Pharmaceutical composition containing anti-LAG-3 antibody and application thereof

Abstract

The invention relates to the technical field of antibody preparations, in particular to a pharmaceutical composition containing an anti-LAG-3 antibody and application of the pharmaceutical composition, and the pharmaceutical composition has good stability.

Description

[0001] This application is a divisional application. The filing date of the original application is November 02, 2023. The application number is 202311446616.7. The name of the invention is “Pharmaceutical composition containing anti-LAG-3 antibodies and its use”. Technical Field

[0002] The present invention relates to the field of antibody preparations, and in particular to a pharmaceutical composition containing an anti-LAG-3 antibody or an antigen-binding fragment thereof and uses thereof. Background Art

[0003] LAG-3, also known as CD223, is the full name of Lymphocyte Activation Gene 3. It is not expressed on naive T cells, but can be expressed on CD4 + and CD8 + LAG-3 is an immunosuppressive receptor that negatively regulates T cell function and plays an important role in maintaining the homeostasis of the body's immune system. However, due to continuous antigen stimulation in the tumor microenvironment, CD4 + and CD8 + The expression of LAG-3 and other immunosuppressive receptors (including PD1, TIM3, and TIGIT) on T cells is upregulated, leading to T cell dysfunction. + It is constitutively expressed on regulatory T cells (Treg). Treg cells that overexpress LAG-3 produce a large amount of immunoregulatory cytokines IL-10 and TGF-β, thereby inhibiting tumor-specific T cells. In addition, LAG-3 is also expressed on γδT cells, natural killer T cells, plasmacytoid dendritic cells (pDC), and activated B cells. Using therapeutic antibodies to inhibit LAG-3 and weaken the T cell inhibition caused by LAG-3 can enhance the body's immune response to tumors.

[0004] Antibodies have large molecular weights, complex structures, and are easily degraded, aggregated, or undergo undesirable chemical modifications, becoming unstable. However, any instability may affect the safety and effectiveness of therapeutic antibodies. Therefore, therapeutic antibodies need to be formulated in appropriate formulations to prevent the destruction of antibody structure and activity caused by denaturation, oxidation, aggregation, etc. during storage, transportation, and administration. Summary of the invention

[0005] The present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) an anti-LAG-3 antibody or an antigen-binding fragment thereof, (b) a buffer, (c) a stabilizer and (d) a surfactant.

[0006] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof can specifically bind to LAG-3 (e.g., human LAG-3) and / or block LAG-3 signaling, including but not limited to polyclonal antibodies, monoclonal antibodies, multispecific antibodies, monospecific antibodies, monovalent antibodies and single-chain antibodies.

[0007] The present application also provides a method for preparing the pharmaceutical composition of the present application, the preparation method comprising the following steps: mixing the anti-LAG-3 antibody or antigen-binding fragment thereof, a buffer, a stabilizer and a surfactant to prepare the pharmaceutical composition. In some embodiments, the preparation method further comprises the step of sterilizing the mixture obtained by mixing.

[0008] The present application also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition of the present application.

[0009] The present application also provides a lyophilized preparation, which can form the pharmaceutical composition of the present application after being reconstituted (ie, reconstituted).

[0010] The present application also provides a product, which comprises the pharmaceutical composition of the present application.

[0011] The present application also provides a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for treating a disease in a subject.

[0012] The present application also provides a method for enhancing the immune response of a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for enhancing the immune response of a subject.

[0013] The present application also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for treating cancer in a subject.

[0014] The present application also provides a method for treating an infectious disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for treating an infectious disease in a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments.

[0016] Figure 1 It is a main peak content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 under the condition of 2-8°C (SEC-HPLC);

[0017] Figure 2 The monomer content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 at 2-8°C (non-reducing CGE);

[0018] Figure 3 The monomer content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 under the condition of 2-8°C (CIEX);

[0019] Figure 4 The main peak content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 under 25°C (SEC-HPLC);

[0020] Figure 5 The monomer content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 at 25°C (non-reducing CGE);

[0021] Figure 6 The monomer content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 at 25°C (CIEX);

[0022] Figure 7 It is the main peak content change curve of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 under light conditions (SEC-HPLC);

[0023] Figure 8 The figure is a curve diagram of the change of monomer content of the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 under light conditions (non-reduced CGE). DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. The purpose of providing these embodiments and examples is to make the disclosure of the present invention more thoroughly understood. Unless otherwise defined, all technical and scientific terms used herein are the same as those generally understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing the embodiments and examples, and are not used to limit the present invention.

[0025] the term

[0026] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0027] The terms "and / or", "or / and", and "and / or" used in this application include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0028] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0029] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0030] In the present application, “further”, “furthermore”, “particularly”, etc. are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present invention.

[0031] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0032] In this application, "one", "another", "another", etc. are used for descriptive purposes only and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, they only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0033] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0034] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0035] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0036] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0037] In this application, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation as practiced in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When specific values ​​or parameters are given herein or in the claims, they are modified by "about" by default unless otherwise indicated.

[0038] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as a reference separately. Unless they conflict with the invention purpose and / or technical solution of the present application, the cited documents involved in the present invention are cited with all contents and all purposes. When the present invention involves cited documents, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves cited documents, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement the present invention. It should be understood that when the content of the citation conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0039] The term "buffer" means a pharmaceutically acceptable buffer, which refers to a substance that helps maintain the pH of the pharmaceutical composition within a desired pH range. Buffers suitable for the present application include phosphate buffers, acetate buffers, citrate buffers, Tris buffers or histidine buffers. In addition, a mixture of two or more buffers can also be used.

[0040] The term "phosphate buffer" is a buffer containing phosphate ions. Examples of phosphate buffers include sodium phosphate buffers. Sodium phosphate buffers can be prepared using disodium hydrogen phosphate or a hydrate thereof (e.g., disodium hydrogen phosphate dodecahydrate) and sodium dihydrogen phosphate or a hydrate thereof (e.g., sodium dihydrogen phosphate dihydrate).

[0041] The term "acetate buffer" is a buffer containing acetate ions. Examples of acetate buffers include sodium acetate buffers. Sodium acetate buffers can be prepared using acetic acid and sodium acetate or a hydrate thereof (e.g., anhydrous sodium acetate or sodium acetate trihydrate). The sodium acetate buffer described in the examples can be prepared from sodium acetate trihydrate and acetic acid (e.g., 20 mmol / L acetic acid-sodium acetate buffer (pH 5.5) can be prepared from about 2.34 g / L sodium acetate trihydrate and about 0.47 g / L acetic acid (36%)), or from anhydrous sodium acetate and acetic acid (e.g., 20 mmol / L acetic acid-sodium acetate buffer (pH 5.5) can be prepared from about 1.41 g / L anhydrous sodium acetate and about 0.17 g / L acetic acid).

[0042] The term "citrate buffer" is a buffer containing citrate ions. Examples of citrate buffers include sodium citrate buffers. Sodium citrate buffers can be prepared using citric acid and sodium citrate or a hydrate thereof (eg, trisodium citrate dihydrate).

[0043] The term "histidine buffer" is a buffer containing histidine ions. Examples of histidine buffers include histidine-histidine hydrochloride buffers. Histidine-histidine hydrochloride buffers can be prepared using histidine (e.g., L-histidine) and histidine hydrochloride or a hydrate thereof (e.g., histidine hydrochloride monohydrate).

[0044] The term "stabilizer" refers to a substance that helps maintain the structural integrity of an active ingredient (e.g., an antibody) in a pharmaceutical composition, particularly during freezing, lyophilization, storage and / or transportation (especially when exposed to stress). In this context, a stabilizer may also function as a viscosity reducer, an antioxidant and / or an isotonic agent.

[0045] The term "antioxidant" refers to a substance that can prevent or reduce oxidation of an active ingredient (eg, an antibody) in a pharmaceutical composition.

[0046] The term "isotonic agent" refers to a substance that helps maintain the osmotic pressure of a pharmaceutical composition at or close to the physiological osmotic pressure.

[0047] The term "antibody" refers to an antigen binding protein having at least one antigen binding domain. The antibody or fragment thereof of the present application may be a whole antibody or any fragment thereof, including a monoclonal antibody or fragment thereof and an antibody variant or fragment thereof. Examples of antibodies or antigen binding fragments thereof of the present application include monospecific antibodies, bispecific antibodies, multispecific antibodies, Fab fragments, Fab' fragments, F(ab)'2 fragments, Fv fragments, isolated CDR regions, single-chain Fv molecules (scFv) and other antibody fragments known in the art. Unless otherwise indicated, the "antibody or antigen binding fragment thereof" of the present application includes a whole antibody and any antigen binding fragment or single chain thereof. Conventional "whole antibodies" are glycoproteins comprising two heavy (H) chains and two light (L) chains, and the heavy chains and light chains are connected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains, namely CH1, CH2 and CH3. Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of a domain CL. The VH and VL regions can also be divided into hypervariable regions, i.e., complementary determining regions (CDRs), and framework regions (FRs) with relatively conservative sequences. Each VH and VL is composed of three CDRs and four FRs, respectively, from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of an antibody comprises a binding domain that interacts with an antigen. The constant region of an antibody can mediate the binding of an immunoglobulin to a host tissue or factor, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. At the same time, as will be appreciated by those skilled in the art, a special "whole antibody", such as a nanobody, has only a heavy (H) chain and no light (L) chain.

[0048] The "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the function of specifically binding to an antigen. It has been demonstrated that the antigen-binding function of an antibody can be implemented by a fragment of the entire antibody. Examples encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragment: a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment consisting of VH and CH1 domains; (iv) Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) dAb fragment consisting of a VH domain (see Ward et al., Nature. 341: 544-546 (1989)); (vi) isolated complementary determining regions (CDRs); and (vii) nanobodies, a heavy chain variable region comprising a single variable domain and two constant domains. In addition, although the two domains VL and VH of the Fv fragment are encoded by different genes, VH and VL can be connected into a single protein chain by a recombinant method through a synthetic linker, wherein VL and VH are paired to form a monovalent molecule (called single-chain Fv (scFv); see, for example, Bird et al., Science. 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. 85: 5879-5883 (1988)). These single-chain antibodies are also encompassed in the term antigen-binding fragment. In addition, recombinant polypeptides, fusion proteins and immunoconjugates comprising the antigen-binding fragment are also encompassed in the term antigen-binding fragment.

[0049] The term "identity" is also known as consistency. The "percentage (%) identity" of an amino acid sequence refers to the percentage of amino acid residues in the sequence to be compared that are identical to the amino acid residues in the specific amino acid sequence shown in this article, after comparing the sequence to be compared with the specific amino acid sequence shown in this article and introducing spaces if necessary to achieve the maximum sequence identity percentage, and not considering any conservative substitutions as part of the sequence identity. The amino acid sequence alignment of identity can be carried out in a variety of ways within the scope of the art, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required to obtain the maximum alignment in the full length of the comparison sequence.

[0050] The term "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient (e.g., antibody) therein to be effectively exerted, and does not contain other components that are unacceptably toxic for the subject to whom the pharmaceutical composition is to be administered. Such pharmaceutical compositions are generally sterile. The pharmaceutical composition of the present application can be prepared into a liquid preparation in aqueous form, such as filled in a prefilled syringe or vial, or prepared into a lyophilized preparation, and reconstituted by dissolving and / or suspending in a physiologically acceptable solution before use. In some embodiments, the pharmaceutical composition of the present application is a liquid preparation. In the present application, "pharmaceutical composition" and "preparation" can be used interchangeably.

[0051] The term "stable" pharmaceutical composition refers to a pharmaceutical composition that substantially retains the physical stability and / or chemical stability and / or biological activity of the active ingredient (eg, antibody) when stored therein.

[0052] The term "high molecular weight impurities" or "aggregates" refers to a general term for impurities having a molecular weight greater than that of the target active ingredient (eg, antibody).

[0053] The term "treatment" generally refers to an operation to obtain a desired pharmacological and / or physiological effect. The effect may be preventive, in terms of completely or partially preventing a disease or its symptoms; and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or side effects produced by the disease. As used herein, "treatment" encompasses any treatment of a patient's disease, including but not limited to preventing the occurrence or recurrence of a disease, alleviating symptoms of a disease, reducing any direct or indirect pathological consequences of a disease, preventing the metastasis of a disease, slowing the progression of a disease, improving or alleviating the state of a disease, extending the frequency and duration of symptom-free periods, and resolving or improving the prognosis of a disease.

[0054] The term "therapeutically effective amount" or "therapeutically effective dose" refers to any amount of a drug that protects a patient from the onset of disease or promotes disease regression, as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of impairment or disability resulting from disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to skilled practitioners, such as in human patients during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in in vitro assays.

[0055] The terms "administering" or "administering" refer to the physical introduction of a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art.

[0056] The terms "subject" or "patient" are used interchangeably. "Subject" or "patient" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates such as non-human primates, sheep, dogs, and rodents (e.g., mice, rats, and guinea pigs). In some embodiments, the subject or patient is a mammal. In some embodiments, the subject or patient is a mouse. In some embodiments, the subject or patient is a human.

[0057] Pharmaceutical composition

[0058] The present application provides a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) an anti-LAG-3 antibody or an antigen-binding fragment thereof, (b) a buffer, (c) a stabilizer and (d) a surfactant.

[0059] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof is at a concentration of 1-50 mg / mL, 2-50 mg / mL, 5-40 mg / mL, 10-40 mg / mL, or 10-30 mg / mL. In some embodiments, the concentration of the anti-LAG-3 antibody or antigen-binding fragment thereof includes but is not limited to 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL or 50 mg / mL. In some embodiments, the concentration of the anti-LAG-3 antibody or antigen-binding fragment thereof is 10-25 mg / mL, 15-23 mg / mL, or 18.5-21.5 mg / mL. In some embodiments, the concentration of the anti-LAG-3 antibody or antigen-binding fragment thereof is 20 mg / mL.

[0060] In some embodiments, the buffer comprises a histidine buffer, an acetate buffer, a Tris buffer, a phosphate buffer, or a citrate buffer. In some embodiments, the buffer comprises an acetate buffer or a citrate buffer. In some embodiments, the buffer comprises an acetate buffer. In some embodiments, the acetate buffer is a sodium acetate buffer.

[0061] In some embodiments, the concentration of the buffer is 1-150 mmol / L, 1-120 mmol / L, 2-100 mmol / L, 5-80 mmol / L, 10-60 mmol / L or 10-40 mmol / L. In some embodiments, the concentration of the buffer includes but is not limited to 2mmol / L, 3mmol / L, 4mmol / L, 5mmol / L, 6mmol / L, 7mmol / L, 8mmol / L, 9mmol / L, 10mmol / L, 11mmol / L, 12mmol / L, 13mmol / L, 14mmol / L, 15mmol / L, 16mmol / L, 17mmol / L, 18mmol / L, 19mmol / L, 20mmol / L, 21mmol / L, 22mmol / L, 23mmol / L, 24mmol / L, 25mmol / L, 26mmol / L, 27mmol / L, 28mmol / L, 29mmol / L, 30mmol / L, 40mmol / L, 50mmol / L, 60mmol / L, 70mmol / L or 80mmol / L. In some embodiments, the concentration of the buffer is 10-30 mmol / L, 10-25 mmol / L, 15-23 mmol / L, or 18.5-21.5 mmol / L. In some embodiments, the concentration of the buffer is 20 mmol / L.

[0062] In some embodiments, the buffer is 1-150mmol / L, 1-120mmol / L, 2-100mmol / L, 5-80mmol / L, 10-60mmol / L or 10-40mmol / L acetate buffer. In some embodiments, the buffer is 10-30mmol / L, 10-25mmol / L, 15-23mmol / L or 18.5-21.5mmol / L acetate buffer. In some embodiments, the buffer is 20mmol / L acetate buffer. In some embodiments, the acetate buffer is sodium acetate buffer.

[0063] In some embodiments, the pH of the buffer is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8. In some embodiments, the pH of the buffer includes, but is not limited to, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7. In some embodiments, the pH of the buffer is 5. In some embodiments, the pH of the buffer is 5.2. In some embodiments, the pH of the buffer is 5.5. In some embodiments, the pH of the buffer is 5.8. In some embodiments, the pH of the buffer is 6.

[0064] In some embodiments, the stabilizer comprises a sugar alcohol (e.g., mannitol, sorbitol), a disaccharide (e.g., trehalose, sucrose, maltose, lactose), a monosaccharide (e.g., dextrose (D-glucose)), an amino acid (e.g., lysine, glycine, proline, arginine or a pharmaceutically acceptable salt thereof), or a salt (e.g., sodium chloride). In some embodiments, the stabilizer comprises trehalose or proline.

[0065] In some embodiments, the concentration of the stabilizer is 1-200 mg / mL, 5-100 mg / mL, 10-80 mg / mL, 15-60 mg / mL, 10-60 mg / mL, or 15-55 mg / mL. In some embodiments, the concentration of the stabilizer includes but is not limited to: 5 mg / mL, 5.8 mg / mL, 6 mg / mL, 10 mg / mL, 11.5 mg / mL, 12 mg / mL, 15 mg / mL, 17 mg / mL, 17.3 mg / mL, 20 mg / mL, 23 mg / mL, 25 mg / mL, 28.8 mg / mL, 29 mg / mL, 30 mg / mL, 34.5 mg / mL, 35 mg / mL, 40 mg / mL, 40.3 mg / mL, 41.7 mg / mL, 43.9 mg / mL, 44.9 mg / mL, 45.7 mg / mL, 46.9 mg / mL, 47.9 mg / mL, 48.9 mg / mL, 49.1 mg / mL, 50. In some embodiments, the concentration of the stabilizer is 20-55 mg / mL, 30-55 mg / mL, or 45-55 mg / mL. In some embodiments, the concentration of the stabilizer is 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL or 55 mg / mL. In some embodiments, the concentration of the stabilizer is 20 mg / mL. In some embodiments, the concentration of the stabilizer is 50 mg / mL. In some embodiments, the concentration of the stabilizer is 15-50 mg / mL, 15-40 mg / mL, or 30-40 mg / mL. In some embodiments, the concentration of the stabilizer is 17 mg / mL, 17.3 mg / mL, 20 mg / mL, 23 mg / mL, 28.8 mg / mL, 29 mg / mL, 30 mg / mL, 34.5 mg / mL or 35 mg / mL. In some embodiments, the concentration of the stabilizer is 17.3 mg / mL. In some embodiments, the concentration of the stabilizer is 34.5 mg / mL.

[0066] In some embodiments, the concentration of the stabilizer is 1-800mmol / L, 10-600mmol / L, 15-500mmol / L, 20-400mmol / L, 50-400mmol / L, or 50-350mmol / L. In some embodiments, the concentration of the stabilizer includes but is not limited to: 10mmol / L, 14.6mmol / L, 15mmol / L, 29mmol / L, 29.2mmol / L, 43.8mmol / L, 44mmol / L, 50mmol / L, 58mmol / L, 58.4mmol / L, 73mmol / L, 87.6mmol / L, 88mmol / L, 100mmol / L, 102mmol / L, 102.3mmol / L, 116.9mmol / L, 117mmol / L, 131.5mmol / L, 132mmol / L, 146mmol / L, In some embodiments, the concentration of the stabilizer is 50-200mmol / L, 50-150mmol / L or 140-150mmol / L. In some embodiments, the concentration of the stabilizer is 50mmol / L, 58mmol / L, 58.4mmol / L, 73mmol / L, 87.6mmol / L, 88mmol / L, 102mmol / L, 102.3mmol / L, 116.9mmol / L, 117mmol / L, 131.5mmol / L, 132mmol / L, 146mmol / L or 146.1mmol / L. In some embodiments, the concentration of the stabilizer is 58mmol / L or 58.4mmol / L. In some embodiments, the concentration of the stabilizer is 146mmol / L or 146.1mmol / L. In some embodiments, the concentration of the stabilizer is 100-350mmol / L, 150-350mmol / L or 250-350mmol / L. In some embodiments, the concentration of the stabilizer is 150 mmol / L, 200 mmol / L, 250 mmol / L, 300 mmol / L or 350 mmol / L.In some embodiments, the concentration of the stabilizer is 150 mmol / L. In some embodiments, the concentration of the stabilizer is 300 mmol / L.

[0067] In some embodiments, the stabilizer is 1-200 mg / mL, 5-100 mg / mL, 10-80 mg / mL, 15-60 mg / mL, 10-60 mg / mL, or 15-55 mg / mL of trehalose or proline. In some embodiments, the stabilizer is 15-55 mg / mL (e.g., 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 31 mg / mL, 32 mg / mL, 33 mg / mL, In some embodiments, the stabilizer is 20-55mg / mL, 30-55mg / mL or 45-55mg / mL of trehalose. In some embodiments, the stabilizer is 20mg / mL, 25mg / mL, 30mg / mL, 35mg / mL, 40mg / mL, 45mg / mL, 50mg / mL or 55mg / mL of trehalose. In some embodiments, the stabilizer is 20 mg / mL of trehalose. In some embodiments, the stabilizer is 50 mg / mL of trehalose. In some embodiments, the stabilizer is 15-50 mg / mL, 15-40 mg / mL, or 30-40 mg / mL of proline. In some embodiments, the stabilizer is 17 mg / mL, 17.3 mg / mL, 20 mg / mL, 23 mg / mL, 28.8 mg / mL, 29 mg / mL, 30 mg / mL, 34.5 mg / mL, or 35 mg / mL of proline. In some embodiments, the stabilizer is 17.3 mg / mL of proline. In some embodiments, the stabilizer is 34.5 mg / mL of proline.

[0068] In some embodiments, the stabilizer is 1-800mmol / L, 10-600mmol / L, 15-500mmol / L, 20-400mmol / L, 50-400mmol / L, or 50-350mmol / L of trehalose or proline. In some embodiments, the stabilizer is 50-200mmol / L, 50-150mmol / L, or 140-150mmol / L of trehalose. In some embodiments, the stabilizer is 50mmol / L, 58mmol / L, 58.4mmol / L, 73mmol / L, 87.6mmol / L, 88mmol / L, 102mmol / L, 102.3mmol / L, 116.9mmol / L, 117mmol / L, 131.5mmol / L, 132mmol / L, 146mmol / L, or 146.1mmol / L of trehalose. In some embodiments, the stabilizer is 58 mmol / L or 58.4 mmol / L trehalose. In some embodiments, the stabilizer is 146 mmol / L or 146.1 mmol / L trehalose. In some embodiments, the stabilizer is 100-350 mmol / L, 150-350 mmol / L or 250-350 mmol / L proline. In some embodiments, the stabilizer is 50-350mmol / L (e.g., 50mmol / L, 60mmol / L, 70mmol / L, 80mmol / L, 90mmol / L, 100mmol / L, 110mmol / L, 120mmol / L, 130mmol / L, 140mmol / L, 150mmol / L, 160mmol / L, 170mmol / L, 180mmol / L, 190mmol / L, 200mmol / L, 210mmol / L, 220mmol / L, 230mmol / L, 240mmol / L, 250mmol / L, 260mmol / L, 270mmol / L, 280mmol / L, 290mmol / L, 300mmol / L, 310mmol / L, 320mmol / L, 330mmol / L, 340mmol / L, 350mmol / L) of proline. In some embodiments, the stabilizer is 150mmol / L, 200mmol / L, 250mmol / L, 300mmol / L or 350mmol / L of proline. In some embodiments, the stabilizer is 150mmol / L of proline. In some embodiments, the stabilizer is 300mmol / L of proline.

[0069] In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant comprises polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85). In some embodiments, the surfactant comprises polysorbate 80 or polysorbate 20. In some embodiments, the surfactant comprises polysorbate 20.

[0070] In some embodiments, the concentration of the surfactant is 0.02-2 mg / mL, 0.04-1.5 mg / mL, 0.06-1 mg / mL, 0.08-0.8 mg / mL, 0.1-0.5 mg / mL, or 0.1-0.3 mg / mL. In some embodiments, the concentration of the surfactant includes but is not limited to: 0.06 mg / mL, 0.08 mg / mL, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL or 1 mg / mL. In some embodiments, the concentration of the surfactant is 0.2 mg / mL.

[0071] In some embodiments, the surfactant is 0.02-2 mg / mL, 0.04-1.5 mg / mL, 0.06-1 mg / mL, 0.08-0.8 mg / mL, 0.1-0.5 mg / mL, or 0.1-0.3 mg / mL of polysorbate 20. In some embodiments, the surfactant is 0.2 mg / mL polysorbate 20.

[0072] In some embodiments, the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8. In some embodiments, the pH of the pharmaceutical composition includes, but is not limited to, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7. In some embodiments, the pH of the pharmaceutical composition is 5. In some embodiments, the pH of the pharmaceutical composition is 5.2. In some embodiments, the pH of the pharmaceutical composition is 5.5. In some embodiments, the pH of the pharmaceutical composition is 5.8. In some embodiments, the pH of the pharmaceutical composition is 6.

[0073] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 1-50 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 1-150 mmol / L buffer (e.g., acetate buffer or citrate buffer), (c) stabilizer (e.g., trehalose or proline), and (d) 0.02-2 mg / mL surfactant (e.g., polysorbate 80 or polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0074] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 2-50 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 1-120 mmol / L buffer (e.g., acetate buffer or citrate buffer), (c) stabilizer (e.g., trehalose or proline), and (d) 0.04-1.5 mg / mL surfactant (e.g., polysorbate 80 or polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0075] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 5-40 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 2-100 mmol / L buffer (e.g., acetate buffer or citrate buffer), (c) stabilizer (e.g., trehalose or proline), and (d) 0.06-1 mg / mL surfactant (e.g., polysorbate 80 or polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0076] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-40 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 5-80 mmol / L buffer (e.g., acetate buffer or citrate buffer), (c) stabilizer (e.g., trehalose or proline), and (d) 0.08-0.8 mg / mL surfactant (e.g., polysorbate 80 or polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0077] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-40 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 10-60 mmol / L buffer (e.g., acetate buffer or citrate buffer), (c) stabilizer (e.g., trehalose or proline), and (d) 0.1-0.5 mg / mL surfactant (e.g., polysorbate 80 or polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0078] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-30 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 10-40 mmol / L buffer (e.g., acetate buffer or citrate buffer), (c) stabilizer (e.g., trehalose or proline), and (d) 0.1-0.3 mg / mL surfactant (e.g., polysorbate 80 or polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0079] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 1-50 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 1-150 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 1-200 mg / mL trehalose or 1-800 mmol / L proline), and (d) 0.02-2 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0080] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 2-50 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 1-120 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 5-100 mg / mL trehalose or 10-600 mmol / L proline), and (d) 0.04-1.5 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0081] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 5-40 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 2-100 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 10-80 mg / mL trehalose or 15-500 mmol / L proline), and (d) 0.06-1 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0082] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-40 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 5-80 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 15-60 mg / mL trehalose or 20-400 mmol / L proline), and (d) 0.08-0.8 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0083] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-40 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 10-60 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 10-60 mg / mL trehalose or 50-400 mmol / L proline), and (d) 0.1-0.5 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0084] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-30 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 10-40 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 15-55 mg / mL trehalose or 50-350 mmol / L proline), and (d) 0.1-0.3 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-7, 5-6.5, 5-6, 5.2-5.8, or 5.5-5.8.

[0085] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 10-25 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 10-25 mmol / L buffer (e.g., acetate buffer, preferably sodium acetate buffer), (c) stabilizer (e.g., 20-55 mg / mL trehalose or 150-350 mmol / L proline), and (d) 0.1-0.3 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-6, 5.2-5.8, or 5.5-5.8.

[0086] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 15-23 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 15-23 mmol / L buffer (e.g., sodium acetate buffer), (c) stabilizer (e.g., 45-55 mg / mL trehalose or 250-350 mmol / L proline), and (d) 0.1-0.3 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-6, 5.2-5.8, or 5.5-5.8.

[0087] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 20 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 20 mmol / L buffer (e.g., sodium acetate buffer), (c) stabilizer (e.g., 50 mg / mL trehalose or 300 mmol / L proline), and (d) 0.2 mg / mL surfactant (e.g., polysorbate 20), and the pH of the pharmaceutical composition is 5-6, 5.2-5.8, or 5.5-5.8.

[0088] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 20 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 20 mmol / L sodium acetate buffer, (c) 50 mg / mL trehalose, and (d) 0.2 mg / mL polysorbate 20, and the pH of the pharmaceutical composition is 5.5-5.8 (e.g., 5.5).

[0089] In some specific embodiments, the pharmaceutical composition of the present application comprises: (a) 20 mg / mL anti-LAG-3 antibody or antigen-binding fragment thereof, (b) 20 mmol / L sodium acetate buffer, (c) 300 mmol / L proline, and (d) 0.2 mg / mL polysorbate 20, and the pH of the pharmaceutical composition is 5.5-5.8 (e.g., 5.5).

[0090] Anti-LAG-3 antibodies or antigen-binding fragments thereof

[0091] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of SEQ ID NO: 7 or 17, and LCDR1, LCDR2 and LCDR3 in the light chain variable region of SEQ ID NO: 8 or 18. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of SEQ ID NO: 7, and LCDR1, LCDR2 and LCDR3 in the light chain variable region of SEQ ID NO: 8. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2 and HCDR3 in the heavy chain variable region of SEQ ID NO: 17, and LCDR1, LCDR2 and LCDR3 in the light chain variable region of SEQ ID NO: 18.

[0092] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1 set forth in SEQ ID NO: 1 or 11, HCDR2 set forth in SEQ ID NO: 2 or 12, HCDR3 set forth in SEQ ID NO: 3 or 13, LCDR1 set forth in SEQ ID NO: 4 or 14, LCDR2 set forth in SEQ ID NO: 5 or 15, and LCDR3 set forth in SEQ ID NO: 6 or 16. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1 set forth in SEQ ID NO: 1, HCDR2 set forth in SEQ ID NO: 2, HCDR3 set forth in SEQ ID NO: 3, LCDR1 set forth in SEQ ID NO: 4, LCDR2 set forth in SEQ ID NO: 5, and LCDR3 set forth in SEQ ID NO: 6. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1 as set forth in SEQ ID NO: 11, HCDR2 as set forth in SEQ ID NO: 12, HCDR3 as set forth in SEQ ID NO: 13, LCDR1 as set forth in SEQ ID NO: 14, LCDR2 as set forth in SEQ ID NO: 15, and LCDR3 as set forth in SEQ ID NO: 16. The CDR sequences of the anti-LAG-3 antibodies or antigen-binding fragments thereof are shown in Table A.

[0093] Table A. CDR sequences of anti-LAG-3 antibodies or antigen-binding fragments thereof

[0094]

[0095] It will be understood by those skilled in the art that, unless otherwise specified, the term "CDR" or "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any known scheme. Although CDR regions have been shown in Table A, however, when referring to antibodies defined by specific CDR sequences, the scope of the antibodies encompasses antibodies defined by CDR sequences defined by any numbering system (e.g., a combination of one or more of the definitions of AbM, Kabat, CCG, Chothia, IMGT or Contact, etc., which are well known in the art).

[0096] In some embodiments, the heavy chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 7 or 17, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.99%, or 100%) identical to SEQ ID NO: 7 or 17. In some embodiments, the sequence of the heavy chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof has no more than 10, optionally no more than 5, 4, or 3 different residues compared to SEQ ID NO: 7 or 17, optionally the different residues are conservative amino acid substitutions. In some embodiments, the heavy chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO:7, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:7. In some embodiments, the heavy chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO:17, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:17.

[0097] In some embodiments, the light chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 8 or 18, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 8 or 18. In some embodiments, the sequence of the light chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof has no more than 10, optionally no more than 5, 4, or 3 different residues compared to SEQ ID NO: 8 or 18, optionally the different residues are conservative amino acid substitutions. In some embodiments, the light chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO:8, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:8. In some embodiments, the light chain variable region of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO:18, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:18.

[0098] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 7 or 17 and a light chain variable region set forth in SEQ ID NO: 8 or 18. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 7 and a light chain variable region set forth in SEQ ID NO: 8. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO: 17 and a light chain variable region set forth in SEQ ID NO: 18.

[0099] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3, wherein HCDR1 comprises the sequence shown in SEQ ID NO:1, HCDR2 comprises the sequence shown in SEQ ID NO:2, HCDR3 comprises the sequence shown in SEQ ID NO:3, LCDR1 comprises the sequence shown in SEQ ID NO:4, LCDR2 comprises the sequence shown in SEQ ID NO:5, and LCDR3 comprises the sequence shown in SEQ ID NO:6, and the heavy chain variable region comprises the sequence shown in SEQ ID NO:7. , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.99% or 100%) identical to SEQ ID NO:7, and the light chain variable region comprises a sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.99% or 100%) identity to SEQ ID NO:8.

[0100] QVQLQQWGAGLLRPSETLSLTCAVYGESFSGYYWNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISSVDTSKTQFSLKLSSVTAADTAVYYCARGWDLLDWNDYWNEYWGQGTLVTVSS(SEQ ID NO:7);

[0101] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIK(SEQ ID NO:8);

[0102] EVQLLESGGGLVQPGGSLRLSCAASGFDFRSYAMMWVRQAPGKGLEWVGGINGEVGGSNTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCVKGAGACGICNDIDIDAWGQGTLVTVSS (SEQ ID NO: 17);

[0103] SYELTQDPAVSVALGQTVRITCSGAGSYAGSYYYGWHQQKPGQAPVTVIYDNDKRPSNIPDRFSGSSSGNTASLTITGAQAEDEADYYCGSTNDNDDGGLFGSGTKVTVL (SEQ ID NO: 18).

[0104] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof is an IgG1, IgG2, or IgG4 antibody or a variant thereof.

[0105] In some embodiments, the heavy chain of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a sequence as set forth in SEQ ID NO: 9 or 19, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 9 or 19. In some embodiments, the sequence of the heavy chain of the anti-LAG-3 antibody or antigen-binding fragment thereof has no more than 10, optionally no more than 8, 7, 6, 5, 4, or 3 different residues compared to SEQ ID NO: 9 or 19, optionally the different residues are conservative amino acid substitutions. In some embodiments, the heavy chain of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO:9, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:9. In some embodiments, the heavy chain of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 19, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 19. The terminal amino acid K of SEQ ID NOs: 9 and 19 may be present or absent.

[0106] In some embodiments, the light chain of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 10 or 20, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10 or 20. In some embodiments, the sequence of the light chain of the anti-LAG-3 antibody or antigen-binding fragment thereof has no more than 10, optionally no more than 8, 7, 6, 5, 4, or 3 different residues compared to SEQ ID NO: 10 or 20, optionally the different residues are conservative amino acid substitutions. In some embodiments, the light chain of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO: 10, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO: 10. In some embodiments, the light chain of the anti-LAG-3 antibody or antigen-binding fragment thereof comprises the sequence set forth in SEQ ID NO:20, or a sequence that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to SEQ ID NO:20.

[0107] In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 9 or 19 and a light chain as set forth in SEQ ID NO: 10 or 20. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 9 and a light chain as set forth in SEQ ID NO: 10. In some embodiments, the anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain as set forth in SEQ ID NO: 19 and a light chain as set forth in SEQ ID NO: 20. The terminal amino acid K of SEQ ID NO: 9 and 19 may be present or absent.

[0108] QVQLQQWGAGLLRPSETLSLTCAVYGESFSGYYWNWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKTQFSLKLSSVTAADTAVYYCARGWDLLDWNDYWNEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPG(SEQ ID NO:9);

[0109] EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO:10);

[0110] EVQLLESGGGLVQPGGSLRLSCAASGFDFRSYAMMWVRQAPGKGLEWVGGINGEVGGSNTYYAPAVKGRATISRDNSKNTLYLQMNSLRAEDTAVYYCVKGAGACGICNDDIDA WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCD KTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:19);

[0111] SYELTQDPAVSVALGQTVRITCSGAGSYAGSYYYGWHQQKPGQAPVTVIYDNDKRPSNIPDRFSGSSSGNTASLTITGAQAEDEADYYCGSTNDNDGLFGSGTKVTVLR TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQID NO:20).

[0112] The anti-LAG-3 antibody of the present application is a fully human anti-lymphocyte activation gene 3 (LAG-3) monoclonal antibody disclosed in Chinese invention patent application CN201780074499.2 (filing date is October 13, 2017). For the purpose of the embodiments of the present application, the entire contents of Chinese invention patent application CN201780074499.2 are hereby incorporated herein by reference.

[0113] "Conservative substitution" refers to an amino acid change that results in a substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. In any embodiment of the present application, in a preferred aspect, the conservative substitution residue is from the following conservative substitution table, optionally the preferred substitution residue shown in Table B.

[0114] Table B. Conservative Substitution Table

[0115] Initial residue Representative replacement Preferred substitutions Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) lle; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0116] Preparation method

[0117] The present application also provides a method for preparing the pharmaceutical composition of the present application, comprising the following steps: mixing the anti-LAG-3 antibody or antigen-binding fragment thereof, a buffer, a stabilizer and a surfactant to prepare a pharmaceutical composition.

[0118] In some embodiments, the preparation method further comprises the step of sterilizing the mixture obtained by mixing. It is understood that the present application does not specifically limit the sterilization method, and membrane filtration can be selected but is not limited to it.

[0119] During the preparation process, there is no particular limitation on the manner and order of mixing the components.

[0120] In one embodiment, the preparation method of the present application comprises:

[0121] mixing the anti-LAG-3 antibody or antigen-binding fragment thereof, a buffer and a stabilizer to prepare a mixture 1; and,

[0122] The mixture 1 and the surfactant are mixed to prepare a pharmaceutical composition.

[0123] Optionally, the step of preparing the mixture 1 comprises: replacing the anti-LAG-3 antibody or antigen-binding fragment thereof into the buffer and stabilizer by ultrafiltration, and optionally concentrating to prepare the mixture 1.

[0124] It is understandable that the preparation method of the present application also includes the step of packaging the prepared pharmaceutical composition (for example, packaging into vials).

[0125] Lyophilized preparations

[0126] The present application also provides a lyophilized preparation, which is obtained by freeze-drying the pharmaceutical composition of the present application. In some embodiments, the freeze-drying is carried out according to methods well known in the art, including but not limited to the steps of pre-freezing, primary drying and secondary drying. It is understood by those skilled in the art that any method of removing water from the pharmaceutical composition of the present application is applicable to the present application.

[0127] The present application also provides a lyophilized preparation, which can form the pharmaceutical composition of the present application after reconstitution. In some embodiments, the solution used for reconstitution includes but is not limited to water for injection, sodium chloride injection, Ringer's solution or glucose injection.

[0128] Products

[0129] The present application also provides a product, which comprises the pharmaceutical composition or lyophilized preparation of the present application. In some embodiments, the product of the present application comprises a container, wherein the pharmaceutical composition or lyophilized preparation of the present application is contained in the container.

[0130] The pharmaceutical composition or product of the present application can be stably stored for a long time at -80°C to room temperature.

[0131] In one embodiment, the pharmaceutical composition or preparation of the present application can be stably stored at about 2°C to about 8°C for at least 24 months, for example, at about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, for at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.

[0132] In one embodiment, the pharmaceutical composition or preparation of the present application can be stably stored at 25°C, for example, stably stored for at least 12 months (e.g., 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).

[0133] In one embodiment, the pharmaceutical composition or preparation of the present application can be stably stored under light conditions, for example, stably stored for at least 1 month (e.g., at least 1 day, at least 3 months, at least 5 days, at least 9 days, at least 11 days, at least 13 days, at least 15 days, at least 17 days, at least 19 days, at least 21 days, at least 23 days, at least 25 days, at least 27 days, at least 29 days, at least 31 days).

[0134] use

[0135] The present application also provides a method for treating a disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for treating a disease in a subject.

[0136] The present application also provides a method for enhancing the immune response of a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for enhancing the immune response of a subject.

[0137] The present application also provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a drug for treating cancer in a subject.

[0138] The term "cancer" is the name of a disease in which body cells become abnormal and divide without control. In some embodiments, the cancer is a recurrent, refractory, metastatic, and / or advanced cancer. In some embodiments, the cancer is a non-solid tumor. In some embodiments, the cancer is a blood tumor. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myeloma, melanoma, non-small cell lung cancer, small cell lung cancer, bladder cancer, head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, Merkel cell carcinoma, fibrosarcoma, glioma, glioblastoma, liver cancer, nasopharyngeal cancer, prostate cancer, breast cancer, pancreatic cancer, endometrial cancer, biliary tract cancer, urinary system cancer, bone cancer, cervical cancer, thyroid cancer, anal cancer, gallbladder cancer, or thymic cancer.

[0139] The present application also provides a method for treating an infectious disease in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition, lyophilized preparation or product of the present application. The present application also provides the use of the pharmaceutical composition, lyophilized preparation or product of the present application in the preparation of a medicament for treating an infectious disease in a subject. In some embodiments, the infectious disease is a disease caused by an organism such as bacteria, fungi, parasites, viruses or other pathogens.

[0140] The pharmaceutical composition, lyophilized preparation or product of the present application can be administered according to methods known in the art. In some embodiments, the lyophilized preparation needs to be reconstituted before administration to obtain a reconstituted solution of the lyophilized preparation. In some embodiments, it can be reconstituted with water for injection, sodium chloride injection, Ringer's solution or glucose injection.

[0141] In some embodiments, the pharmaceutical composition, lyophilized preparation or product of the present application are suitable for parenteral administration. In some embodiments, the pharmaceutical composition, lyophilized preparation and product of the present application are suitable for intravenous, intramuscular, intraperitoneal, subcutaneous, epidermal, spinal, intralesional injection or infusion. In some embodiments, the pharmaceutical composition, lyophilized preparation and product of the present application are suitable for topical application, suction, oral administration or administration by sustained release or delayed release delivery system. In some embodiments, the pharmaceutical composition, lyophilized preparation and reconstituted solution of the present application can be diluted with a suitable diluent before administration. In some embodiments, water for injection, sodium chloride injection, Ringer's solution or glucose injection can be diluted.

[0142] In some embodiments, the pharmaceutical composition, lyophilized preparation or product of the present application can be administered alone or in combination with other drugs, for example, it can be administered together with cancer therapeutic agents known in the art, such as immunostimulants, chemotherapeutic agents, anti-tumor agents, anti-angiogenic agents or radiation therapy.

[0143] The embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance given in the present invention, and can also be based on the experimental manual or conventional conditions in the art, or can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art. In the following examples, the measurement parameters of the raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. Involving temperature and time parameters, acceptable deviations caused by instrument test accuracy or operating accuracy are allowed.

[0144] Example

[0145] The vector containing the gene encoding the heavy chain (SEQ ID NO: 9) and the light chain (SEQ ID NO: 10) of the anti-LAG-3 antibody was transfected into CHO cells, the CHO cells were cultured, and the cell culture supernatant was then purified by a standard purification method to obtain the anti-LAG-3 antibody in the following example, which has the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8.

[0146] The English codes and corresponding abbreviations in the following embodiments are as follows: SEC-HPLC is size exclusion high performance liquid chromatography; CGE is capillary gel electrophoresis; iCIEF is full column imaging capillary isoelectric focusing; CIEX is cation exchange chromatography; MFI is microfluidic imaging; kDa is kilodaltons; A2 is the second virial coefficient.

[0147] Example 1

[0148] A pharmaceutical composition containing an anti-LAG-3 antibody and a buffer was prepared according to Table 1, and the preparation steps included: replacing the anti-LAG-3 antibody into the buffer by ultrafiltration, with the final concentration of the anti-LAG-3 antibody being 20 mg / mL; after uniform mixing, the mixture was sterilized by filtration through a 0.22 μm filter membrane, and dispensed into vials, and stoppered and capped.

[0149] The CG-MALS method of ProJect Pharmaceutics was used to determine the second virial coefficient (A2) value of the pharmaceutical composition, and the pharmaceutical composition with a higher A2 value was screened out (A2 < 0 indicates mutual attraction between molecules, and A2 > 0 indicates mutual repulsion between molecules). The A2 value detection results are shown in Table 2.

[0150] Table 1. Composition of the pharmaceutical composition

[0151]

[0152] Table 2. Test results

[0153] Drug Composition No. <![CDATA[A2*10 -4 (mol·mL / g 2 )]]> MW(kDa) F1 1.92 114 F2 -0.33 114 F3 -0.39 114

[0154] The results showed that when the concentration of anti-LAG-3 antibody was 20 mg / mL, the A2 value in the pharmaceutical composition containing citrate buffer (corresponding to F2 and F3) was the lowest, and the attraction between proteins was dominant, which was not conducive to the stability of anti-LAG-3 antibody; the A2 value in the pharmaceutical composition containing acetate buffer (corresponding to F1) was higher, and the repulsion between proteins was dominant, which was conducive to the stability of anti-LAG-3 antibody.

[0155] Example 2

[0156] A pharmaceutical composition containing an anti-LAG-3 antibody, a buffer and a stabilizer was prepared according to Table 3, and the preparation steps included: replacing the anti-LAG-3 antibody into the buffer and the stabilizer by ultrafiltration, wherein the final concentration of the anti-LAG-3 antibody was 20 mg / mL; after uniform mixing, the mixture was sterilized by filtration through a 0.22 μm filter membrane, and dispensed into vials, and stoppered and capped.

[0157] The CG-MALS method of ProJect Pharmaceutics was used to determine the second virial coefficient (A2) value of the pharmaceutical composition, and the pharmaceutical composition with a higher second virial coefficient (A2) value was screened out (A2 < 0 indicates mutual attraction between molecules, and A2 > 0 indicates mutual repulsion between molecules). The A2 value detection results are shown in Table 4.

[0158] Table 3. Composition of the pharmaceutical composition

[0159]

[0160] Table 4. Test results

[0161] Drug Composition No. <![CDATA[A2*10 -4 (mol·mL / g 2 )]]> MW(kDa) F1a-b1 -0.25 109 F1a-b2 1.14 108 F1a-b3 1.23 109 F1a-b4 -0.23 107 F1a-b5 -0.03 109 F1a-b6 1.21 108 F1a-b7 1.31 105 F1a-b8 -0.03 121

[0162] The results showed that in 20 mmol / L acetate buffer (pH 5.5), the A2 values ​​in the pharmaceutical compositions containing sodium chloride (corresponding to F1a-b1, F1a-b4, F1a-b5 and F1a-b8) were all <0, and the attraction between proteins was dominant, indicating that sodium chloride was not conducive to the stability of anti-LAG-3 antibodies; the A2 values ​​in the pharmaceutical compositions containing 2-5% (w / v) trehalose (corresponding to F1a-b2 and F1a-b3) and the pharmaceutical compositions containing 150-300 mmol / L proline (corresponding to F1a-b6 and F1a-b7) were higher, and the repulsion between proteins was dominant, indicating that trehalose and proline were conducive to the stability of anti-LAG-3 antibodies.

[0163] Example 3

[0164] A pharmaceutical composition containing an anti-LAG-3 antibody, a buffer, a stabilizer and a surfactant was prepared according to Table 5, and the preparation steps included: replacing the anti-LAG-3 antibody into the buffer and the stabilizer by ultrafiltration, concentrating after the replacement, and then adding polysorbate 20 according to Table 5, with the final concentration of the anti-LAG-3 antibody being 20 mg / mL, mixing evenly, filtering and sterilizing through a 0.22 μm filter membrane, and dispensing into vials, and stoppering and capping.

[0165] The pharmaceutical composition sample was frozen at -80°C for at least 1 hour, and thawed at room temperature in the dark until the sample was completely thawed. This was considered one cycle. Freeze-thaw cycles were performed 0, 3, and 6 times to prepare freeze-thaw cycle samples and investigate freeze-thaw cycle (FT) stability. After freeze-thaw cycles, the SEC-HPLC, non-reducing CGE, iCIEF, and MFI results of the pharmaceutical composition were tested. The specific data are shown in Tables 6.1 and 6.2.

[0166] Table 5. Composition of the pharmaceutical composition

[0167]

[0168] Note: “-” means none.

[0169] Table 6.1. Test results I

[0170]

[0171] Note: “-” means none.

[0172] Table 6.2. Test results II

[0173]

[0174] Note: “-” means none.

[0175] The results showed that after freeze-thaw cycles, there was no significant change in the visible foreign matter, anti-LAG-3 antibody content, non-reducing CGE and iCIEF in all pharmaceutical compositions; the SEC-HPLC test results showed that the polymer impurities in the pharmaceutical compositions without any stabilizer (corresponding to F1a and F1a-c1) increased the most; compared with the pharmaceutical compositions without polysorbate 20 (corresponding to F1a, F1a-b3 and F1a-b7), the number of insoluble particles (2-100μm) in the pharmaceutical compositions containing polysorbate 20 (corresponding to F1a-c1, F1a-b3-c1 and F1a-b7-c1) was significantly reduced; compared with the pharmaceutical compositions containing only polysorbate 20 (corresponding to F1a-c1), the number of insoluble particles (2-100μm) in the pharmaceutical compositions containing trehalose or proline (corresponding to F1a-b3-c1 and F1a-b7-c1) was significantly reduced.

[0176] Example 4

[0177] A pharmaceutical composition containing an anti-LAG-3 antibody, a buffer, a stabilizer and a surfactant was prepared according to Table 7, and the preparation steps included: replacing the anti-LAG-3 antibody into the buffer and the stabilizer by ultrafiltration, concentrating after the replacement, and then adding polysorbate 20 according to Table 7, with the final concentration of the anti-LAG-3 antibody being 20 mg / mL, mixing evenly, filtering and sterilizing through a 0.22 μm filter membrane, and dispensing into vials, and stoppering and capping.

[0178] Using -80℃ stability sampling as a control, the long-term stability at 2-8℃, the accelerated stability at 20-25℃ and the light stability were investigated.

[0179] Table 7. Composition of the pharmaceutical composition

[0180]

[0181] The pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 were subjected to long-term 2-8°C, accelerated 25°C and light stability tests. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 .

[0182] (1) Long-term stability study at 2-8℃

[0183] The pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 were stored at 2-8°C for 24 months. Figures 1 to 3 .

[0184] The results showed that there were no significant changes and differences in the pH value, anti-LAG-3 antibody concentration, osmotic pressure molar concentration, SEC-HPLC and other results of F1a-b3-c1 and F1a-b7-c1; the non-reducing CGE assay showed that the monomer content of F1a-b3-c1 and F1a-b7-c1 decreased slightly during storage, and there was no difference between the two pharmaceutical compositions; the CIEX assay showed that the monomer content of F1a-b3-c1 and F1a-b7-c1 decreased slightly during storage, the alkaline peak increased, and there was no difference between the two pharmaceutical compositions. The results of the long-term stability study at 2-8°C showed that the pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 could remain stable for at least 24 months under 2-8°C conditions.

[0185] (2) Accelerated stability test at 25°C

[0186] The pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 were stored at 25°C for 12 months. Figures 4 to 6 .

[0187] The results showed that there were no significant changes and differences in the pH value, anti-LAG-3 antibody concentration, osmotic pressure molar concentration and other results of F1a-b3-c1 and F1a-b7-c1; SEC-HPLC determination showed that the main peak content of F1a-b3-c1 and F1a-b7-c1 decreased during storage, and F1a-b7-c1 was slightly better than F1a-b3-c1; non-reducing CGE determination showed that the monomer content of F1a-b3-c1 and F1a-b7-c1 decreased during storage, and F1a-b3-c1 was slightly better than F1a-b7-c1; CIEX determination showed that the monomer content of F1a-b3-c1 and F1a-b7-c1 decreased during storage, and there was no significant difference between the two pharmaceutical compositions.

[0188] (3) Light stability study

[0189] The pharmaceutical compositions F1a-b3-c1 and F1a-b7-c1 were exposed to light for 1 month at room temperature. Figures 7 and 8 .

[0190] The results showed that: by visual inspection, both F1a-b3-c1 and F1a-b7-c1 were yellow clear solutions, and there was no difference between the two pharmaceutical compositions; by SEC-HPLC, the main peak contents of F1a-b3-c1 and F1a-b7-c1 were reduced, and F1a-b3-c1 was superior to F1a-b7-c1; by non-reduced CGE, the monomer contents of F1a-b3-c1 and F1a-b7-c1 were reduced, F1a-b3-c1 is better than F1a-b7-c1; peptide mapping analysis was performed on the irradiated drug compositions. Compared with the drug compositions not irradiated, the deamidation of anti-LAG-3 antibodies in F1a-b3-c1 and F1a-b7-c1 did not increase, and the M (methionine), H (histidine) and W (tryptophan) oxidation sites of the anti-LAG-3 antibodies increased significantly, among which the oxidation of F1a-b3-c1 was lower than that of F1a-b7-c1.

[0191] Example 5

[0192] The pharmaceutical composition provided in this embodiment is shown in Table 8 below. The preparation method refers to Example 4, and the long-term stability at 2-8°C, the accelerated stability at 20-25°C, and the light stability are examined.

[0193] Table 8. Composition of the pharmaceutical composition

[0194]

[0195] Example 6

[0196] The pharmaceutical composition provided in this embodiment is shown in Table 9 below. The preparation method refers to Example 4, and the long-term stability at 2-8°C, the accelerated stability at 20-25°C, and the light stability are examined.

[0197] Table 9. Composition of the pharmaceutical composition

[0198]

[0199] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0200] The above-described embodiments only express several implementation methods of the present invention, which is convenient for understanding the technical solutions of the present invention in detail, but cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that, for those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can be made, which all belong to the protection scope of the present invention. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and the equivalent forms obtained also fall within the protection scope of this application. It should also be understood that the technical solutions obtained by those skilled in the art on the basis of the technical solutions provided by the present invention through logical analysis, reasoning or limited experiments are all within the protection scope of the claims attached to the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an anti-LAG-3 antibody or an antigen-binding fragment thereof, a buffer, a stabilizer and a surfactant; wherein, The buffer is sodium acetate buffer, and the concentration of the sodium acetate buffer is 10-25 mmol / L; The stabilizer is trehalose or proline, the concentration of the trehalose is 20-55 mg / mL, and the concentration of the proline is 150-350 mmol / L; The surfactant is polysorbate 20, and the concentration of polysorbate 20 is 0.1-0.3 mg / mL; The anti-LAG-3 antibody or antigen-binding fragment thereof comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6; The concentration of the anti-LAG-3 antibody or antigen-binding fragment thereof is 10-25 mg / mL; Furthermore, the pH of the pharmaceutical composition is 5-6.

2. The pharmaceutical composition according to claim 1, characterized in that The concentration of the sodium acetate buffer is 15-23 mmol / L; The concentration of trehalose is 45-55 mg / mL, and the concentration of proline is 250-350 mmol / L; The concentration of polysorbate 20 is 0.1-0.3 mg / mL; The concentration of the anti-LAG-3 antibody or antigen-binding fragment thereof is 15-23 mg / mL; Furthermore, the pH of the pharmaceutical composition is 5-6.

3. The pharmaceutical composition according to claim 1, characterized in that The concentration of the sodium acetate buffer is 20mmol / L; The concentration of trehalose is 50 mg / mL, and the concentration of proline is 300 mmol / L; The concentration of polysorbate 20 is 0.2 mg / mL; The concentration of the anti-LAG-3 antibody or antigen-binding fragment thereof is 20 mg / mL; Furthermore, the pH of the pharmaceutical composition is 5-6.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain variable region shown in SEQ ID NO:7 and a light chain variable region shown in SEQ ID NO:

8.

5. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that The anti-LAG-3 antibody or antigen-binding fragment thereof comprises a heavy chain shown in SEQ ID NO:9 and a light chain shown in SEQ ID NO:

10.

6. A freeze-dried preparation, characterized in that The lyophilized preparation is obtained by freeze-drying the pharmaceutical composition according to any one of claims 1 to 5, or the lyophilized preparation can form the pharmaceutical composition according to any one of claims 1 to 5 after being reconstituted.

7. A product, characterized in that The preparation comprises the pharmaceutical composition according to any one of claims 1 to 5.

8. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for treating cancer.

9. The use according to claim 8, characterized in that The cancer is relapsed, refractory, metastatic and / or advanced cancer; Optionally, the cancer is a hematological tumor or a solid tumor; Optionally, the cancer is leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, myeloma, melanoma, non-small cell lung cancer, small cell lung cancer, bladder cancer, head and neck cancer, ovarian cancer, colorectal cancer, renal cell carcinoma, Merkel cell carcinoma, fibrosarcoma, glioma, glioblastoma, liver cancer, nasopharyngeal cancer, prostate cancer, breast cancer, pancreatic cancer, endometrial cancer, biliary tract cancer, urinary system cancer, bone cancer, cervical cancer, thyroid cancer, anal cancer, gallbladder cancer or thymic cancer.

10. The use according to claim 8 or 9, characterized in that The pharmaceutical composition is for parenteral administration.

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