (OD) 350nm Law)

[0213]

[0214] (4) Purity

[0215] The purity results (SEC-HPLC method and non-reducing CE-SDS method) are shown in Table 5. The results indicate that after being stored at 40℃±2℃ for 2 weeks, the purity of samples at pH 5.0, pH 5.5, and pH 6.0 did not change significantly.

[0216] Table 5. pH screening purity results

[0217]

[0218] (5) Charge variants

[0219] The results of charge variant analysis (CEX-HPLC method) are shown in Table 6, and their trends are shown in Figures 1 and 2. The results indicate that after two weeks of storage at 40℃±2℃, significant changes occurred in both the principal components and acidic components of samples at pH 5.0, pH 5.5, and pH 6.0. Compared to day 0, the principal components of samples at pH 5.0, pH 5.5, and pH 6.0 decreased by 4.8%, 4.4%, and 7.2%, respectively, while the acidic components increased by 3.6%, 4.3%, and 7.4%, respectively. In conclusion, based on the charge variant detection results, the antibody protein is more stable under pH 5.0 and pH 5.5 conditions.

[0220] Table 6. Results of pH screening for charge variants (CEX-HPLC method, %)

[0221]

[0222]

[0223] In summary, the pH screening results indicate that a pH between 5.0 and 5.5 is suitable for the histidine buffer system in the antibody formulation. pH 5.2 was selected for the next round of formulation determination experiments.

[0224] Example 3. Prescription Determination Experiment

[0225] 3.1 Experimental Procedure

[0226] Based on the pH screening results and the experience of the formulation development platform, the effects of different stabilizers (sorbitol, sucrose, and arginine hydrochloride) and polysorbate 80 content on antibody protein stability were investigated. Three formulations were designed, and detailed formulation information is shown in Table 7.

[0227] Table 7. Prescription Information Table

[0228]

[0229] Prepare the buffer solutions for each formulation according to Table 7, and ultrafilter the antibody proteins into their respective formulation solutions. After replacement, adjust the protein concentration of each formulation to approximately 50 mg / ml; add polysorbate 80. Filter and dispense into vials, stopper, and crimp cap. Perform stability studies on the above samples under shaking, freeze-thaw cycles, and different temperature conditions. See Table 8 for specific procedures.

[0230] Table 8. Experimental conditions and sampling plan

[0231]

[0232] Note: "*" indicates that the point was set for observation but was not tested.

[0233] 3.2 Judgment Criteria

[0234] For specific judgment criteria, please refer to Table 2 in Example 2.

[0235] 3.3 Experimental Results

[0236] (1) Oscillation Experiment

[0237] The results of the oscillation experiment are detailed in Table 9. The results show that after oscillation for 5 days at room temperature and in the dark at 650 r / min, the appearance and visible foreign matter of the three groups of prescription samples were all qualified; the protein content, purity, charge variants and relative binding activity did not change significantly.

[0238] Table 9. Results of the oscillation experiment

[0239]

[0240] Note: N / A indicates that this detection is not set.

[0241] (2) Freeze-thaw experiment

[0242] The results of the freeze-thaw experiment are detailed in Table 10. After six freeze-thaw cycles, the appearance and visible foreign matter of the three groups of formulation samples were all qualified; the protein content, purity, charge variants, and relative binding activity did not change significantly.

[0243] Table 10. Results of freeze-thaw experiment

[0244]

[0245] The above experimental results show that there is no significant difference in the stability of formulations 1, 2 and 3 in the shaking and freeze-thaw experiments.

[0246] Considering that the antibody is administered via intravenous infusion, the low polysorbate 80 content after sample dilution with physiological saline would affect protein stability. Furthermore, given that sorbitol and sucrose have no difference in their protective effects on proteins, non-reducing sorbitol was chosen to reduce the risk of saccharification reactions between excipients during long-term storage. Therefore, stability confirmation experiments were conducted on Formulation 2 at 40°C and 25°C.

[0247] (3) Stability confirmation test at 40℃

[0248] The results of the 40℃ stability confirmation experiment are detailed in Table 11. After being placed at 40℃ for one month, the appearance and visible foreign matter of the samples were acceptable. There were no significant changes in protein content, purity, and relative binding activity; however, the charge variants showed more significant changes, with the main component decreasing by 13.5%, the acidic component increasing by 8.4%, and the basic component increasing by 5.1%, as shown in Figure 3. Based on experience from the formulation development platform, these charge variant changes are within an acceptable range.

[0249] Table 11. Results of stability confirmation experiment at 40℃

[0250]

[0251] Note: N / A indicates that this detection is not set.

[0252] (4) Stability confirmation test at 25℃

[0253] The results of the 25℃ stability confirmation experiment are detailed in Table 12. After being placed at 25℃ for 2 months, the appearance and visible foreign matter of the samples were acceptable. There were no significant changes in protein content and purity; however, the charge variants showed more significant changes, with the main component decreasing by 6.4%, the acidic component increasing by 3.6%, and the basic component increasing by 2.9%, as shown in Figure 4. Based on experience from the formulation development platform, these charge variant changes are within an acceptable range.

[0254] Table 12. Results of the stability confirmation experiment at 25℃

[0255]

[0256] Based on the above experimental results and the experience of the formulation development platform, Formulation 2 was finally selected as the antibody formulation. Its composition is: 50.0 mg / ml recombinant fully human anti-TIGIT antibody, 0.21 mg / ml histidine, 1.81 mg / ml histidine hydrochloride, 25.00 mg / ml sorbitol, 17.91 mg / ml arginine hydrochloride, 0.50 mg / ml polysorbate 80, pH 5.2.

[0257] The foregoing describes exemplary embodiments of the present invention. Those skilled in the art should understand that these disclosures are merely exemplary, and various other substitutions, adaptations, and modifications can be made within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments listed herein. sequence list <110> Sherpa Biotechnology (Hangzhou) Co., Ltd. <120> Recombinant fully human anti-TIGIT monoclonal antibody formulation, its preparation method and uses <130> PF 200947CNP <160> 10 <170>PatentIn version 3.3 <210>1 <211>9 <212>PRT <213>artificial <220> <223>synthetic construct <400>1 Tyr Thr Phe Thr Glu Tyr Tyr Met His 1 5 <210>2 <211>17 <212>PRT <213>artificial <220> <223>synthetic construct <400>2 Ile Ile Ser Pro Ser Ala Gly Ser Thr Lys Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly <210>3 <211>15 <212>PRT <213>artificial <220> <223>synthetic construct <400>3 Ala Arg Asp His Asp Ile Arg Leu Ala Gly Arg Leu Ala Asp Tyr 1 5 10 15 <210>4 <211>11 <212>PRT <213>artificial <220> <223>synthetic construct <400>4 Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala 1 5 10 <210>5 <211>7 <212>PRT <213>artificial <220> <223>synthetic construct <400>5 Ala Ala Ser Ser Leu Gln Ser 1 5 <210>6 <211>9 <212>PRT 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Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Val Ile Leu Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210>9 <211>448 <212>PRT <213>artificial <220> <223>synthetic construct <400>9 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Glu Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Ser Pro Ser Ala Gly Ser Thr Lys Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Asp Ile Arg Leu Ala Gly Arg Leu Ala Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr 210 215 220 Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270 Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440 445 <210>10 <211>214 <212>PRT <213>artificial <220> <223>synthetic construct <400>10 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Ser Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Val Ile Leu Pro Ile 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210

Claims

1. A liquid antibody formulation comprising (i) 10-60 mg / ml of an anti-TIGIT antibody protein; (ii) 10 mM of a histidine buffer, (iii) 20-30 mg / ml of sorbitol, (iv) 0.3-0.6 mg / ml of polysorbate 80, and (v) 70-90 mM of arginine, wherein the anti-TIGIT antibody protein comprises a heavy chain VH CDR1 of YTFTEYYMH (SEQ ID NO: 1); a heavy chain VH CDR2 of IISPSAGSTKYAQKFQG (SEQ ID NO: 2); a heavy chain VH CDR3 of ARDHDIRLAGRLADY (SEQ ID NO: 3); a light chain VL CDR1 of RASQGISSWLA (SEQ ID NO: 4); a light chain VL CDR2 of AASSLQS (SEQ ID NO: 5); and a light chain VL CDR3 of QQAVILPIT (SEQ ID NO: 6), wherein the pH of the liquid antibody formulation being 5.2 ± 0.

2.

2. The liquid antibody formulation according to claim 1, wherein the pH is 5.

2.

3. The liquid antibody formulation according to claim 1, wherein the anti-TIGIT antibody protein is 50 mg / ml.

4. The liquid antibody formulation according to claim 1, wherein the histidine buffer is a histidine-histidine hydrochloride buffer system.

5. The liquid antibody formulation according to claim 1, wherein the liquid antibody formulation comprises 85 mM of arginine.

6. The liquid antibody formulation according to claim 1, wherein the polysorbate 80 is 0.5 mg / ml.

7. The liquid antibody formulation according to claim 1, wherein the anti-TIGIT antibody comprises a heavy chain variable region VH and a light chain variable region VL, wherein the heavy chain variable region comprises the sequence of SEQ ID NO: 7 or a sequence at least 90% identical thereto, and the light chain variable region comprises the sequence of SEQ ID NO: 8 or a sequence at least 90% identical thereto.

8. The liquid antibody formulation according to claim 1, wherein the anti-TIGIT antibody comprises a heavy chain variable region VH of the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region VL of the amino acid sequence set forth in SEQ ID NO:

8.

9. The liquid antibody formulation according to claim 1, wherein the anti-TIGIT antibody is an IgG type antibody.

10. The liquid antibody formulation according to claim 1, wherein the anti-TIGIT antibody is an IgG4 subtype antibody.

11. The liquid antibody formulation according to claim 1, wherein the anti-TIGIT antibody comprises a heavy chain sequence of SEQ ID NO: 9 or at least 90% identical thereto and a light chain sequence of SEQ ID NO: 10 or at least 90% identical thereto.

12. The liquid antibody formulation of claim 1, wherein the anti-TIGIT antibody comprises a heavy chain sequence of SEQ ID NO: 9 and a light chain sequence of SEQ ID NO:

10.

13. The liquid antibody formulation of claim 1, wherein the anti-TIGIT antibody is recombinantly expressed in 293 cells or CHO cells.

14. The liquid antibody formulation of claim 1, wherein the liquid formulation is an injection.

15. The liquid antibody formulation of claim 1, wherein the liquid formulation is a subcutaneous injection or an intravenous injection.

16. The liquid antibody formulation of claim 1, wherein the liquid formulation is an infusion.

17. The liquid antibody formulation of claim 1, wherein the liquid formulation is an intravenous infusion.

18. The liquid antibody formulation of any one of claims 1-17, wherein the liquid antibody formulation comprises 50 mg / ml of anti-TIGIT antibody protein, 0.21 mg / ml histidine, 1.81 mg / ml histidine hydrochloride, 25.00 mg / ml sorbitol, 17.91 mg / ml arginine hydrochloride, 0.50 mg / ml polysorbate 80, pH 5.

2.

19. The liquid antibody formulation of claim 1, wherein, The formulation is stable after storage at 25°C for at least 2 months or at 40°C ± 2°C for 1 month, with one or more of the following characteristics: (i) a change in the main peak of less than 1% as measured by SEC-HPLC method, and / or the formulation has a purity of greater than 96%; (ii) a change in the main peak of less than 2% as measured by non-reduced CE-SDS method, and / or the formulation has a purity of greater than 96%; (iii) a change in the main component, acidic component and basic component of the anti-TIGIT antibody protein in the formulation of no more than 40% and / or a change in the main component of no more than 20% relative to the initial value on the 0th day of storage as measured by CEX-HPLC method; (iv) a relative binding activity of the anti-TIGIT antibody protein in the formulation of 90-110% relative to the initial value on the 0th day of storage as measured by ELISA method.

20. The liquid antibody formulation of claim 19, wherein the formulation is stable under shaking and / or repeated freeze-thawing.

21. A solid antibody formulation obtained by solidifying the liquid antibody formulation of any one of claims 1-20.

22. The solid antibody formulation of claim 21, wherein the solid antibody formulation is in the form of a lyophilized powder.

23. A delivery device comprising the liquid antibody formulation of any one of claims 1-20 or the solid antibody formulation of claim 21 or 22.

24. A pre-filled syringe comprising the liquid antibody formulation of any one of claims 1-20 or the solid antibody formulation of claim 21 or 22 for intravenous injection or intramuscular injection.

25. The delivery device of claim 23 or the pre-filled syringe of claim 24, further comprising a second therapeutic agent.

26. The delivery device or pre-filled syringe of claim 25, wherein, The second therapeutic agent is an anti-PD-1 antibody.

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