TW202333788A - Pharmaceutical composition comprising anti tnfr2 antibody - Google Patents

Abstract

The present invention relates to a TNFR2 antibody preparation. Specifically, the present invention provides a TNFR2 antibody pharmaceutical composition, which comprises TNFR2 antibody or its antigen binding segment, a buffer, a protein stabilizer and a surfactant. The pharmaceutical composition of the present invention can maintain the stability of TNFR2 antibody during the long-term storage and transportation of the product, thereby ensuring the stability, safety and effectiveness of the drug.

Description

An anti-TNFR2 antibody pharmaceutical composition

The present invention relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition containing a TNFR2 antibody or an antigen-binding fragment thereof.

The present invention claims the priority of the Chinese patent application submitted to the China Patent Office on December 28, 2021, with the application number 202111624712.7 and the invention name "An anti-TNFR2 antibody pharmaceutical composition", the entire content of which is incorporated into the present invention by reference. .

The preparations of macromolecular protein drugs (such as antibodies, etc.) are usually injections, which are suitable for parenteral administration. The administration methods usually include subcutaneous, intravenous, intramuscular, etc. Since proteins are relatively unstable in solution and can easily form particles and aggregate, stability has become a difficult problem in the development of macromolecular protein drugs. Therefore, the development of liquid preparations for macromolecular protein drugs (such as antibodies, etc.) first needs to solve the problem of protein stability.

In view of this, the present invention provides an anti-TNFR2 antibody pharmaceutical composition.

In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

In a first aspect, the present disclosure provides a pharmaceutical composition comprising an anti-TNFR2 antibody or an antigen-binding fragment thereof, a buffer, a protein stabilizer and a surfactant.

In one embodiment, the anti-TNFR2 antibody or antigen-binding fragment thereof comprises:

CDR1-VH is selected from SEQ ID NO. 1, 7, or 13;

CDR2-VH is selected from SEQ ID NO. 2, 8, or 14;

CDR3-VH is selected from SEQ ID NO. 3, 9, or 15;

CDR1-VL is selected from SEQ ID NO. 4, 10, or 16;

CDR2-VL is selected from SEQ ID NO. 5, 11, or 17; and

CDR3-VL is selected from SEQ ID NO. 6, 12, or 18.

In a specific embodiment, the anti-TNFR2 antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region:

(1) The heavy chain variable region includes:

a. Selected from the VH sequence shown in any one of SEQ ID NO: 19-23,

b. Selected from the group consisting of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 compared to any sequence of SEQ ID NO: 19-23 %, 99% or 100% sequence identity of the VH sequence, or,

c. Selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or compared to any sequence of SEQ ID NO: 19-23 Replacement VH sequence;

(2) The light chain variable region includes:

a. Selected from the VL sequence shown in any one of SEQ ID NO: 24-28,

b. Selected from the group consisting of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 compared to any sequence of SEQ ID NO: 24-28 %, 99% or 100% sequence identity of the VL sequence, or,

c. Selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or compared to any sequence of SEQ ID NO: 24-28 Replaced VL sequence;

Optionally, the amino acid insertion, deletion and/or substitution occurs in the heavy chain variable region or/and the framework region (FR) of the light chain variable region;

Optionally, the substitution is a substitution of a conservative amino acid.

In another specific embodiment, the heavy chain variable region and light chain variable region are selected from the group consisting of:

(1) Having VH shown in SEQ ID NO. 19 and VH shown in SEQ ID NO. 24 VL;

(2) Having VH shown in SEQ ID NO. 19 and VL shown in SEQ ID NO. 25;

(3) Having VH shown in SEQ ID NO. 20 and VL shown in SEQ ID NO. 26;

(4) Having VH shown in SEQ ID NO. 21 and VL shown in SEQ ID NO. 26;

(5) Having VH shown in SEQ ID NO. 22 and VL shown in SEQ ID NO. 27;

(6) Having VH shown in SEQ ID NO. 23 and VL shown in SEQ ID NO. 28;

(7) It has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% compared to any of the above sequence combinations (1) to (6). , VH and VL combinations with 98%, 99% or 100% sequence identity;

(8) Having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions or deletions compared to any of the above sequence combinations (1) to (7) and/or alternative VH and VL combinations; or

(9) It has a complementarity-determining region (CDR) that is completely identical to any of the above sequence combinations (1) to (8), and the FR has at least 85%, 90%, 91%, 92%, 93 VH and VL combinations with %, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity;

Alternatively, the amino acid insertion, deletion and/or substitution occurs in the heavy chain variable region or/and the FR region of the light chain variable region;

Optionally, the substitution is a substitution of a conservative amino acid.

In a specific embodiment, the anti-TNFR2 antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies ), monovalent antibodies, multiple valent antibody, full-length antibody, antibody fragment, Fab, Fab', F(ab')2, Fd, Fv, scFv, or diabody.

In another embodiment, the concentration of the anti-TNFR2 antibody is 1 mg/mL to 100 mg/mL, preferably 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70 mg/mL, 80 mg /mL, 90mg/mL, more preferably 30mg/mL.

In another embodiment, the buffer is selected from the group consisting of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, histidine-hydrochloride histidine buffer or disodium hydrogen phosphate-diphosphate The sodium hydrogen buffer is preferably histidine-histidine hydrochloride buffer.

In a specific embodiment, the buffer concentration is 10-100mM, preferably 10-60mM, and more preferably 20mM.

In another embodiment, the protein stabilizer is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose or sorbitol, preferably sorbitol.

In a specific embodiment, the concentration of the protein stabilizer is 1-10% (w/v), preferably 2-8% (w/v), and more preferably 4.7% (w/v).

In another embodiment, the surfactant is selected from polysorbate 80, poloxamer or glyceryl fatty acid ester, preferably polysorbate 80.

In a specific embodiment, the concentration of the surfactant is 0.02~0.1% (w/w), preferably 0.02% (w/w), 0.04% (w/w), 0.06% (w/ w), 0.08% (w/w), 0.1% (w/w), and more preferably 0.04% (w/w).

In another embodiment, the pH value of the pharmaceutical composition is 4.5-7.5, preferably 4.5, 5, 5.5, 6, 6.5, 7, 7.5, more preferably 5.5.

In a second aspect, the present disclosure provides a pharmaceutical composition, said pharmaceutical composition comprising:

a) anti-TNFR2 antibody at a concentration of 1 mg/mL to 100 mg/mL, b) histidine-histidine hydrochloride buffer at a concentration of 10-100 mM, pH 4.5-7.5, c) a concentration of 1-10% (w/v) sorbitol, and d) polysorbate 80 at a concentration of 0.02~0.1% (w/w);

Preferably, the pharmaceutical composition contains:

a) anti-TNFR2 antibody at a concentration of 10 mg/mL to 60 mg/mL, b) histidine-histidine hydrochloride buffer at a concentration of 10-60 mM, pH 5.0-6.0, c) a concentration of 2-8% (w/v) sorbitol, and d) polysorbate 80 at a concentration of 0.04% (w/w);

More preferably, the pharmaceutical composition contains:

a) anti-TNFR2 antibody at a concentration of 30 mg/mL, b) histidine-histidine hydrochloride buffer at a concentration of 20 mM, pH 5.5, c) sorbitol at a concentration of 4.7% (w/v), and d) Polysorbate 80 at a concentration of 0.04% (w/w).

In a specific embodiment, the pharmaceutical composition is in the form of a liquid preparation.

In a third aspect, the present disclosure provides a method for preparing the pharmaceutical composition as described in the first and second aspects, including the step of replacing the anti-TNFR2 antibody solution with a buffer, and the buffer is preferably histidine. -Histidine hydrochloride buffer, the concentration of the buffer is preferably 20mM, and the pH of the buffer is preferably 5.5.

In a fourth aspect, the present disclosure provides a pharmaceutical composition of an anti-TNFR2 antibody or an antigen-binding fragment thereof, which is prepared by the method described in the third aspect.

In a fifth aspect, the present disclosure provides pharmaceutical compositions described in the first to second aspects, products prepared by the method described in the third aspect, or pharmaceutical compositions described in the fourth aspect in the preparation of treatments and / Or use in drugs to prevent immune abnormality-related diseases; Preferably, the immune abnormality-related disease is a disease related to Treg cells and/or MDSC function; Preferably, the disease is cancer or an autoimmune disease; More preferably, the disease is a cancer or an autoimmune disease. , the cancer is selected from the group consisting of ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or response to CTLA-4 and PD-1 Therapy-resistant advanced solid tumors, such as metastatic melanoma; more preferably, the autoimmune disease can be selected from the group consisting of rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disease, systemic Lupus erythematosus, myasthenia gravis, IgG4-related diseases sick.

In a sixth aspect, the present disclosure provides a method for treating and/or preventing immune abnormality-related diseases, the method comprising administering to a subject an effective amount of the pharmaceutical composition described in the first to second aspects, and a third The product prepared by the method described in the fourth aspect, or the pharmaceutical composition described in the fourth aspect; Preferably, the immune abnormality-related disease is a disease related to Treg cells and/or MDSC function; Preferably, the disease is cancer or autologous Immune disease; more preferably, the cancer is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or Advanced solid tumors that are resistant to CTLA-4 and PD-1 therapy, such as metastatic melanoma; more preferably, the autoimmune disease can be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, Neuromyelitis optica spectrum disease, systemic lupus erythematosus, myasthenia gravis, IgG4-related diseases.

Beneficial effects: The TNFR2 antibody preparation of the present invention can maintain the stability of the anti-TNFR2 antibody during long-term storage and transportation of the product, thereby ensuring the stability, safety and effectiveness of the drug.

Figure 1 shows the trend chart of the summary of CEX-HPLC results when samples were placed at 2-8°C and 25°C to confirm the stability of the prescription;

Figure 2 shows the trend chart summarizing the SE-HPLC results when the sample was placed at 2-8°C and 25°C to confirm the stability of the prescription;

Figure 3 shows the summary trend chart of CE-SDS (NR) results when samples were placed at 2-8°C and 25°C to confirm the stability of the prescription;

Figure 4 shows the trend chart of the CE-SDS(R) results when the sample was placed at 2-8°C and 25°C to confirm the stability of the prescription.

The invention discloses an anti-TNFR2 antibody pharmaceutical composition. Technical personnel can learn from the content of this article and appropriately improve the implementation of process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make modifications or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.

The raw materials and reagents used in the anti-TNFR2 antibody pharmaceutical composition provided by the invention can be purchased from the market.

The present invention will be further described below in conjunction with the examples:

Terminology explanation:

Unless otherwise stated, terms used herein have the meanings commonly understood by those of ordinary skill in the art. For terms that are expressly defined herein, the meaning of that term shall be governed by the stated definition.

As used herein, the term "TNFR2" refers to tumor necrosis factor receptor 2, also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF1B) or CD120b, a membrane receptor that binds tumor necrosis factor-alpha (TNFα). body. The TNFR2 is preferably human TNFR2.

As used herein, the terms "anti-tumor necrosis factor receptor 2 antibody", "tumor necrosis factor receptor 2 antibody", "anti-TNFR2 antibody", "TNFR2 antibody", "anti-TNFR2 antibody portion" and/or "anti-TNFR2 antibody" -TNFR2 antibody fragment" and the like refers to any protein containing at least a portion of an immunoglobulin molecule capable of specifically binding to TNFR2 (such as, but not limited to, at least one complementarity determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy chain or light chain variable region, heavy chain or light chain constant region, framework region or any part thereof) protein or peptide-containing molecule. TNFR2 antibodies also include antibody-like protein scaffolds such as the tenth fibronectin type III domain (10Fn3), which contain BC, DE and FG structural loops similar in structure and solvent accessibility to the antibody CDRs. The tertiary structure of the 10Fn3 domain is similar to the tertiary structure of the IgG heavy chain variable region, and is obtained by combining the residues of the BC, DE and FG loops of 10Fn3. By replacing the bases with residues from the CDR-H1, CDR-H2 or CDR-H3 region of a TNFR2 monoclonal antibody, one skilled in the art can graft, for example, the CDRs of a TNFR2 monoclonal antibody onto a fibronectin scaffold.

As used herein, the term "antibody" (Ab) refers to an immunoglobulin molecule that specifically binds or is immunoreactive to a target antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies (including but not Limited to chimeric antibodies, humanized antibodies, fully human antibodies, heterologous conjugated antibodies (such as bispecific, trispecific and tetraspecific antibodies, diabodies, tribodies and tetrabodies), antibody conjugates) and antigen-binding fragments of antibodies (including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments). Furthermore, unless otherwise stated, the term "monoclonal antibody" (mAb) is intended to include intact antibody molecules capable of specifically binding to a target protein as well as incomplete antibody fragments (e.g., Fab and F(ab')2 fragments, which lack The Fc fragment of the intact antibody (clears more quickly from the animal circulation) and therefore lacks Fc-mediated effector function (see Wahl et al., J. Nucl. Med. 24:316, 1983; cited in Add to this article).

As used herein, the term "monoclonal antibody" refers to an antibody derived from a single clone (including any eukaryotic, prokaryotic, or phage clone) and is not limited to the method of production of the antibody.

As used herein, the terms "antigen-binding fragment" and "antibody fragment" are interchangeable and refer to one or more antibody fragments that retain the ability to specifically bind a target antigen. The antigen-binding function of an antibody can be performed by fragments of the full-length antibody. The antibody fragment may be a Fab, F(ab')2, scFv, SMIP, diabody, tribody, affibody, Nanobody, aptamer or domain antibody. Examples of binding fragments encompassed by the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragments, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab)2 fragments , a bivalent fragment containing two Fab fragments connected by a disulfide bond at the hinge region; (iii) an Fd fragment consisting of VH and CHl domains; (iv) consisting of the VL and VH domains of an antibody single arm Fv fragment; (V) dAb containing VH and VL domains; (vi) dAb fragment consisting of VH domain (Ward et al., Nature 341:544-546, 1989); (vii) a dAb consisting of a VH or VL domain; (viii) an isolated complementarity determining region (CDR); and (ix) two or more A combination of isolated CDRs that may optionally be linked by synthetic linkers. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, the two domains can be joined using recombinant methods through a linker that enables them to be made in which the VL and VH regions pair to form A single protein chain of a monovalent molecule (termed a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988 and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 ,1988). These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and these fragments are screened for use in the same manner as intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact immunoglobulins, or in some embodiments by chemical peptide synthesis procedures known in the art.

As used herein, the term "complementarity determining region" (CDR) refers to the hypervariable regions found in both light and heavy chain variable domains. The more conserved portion of the variable domain is called the framework region (FR). As is understood in the art, the amino acid positions representing the hypervariable regions of an antibody may vary depending on the context and various definitions known in the art. Some positions within the variable domain can be considered hybrid hypervariable positions because these positions can be considered to be within the hypervariable region under one set of criteria (such as IMGT or KABAT) and within a different group. outside the hypervariable zone under standards such as KABAT or IMGT. One or more of these locations may also be found in the extended hypervariable zone. The invention includes antibodies comprising modifications in these hybrid hypervariable positions. The variable domains of native heavy and light chains each contain four framework regions that primarily adopt a sheet configuration, connected by three CDRs (CDR1, CDR2, and CDR3) that form loops that connect the sheet structure , and in some cases form part of the lamellar structure. The CDRs in each chain are held closely together by the FR region in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and with CDRs from other antibody chains contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Protein sof Immunological Interest, National Institute of Health, Bethesda, MD. 1987; which is incorporated herein by reference). For example, in this article, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, second CDR and third CDR of the heavy chain variable region (VH) respectively. These three CDRs constitute the heavy chain variable region (VH). The CDR combination of the light chain (or its variable region) (VHCDR combination); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and third CDR of the light chain variable region (VL) respectively. Three CDRs, these three CDRs constitute the CDR combination of the light chain (or its variable region) (VLCDR combination).

As used herein, the term "Kabat numbering system" generally refers to the immunoglobulin alignment and numbering system proposed by Elvin A. Kabat (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service , National Institutes of Health, Bethesda, Md., 1991).

As used herein, the term "VH" refers to the variable region of an immunoglobulin heavy chain of an antibody (including the heavy chain of a Fv, scFv, or Fab). The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of an Fv, scFv, dsFv or Fab).

The term "heavy chain constant region" herein refers to the carboxyl-terminal portion of the antibody heavy chain that is not directly involved in the binding of the antibody to the antigen, but exhibits effector functions, such as interaction with Fc receptors, that are relative to the antibody's Variable domains have more conserved amino acid sequences. "Heavy chain constant region" at least includes: CH1 domain, hinge region, CH2 domain, CH3 domain, or variants or fragments thereof. “Heavy chain constant region” includes “full-length heavy chain constant region” and “heavy chain constant region fragment”, the former having a structure substantially similar to a natural antibody constant region, and “heavy chain constant region fragment” including only “a portion of the full-length heavy chain constant region” . Illustratively, a typical "full-length antibody heavy chain constant region" consists of CH1 domain-hinge region-CH2 domain-CH3 domain; when the antibody is IgE, it also includes a CH4 domain; when the antibody is a heavy chain antibody , then it does not include the CH1 domain. Illustratively, a typical "heavy chain constant region fragment" can be selected from CH1, Fc or CH3 domains.

The term "light chain constant region" herein refers to the carboxyl-terminal portion of the antibody light chain, which is not directly involved in the binding of the antibody to the antigen. The light chain constant region may be selected from the constant kappa structure. domain or constant lambda domain.

As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable and refer to the alignment of sequences and the introduction of gaps (if necessary) to achieve maximum percent sequence identity (e.g. , for optimal alignment, gaps can be introduced in one or both of the candidate and reference sequences, and for comparison purposes, non-homologous sequences can be ignored) followed by the amino acids (or nucleosides) of the candidate sequence Acid) residues are identical to the amino acid (or nucleotide) residues of the reference sequence. For the purpose of determining percent sequence identity, alignment can be accomplished in a variety of ways well known to those skilled in the art, such as using publicly available computer software, such as BLAST, ALIGN or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximal alignment over the full length of the sequences being compared. For example, a reference sequence aligned for comparison with a candidate sequence may show that the candidate sequence exhibits a 50%/( to 100% sequence identity. The length of the candidate sequences aligned for comparison purposes may be, for example, at least 30% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of the length of the reference sequence. . When a position in the candidate sequence is occupied by the same amino acid (or nucleotide) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

The term "conserved amino acids" herein generally refers to amino acids that belong to the same class or have similar characteristics (eg, charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). For example, the amino acids in each of the following groups belong to each other's conserved amino acid residues, and the substitution of amino acid residues within the group belongs to the substitution of conservative amino acids:

(1) Acidic amino acids: Asp (D) and Glu (E);

(2) Basic amino acids: Lys(K), Arg(R) and His(H);

(3) Hydrophilic uncharged amino acids: Ser(S), Thr(T), Asn(N) and Gln(Q);

(4) Aliphatic uncharged amino acids: Gly(G), Ala(A), Val(V), Leu (L) and Ile(I);

(5) Non-polar uncharged amino acids: Cys(C), Met(M) and Pro(P);

(6) Aromatic amino acids: Phe (F), Tyr (Y) and Trp (W).

As used herein, the term "specific binding" refers to a binding reaction that determines the presence of an antigen in a heterogeneous population of proteins and other biomolecules, e.g., by antibodies or antigen-binding fragments thereof Specific recognition. An antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of less than 100 nM. For example, an antibody or antigen-binding fragment thereof that specifically binds to an antigen will bind to the antigen with a KD of up to 100 nM (eg, between 1 pM and 100 nM). An antibody or antigen-binding fragment thereof that does not exhibit specific binding to a particular antigen or epitope thereof will exhibit a KD greater than 100 nM (eg, greater than 500 nM, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen or epitope thereof. A variety of immunoassay formats can be used to select antibodies that specifically immunoreact with a specific protein or carbohydrate. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with proteins or carbohydrates. See, Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), which describe methods that can be used to determine specific Immunoassay formats and conditions for immunoreactivity.

As used herein, "pharmaceutical composition" means a mixture containing one or more compounds described herein, or physiologically/pharmaceutically acceptable salts or prodrugs thereof, together with other chemical components, such as physiologically/ Pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active ingredients of the antibody, promote administration to the organism, and facilitate the absorption of the active ingredients to exert biological activity. As used herein, "pharmaceutical composition" and "preparation" are not mutually exclusive.

The present invention will be further described below in conjunction with the examples:

Example 1 Humanization, expression and purification of anti-TNFR2 monoclonal antibody

1.1 Humanization of anti-TNFR2 monoclonal antibody

The "CDRs transplantation" method is used for antibody humanization, that is, the humanized antibody with the highest homology is selected based on the sequence to provide the antibody framework regions (FRs), and the antigen-binding fragment complementarity determining regions (CDRs) of the target antibody based on the Kabat naming method are ), transplanted to the former to form a humanized antibody variable region sequence in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Among them, the complementarity determining regions (CDRs) of the antigen-binding fragments of the Kabat nomenclature of the TNFR2 antibody are detailed in Table 1, Table 2 shows the VH and VL sequences of the humanized antibody molecule, and Table 3 shows the pairing of the VH and VL sequences of the humanized anti-TNFR2 antibody. .

1.2 Expression of humanized anti-TNFR2 antibody

Humanized anti-TNFR2 antibodies are expressed using CHO as host cells and cultured in Dynamis AGT medium (Manufacturer: gibco, Cat. No.: A26615-01). The culture period does not exceed 15 days. The reactor control parameters are pH 6.8~7.6. Dissolved oxygen (DO) is 20%~80%, rotation speed is 79RPM~81RPM, and initial culture temperature 36.0℃~37.0℃; when culture reaches the 8th day, reduce the culture temperature to 32.0±0.5℃ until harvest.

1.3 Purification of humanized anti-TNFR2 antibody

The purification of humanized anti-TNFR2 antibodies uses multi-step chromatography, concentration and filtration unit operations to purify the antibodies in sequence. The supernatant harvested liquid was captured by Protein A affinity chromatography (Mabselect PrismA). The captured antibody solution is incubated at low pH to inactivate potential viruses. The antibody solution is neutralized and the precipitate is removed by depth filtration. Then, anion exchange chromatography (Capto Q) is performed sequentially to remove HCD, HCP, protein A and other impurities, and cation exchange (Capto S Impact) chromatography is performed to remove impurities such as HCP and aggregates. Filtration through nanofiltration membrane bags to remove potential endogenous and endogenous viruses. The antibody solution is then concentrated using an ultrafiltration membrane bag and the buffer is replaced to complete the purification and obtain the humanized anti-TNFR2 antibody protein stock solution.

Example 2 Buffer system screening

2.1 Buffer system screening plan

The experiment covered 4 kinds of buffer salts (acetate, citrate, histamine and phosphate), with pH values from 4.5 to 7.5, and a total of 12 preparation buffer systems were set up for screening (see Table 4). The protein stock solution obtained in Example 1 was subjected to ultrafiltration, concentration, and liquid replacement. The stock solution was replaced into the corresponding 12 preparation buffer systems: the protein stock solution was added into the corresponding ultrafiltration tubes in 12 groups, and a centrifuge was used for ultrafiltration and concentration. , the protein is retained, and the buffer of the original protein solution flows through the ultrafiltration membrane to achieve the purpose of protein concentration. Then the corresponding 12 preparation buffers are added respectively. The buffer of the original protein solution is diluted, and the ultrafiltration and concentration are continued. The concentration is completed. Then continue to add the corresponding 12 preparation buffers respectively, continue ultrafiltration and concentration, and repeat the operation until the liquid change is completed. The humanized TNFR2 antibody #2-1 described in Example 1 was used in each of the above prescriptions, and the protein concentration was 30 mg/mL. Through the 40℃ high temperature accelerated test, the appearance, pH value, concentration, purity (size exclusion chromatography (SE-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis (CE-SDS (NR))), charge isomerism (ion exchange chromatography (CEX-HPLC)), dynamic light scattering (DLS), thermal stability (poor Using Display Scanning Fluorescence (DSF) as an indicator, the stability of this protein in 12 buffer systems was comprehensively investigated and compared to select the optimal buffer system. The specific prescription composition and investigation plan are shown in Table 4 below.

2.2 Buffer system screening results

The main results of buffer system screening are summarized in Table 5-Table 6 below.

2.3 Conclusion of buffer system screening

In the 12 buffer systems, after 4 weeks of inspection at 40°C, the appearance was slightly yellow, slightly opalescent liquid, and the pH and concentration were within the target values without significant changes;

Thermal stability test, protein melting temperature (Tm): Tm1 of F4 prescription is 68.46°C, which is significantly lower than other prescriptions. Tm1 of other prescriptions are all greater than 69°C, with no significant difference;

DLS: Particle size sorting (from small to large): F7, F8, F9>F3, F10, F11, F12>F1>F6, F2>F5, F4;

SE-HPLC: Purity ranking F7>F8>F2, F9, F3>F1, F5, F6>F10>F11, F4>F12;

CE-SDS(NR): Main peak purity ranking F8, F9, F6, F7, F3>F10>F11, F5>F2>F12, F4>F1;

CEX-HPLC: Main peak purity F8>F7, F9, F6>F3>F2, F1, F5, F10>F4>F11>F12;

The acidic peaks increase, and the order of advantages and disadvantages is F7, F1>F8, F2>F3, F4, F5, F6, F9>F10>F11>F12;

To sum up, after screening the buffer system, the ranking of the advantages and disadvantages of each prescription is as follows: F7>F8>F9, F3>F2>F1>F4, F5, F6, F10, F11, F12. The F7 formula was comprehensively superior to other formulas in terms of purity, degradation, aggregation, charge heterogeneity, etc., and showed good stability in this 40°C accelerated experimental investigation. Therefore, the F7 formula (20mM histidine acid-histidine hydrochloride, pH 5.5) was initially selected as the optimal formula as the buffer system for the preparation of this product for the next step of excipient screening research.

Example 3 Excipient screening

3.1 Excipient screening plan

Based on the buffer system screening results and the goal of developing freeze-dried preparations, corresponding excipients were added to the optimal buffer system, including sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol, and sorbitol, a total of 7 Prescription, protein concentration is 30mg/mL. Through the freeze-thaw test (one round of freeze-thaw from -20°C to 25°C, 3-5 rounds of continuous freeze-thaw), and the high-temperature accelerated test at 40°C, the appearance, pH value, concentration, and purity (size exclusion chromatography (SE-HPLC) ), non-reducing sodium dodecyl sulfate capillary electrophoresis size exclusion chromatography (CE-SDS (NR)), charge isomerization (ion exchange chromatography (CEX-HPLC)), dynamic light scattering (DLS), thermal stability The stability (differential scanning fluorescence (DSF)) was used as an indicator to examine the stability of each formulation, and an optimal formulation was selected for the next round of surfactant screening. The specific formulation composition and investigation plan are shown in Table 7 below.

3.2 Excipient screening results

The main results of excipient screening are summarized in Table 8-Table 11 below.

Table 9 Excipient screening 40℃ accelerated test results

Table 11 Excipient screening-20℃ to 25℃ freeze-thaw test results

3.3 Conclusions on screening of excipients

The sample concentration is 30 mg/mL, and five freeze-thaw experiments from -20°C to 25°C and excipient screening at 40°C for 4 weeks were conducted.

The appearance is yellowish, slightly opalescent liquid, and the pH and concentration are within the target values without significant changes;

Thermal stability test (DSF), melting temperature of protein (Tm1 and Tm2): F7-4, F7-5>F7-2, F7-6, F7-7, without excipients (F7)>F7-1, F7 -3;

DLS: After 4 weeks of inspection at 40°C, the particle size of F7-1 increased significantly relative to T0; the particle size of F7-2 and F7-6 increased significantly relative to T0 after freezing and thawing for 5 times;

SE-HPLC: 4 weeks at 40°C, purity F7-2, F7-3, F7-4, F7-5, F7-6, F7-7>F7-1; freeze and thaw 5 times, purity F7-1, F7 -3, F7-4, F7-5, F7-7>F7-2>F7-6;

CE-SDS(NR): 4 weeks of investigation at 40°C, CE(NR) main peak purity F7-7, F7-5, F7-6, F7-4, F7-2>F7-1, F7-3; freeze-thaw 5 No significant changes;

CEX-HPLC: 4 weeks of investigation at 40°C, main peak purity F7-3>F7-1, F7-6, F7-7>F7-2, F7-4, F7-5; acidic peaks increased, ranking F7-1, F7-3>F7-6, F7-7>F7-4, F7-5, F7-2; there was no significant difference among the prescriptions after freezing and thawing 5 times.

In summary, excipient screening has evaluated various stability indicators. The F7-7 prescription has improved in terms of purity, degradation, aggregation, charge heterogeneity, etc. in this 40°C accelerated experiment and 5 freeze-thaw inspections from -20 to 25°C. It is comprehensively better than other prescriptions and shows good stability. Therefore, the F7-7 formula (4.7% sorbitol (W/V), 20mM histidine acid-histidine hydrochloride, pH 5.5) was initially selected as the optimal formula for the next step of surfactant screening research.

Example 4 Surfactant Screening

4.1 Surfactant screening scheme

Add different concentrations of surfactants to the optimal buffer system and excipients, including polysorbate 80-free, 0.02%, 0.04%, 0.06%, 0.08%, and 0.1% polysorbate 80 (W/W) for a total of 6 A prescription, with a protein concentration of 30 mg/mL, passed the 40°C acceleration test and 300 rpm shaking test, and was evaluated for appearance, pH value, concentration, purity (size exclusion chromatography (SE-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis size exclusion chromatography Method (CE-SDS (NR)), charge isomerization (ion exchange chromatography (CEX-HPLC)), dynamic light scattering (DLS), and insoluble particles were used as indicators to examine the stability of each formulation and select an optimal formulation. The prescription was subjected to a prescription confirmation stability test to confirm the final prescription. The specific prescription composition and investigation plan are shown in Table 12 below.

T0=starting point of investigation; W=week; D=day.

4.2 Surfactant screening results

The main results of surfactant screening are summarized in Table 13-Table 14 below.

備註：NA=不適用。

Note: NA=not applicable.

4.3 Conclusions on surfactant screening

The sample concentration is 30 mg/mL, and the test is conducted at 40°C for 4 weeks. The appearance is colorless slightly opalescent liquid; the pH and concentration are within the target values without significant changes;

SE-HPLC: pros and cons of each prescription, F7-7-3, F7-7-5>F7-7-2, F7-7-6>F7-7-4>F7-7-1;

CE-SDS(NR): pros and cons of each prescription, F7-7-6, F7-7-2>F7-7-4, F7-7-3>F7-7-5, F7-7-1;

DLS: The advantages and disadvantages of each particle size prescription, F7-7-1, F7-7-6>F7-7-5>F7-7-2>F7-7-4>F7-7-3;

10μm的顆粒始終明顯多於其他處方；其他處方均無明顯差異； Insoluble particles: pros and cons of each prescription, F7-7-1 prescription

There are always significantly more particles of 10 μm than other prescriptions; there is no significant difference in other prescriptions;

CEX-HPLC: The F7-7-3 prescription is slightly better than other prescriptions.

10μm的顆粒始終明顯多於其他處方；其他處方均無明顯差異CE-SDS(NR)：主峰含量F7-7-3、F7-7-6>F7-7-4>F7-7-1>F7-7-2、F7-7-5。 Shake at 25℃, 300rpm for 7 days. SE-HPLC, DLS, CEX-HPLC: there is no significant difference in each prescription; insoluble particles: F7-7-1 prescription

There are always more 10μm particles than other prescriptions; there is no significant difference in other prescriptions. CE-SDS(NR): main peak content F7-7-3, F7-7-6>F7-7-4>F7-7-1>F7 -7-2, F7-7-5.

In summary, excipient screening passed the evaluation of various stability indicators: F7-7-3, F7-7-6>F7-7-2, F7-7-4>F7-7-5>F7-7-1, That is, the optimal prescription is F7-7-3 or F7-7-6.

However, the polysorbate 80 content in prescriptions F7-7-2 and F7-7-4 is at the upper and lower edges of the polysorbate 80 content in prescription F7-7-3, and the polysorbate 80 content in prescription F7-7-5 is The sorbide 80 content is on the edge of the polysorbate 80 content in prescription F7-7-6, while prescriptions F7-7-2 and F7-7-4 are both better than prescription F7-7-5.

Therefore, considering the operable space of production, the F7-7-3 formula (0.04% polysorbate 80 (W/W), 20mM histidine acid-histidine hydrochloride, 4.7% sorbitol, pH5.5) was selected to be more as appropriate. Therefore, F7-7-3 prescription (0.04% polysorbate 80 (W/W), 20mM histidine-histidine hydrochloride, 4.7% sorbitol (W/V), pH5.5) is the optimal prescription for this screening, and we are preparing to conduct the next step of prescription confirmation and stability studies, and conduct final testing on this prescription. Confirm.

Example 5 Prescription Confirmation Stability

5.1 Prescription Confirmation Stability Plan

The stability confirmation and packaging material selection research experiments of this prescription are based on the results of screening of buffer systems, excipients and surfactants. One prescription is determined: 30mg/mL TNFR2 monoclonal antibody (#2-1), 20mM histidine-hydrochloric acid Histidine, 4.7% sorbitol (W/V), 0.04% polysorbate 80 (W/W), pH 5.5, 5mL/bottle, dispense into vials, use rubber stopper and aluminum-plastic cap to form a seal The packaging system was placed upright and inverted respectively, and immediate stability and accelerated stability tests were carried out for 6 months, and stability confirmation studies were conducted on the final prescription.

The specific inspection plan is shown in Table 15 below.

In this scheme, the activity detection method uses Elisa and Cell assay blocking methods, the details are as follows:

Elisa method: Use the binding effect of anti-TNFR2 monoclonal antibody to human TNFR2 to detect the half-effect concentration of anti-TNFR2 monoclonal antibody on 50ng of TNFR2. The 96-well plate is pre-coated with the antigen (human TNFR2-his) (Sino Biological, 10417-H08H). The anti-TNFR2 monoclonal antibody binds to the antigen on the enzyme plate. The plate is washed to remove various unbound components, and then Peroxidase- conjugated Mouse anti Human IgG Fcγ (Jackson Immuno, 209-035-098). After incubation, wash the plate to remove excess enzyme-labeled antibodies; finally add HRP substrate TMB (Thermo, 34029) to produce a blue product, the color depth is the same as Anti-TNFR2 monoclonal antibody concentration is directly proportional. After the stop solution is terminated, the blue product turns to yellow, and the OD value is read with a microplate reader.

Cell blocking assay method: Humanized anti-TNFR2 monoclonal antibody is a monoclonal antibody that specifically binds to Human TNFR2 and can inhibit the binding of Human TNFα to the receptor Human TNFR2. This method uses the monoclonal cell line CT26-2A03 overexpressing TNFR2 obtained through self-screening as the detection cell. First add humanized anti-TNFR2 monoclonal antibody, then add biotin-labeled TNFα (TNFα-biotin) (AcroBiosystems, TNA-H82E3), the ligand TNFα interacts with the receptor TNFR2, and then add PE-labeled Streptavidin (Biolegend , 405204) is incubated to form a CHO-TNFR2~TNFα-biotin~Streptavidin-PE complex. At this time, the cells are indirectly labeled with PE dye, and obvious positive signals can be seen by microplate reader detection. If the binding site of the humanized anti-TNFR2 monoclonal antibody and Human TNFR2 conflicts with the binding site of Human TNFα, it will affect the binding of Human TNFα to Human TNFR2, ultimately affecting the intensity of the PE signal and the detection performance on the microplate reader. This is the weakening of the PE signal. Using this principle, the function of humanized anti-TNFR2 monoclonal antibodies in inhibiting the binding of Human TNFR2 to Human TNFα at the cellular level can be detected.

5.2 Prescription confirmation stability results

The summary of the main results of prescription confirmation stability are shown in Table 16-Table 17 below, Figure 1-Figure 4.

Table 16 Prescription confirmation stability results

5.3 Conclusion of stability study on prescription confirmation

After being placed at a low temperature of 2~8°C for 6 months, there was no significant change in conventional testing indicators, insoluble particles, purity, charge heterogeneity, activity, etc. between the upright and inverted samples, and there was no significant difference between the upright and inverted samples.

After 6 months of accelerated stability testing at 25°C, the upright and inverted samples were within the quality standards in terms of conventional testing indicators, insoluble particles, purity, charge heterogeneity, activity, etc., and there was no significant difference between the upright and inverted samples. .

After 3 months of accelerated stability testing at 40°C, only the CE-SDS (NR) and CEX-HPLC purity indicators of the upright and inverted samples exceeded the quality standards. Other purity indicators, conventional detection indicators, insoluble particles, and charge heterogeneous bodies , activity, etc. are all within the range of quality standards, and there is no obvious difference between the upright and inverted samples.

To sum up, during the instant stability investigation at low temperature (2~8℃), The conventional testing indicators, insoluble particles, purity, charge heterogeneity, activity, etc. of the sample did not change significantly. In the 25°C accelerated experiment, all indicators were within the quality standard range, indicating that the protein in this prescription has good stability. The purpose of the invention is achieved. In the accelerated stability test at high temperature of 40°C, there was a downward trend in purity and charge heterogeneity, which is consistent with the trend of the quality indicators of this prescription during the prescription screening stage. Under high temperature conditions, some key indicators show a downward trend, which also indicates that this product is sensitive to high temperatures. It is recommended that this product be stored and transported at low temperature (2~8℃).

The above describes in detail an anti-TNFR2 antibody pharmaceutical composition provided by the present invention. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only used to help understand the method and its core idea of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims (19)

A pharmaceutical composition, characterized in that the pharmaceutical composition contains an anti-TNFR2 antibody or an antigen-binding fragment thereof, a buffer, a protein stabilizer and a surfactant. The pharmaceutical composition according to claim 1, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof contains: CDR1-VH is selected from SEQ ID NO. 1, 7, or 13; CDR2-VH is selected from SEQ ID NO. 2, 8, or 14; CDR3-VH is selected from SEQ ID NO. 3, 9, or 15; CDR1-VL is selected from SEQ ID NO. 4, 10, or 16; CDR2-VL is selected from SEQ ID NO. 5, 11, or 17; and CDR3-VL is selected from SEQ ID NO. 6, 12, or 18. The pharmaceutical composition according to claim 1 or 2, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof contains a heavy chain variable region and a light chain variable region: (1) The heavy chain variable region includes: a. Selected from the VH sequence shown in any one of SEQ ID NO: 19-23, b. Selected from the group consisting of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 compared to any sequence of SEQ ID NO: 19-23 %, 99% or 100% sequence identity of the VH sequence, or, c. Selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or compared to any sequence of SEQ ID NO: 19-23 Replacement VH sequence; (2) The light chain variable region includes: a. Selected from the VL sequence shown in any one of SEQ ID NO: 24-28, b. Selected from the group consisting of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 compared to any sequence of SEQ ID NO: 24-28 %, 99% or 100% sequence identity of the VL sequence, or, c. Selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or compared to any sequence of SEQ ID NO: 24-28 Replaced VL sequence; Alternatively, the amino acid insertion, deletion and/or substitution occurs in the heavy chain variable region or/and the FR region of the light chain variable region; Optionally, the substitution is a substitution of a conservative amino acid. The pharmaceutical composition according to claim 3, wherein the heavy chain variable region and the light chain variable region are selected from the following group: (1) Having VH shown in SEQ ID NO. 19 and VL shown in SEQ ID NO. 24; (2) Having VH shown in SEQ ID NO. 19 and VL shown in SEQ ID NO. 25; (3) Having VH shown in SEQ ID NO. 20 and VL shown in SEQ ID NO. 26; (4) Having VH shown in SEQ ID NO. 21 and VL shown in SEQ ID NO. 26; (5) Having VH shown in SEQ ID NO. 22 and VL shown in SEQ ID NO. 27; (6) Having VH shown in SEQ ID NO. 23 and VL shown in SEQ ID NO. 28; (7) It has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% compared to any of the above sequence combinations (1) to (6). , VH and VL combinations with 98%, 99% or 100% sequence identity; (8) Having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid insertions or deletions compared to any of the above sequence combinations (1) to (7) and/or alternative VH and VL combinations; or (9) It has a CDR that is completely identical to any of the above sequence combinations (1) to (8), and a FR of at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, VH and VL combinations with 96%, 97%, 98%, 99% or 100% sequence identity; Alternatively, the amino acid insertion, deletion and/or substitution occurs in the heavy chain variable region or/and the FR region of the light chain variable region; Optionally, the substitution is a substitution of a conservative amino acid. The pharmaceutical composition according to any one of claims 1 to 4, wherein the anti-TNFR2 antibody or antigen-binding fragment thereof is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, natural antibodies, engineered antibodies, and monospecific antibodies. , multispecific antibody (e.g., bispecific antibody), monovalent antibody, multivalent antibody, full-length antibody, antibody fragment, Fab, Fab', F(ab')2, Fd, Fv, scFv, or diabody . The pharmaceutical composition according to any one of claims 1 to 5, wherein the concentration of the anti-TNFR2 antibody is 1 mg/mL to 100 mg/mL, preferably 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL. mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, and more preferably 30 mg/mL. The pharmaceutical composition according to any one of claims 1 to 6, wherein the buffer is selected from the group consisting of acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, and histidine-histamine hydrochloride. Acid buffer or disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably histidine acid-histidine hydrochloride buffer. The pharmaceutical composition according to claim 7, wherein the buffer concentration is 10-100mM, preferably 10-60mM, more preferably 20mM. The pharmaceutical composition according to any one of claims 1 to 8, wherein the protein stabilizer is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose or sorbitol, preferably sorbitol . The pharmaceutical composition according to claim 9, wherein the concentration of the protein stabilizer is 1-10% (w/v), preferably 2-8% (w/v), more preferably 4.7% (w/v) /v). The pharmaceutical composition according to any one of claims 1 to 10, wherein the surfactant is selected from polysorbate 80, poloxamer or glyceryl fatty acid ester, preferably polysorbate 80. The pharmaceutical composition according to claim 11, wherein the concentration of the surfactant is 0.02~0.1% (w/w), preferably 0.02% (w/w), 0.04% (w/w), 0.06% (w/w), 0.08% (w/w), 0.1% (w/w), more preferably 0.04% (w/w). The pharmaceutical composition according to any one of claims 1 to 12, wherein the pH value of the pharmaceutical composition is 4.5-7.5, preferably 4.5, 5, 5.5, 6, 6.5, 7, 7.5, more preferably is 5.5. The pharmaceutical composition according to any one of claims 1 to 13, which includes: a) anti-TNFR2 antibody at a concentration of 1 mg/mL to 100 mg/mL, b) Histidine-HCl histidine buffer with a concentration of 10-100mM and a pH value of 4.5-7.5, c) Sorbitol at a concentration of 1-10% (w/v), and d) Polysorbate 80 with a concentration of 0.02~0.1% (w/w); Preferably, the pharmaceutical composition contains: a) anti-TNFR2 antibody at a concentration of 10 mg/mL to 60 mg/mL, b) Histidine-HCl histidine buffer with a concentration of 10-60mM and a pH value of 5.0-6.0, c) Sorbitol at a concentration of 2-8% (w/v), and d) Polysorbate 80 with a concentration of 0.04% (w/w); More preferably, the pharmaceutical composition contains: a) Anti-TNFR2 antibody at a concentration of 30 mg/mL, b) Histidine-HCl histidine buffer with a concentration of 20mM and a pH value of 5.5, c) Sorbitol at a concentration of 4.7% (w/v), and d) Polysorbate 80 at a concentration of 0.04% (w/w). The pharmaceutical composition according to any one of claims 1 to 14, wherein the pharmaceutical composition is in the form of a liquid preparation. The method for preparing the pharmaceutical composition according to any one of claims 1 to 15, which includes the step of subjecting the anti-TNFR2 antibody solution to buffer replacement, The buffer is preferably histidine-histidine hydrochloride buffer, the concentration of the buffer is preferably 20mM, and the pH of the buffer is preferably 5.5. A pharmaceutical composition containing an anti-TNFR2 antibody or an antigen-binding fragment thereof, characterized in that the pharmaceutical composition is prepared by the method described in claim 16. The pharmaceutical composition according to any one of claims 1 to 15, the product prepared by the method of claim 16, or the pharmaceutical composition according to claim 17, in the preparation of drugs for the treatment and/or prevention of diseases related to immune abnormalities. use; Preferably, the immune abnormality-related disease is a disease related to Treg cell and/or MDSC function; Preferably, the disease is cancer or an autoimmune disease; More preferably, the cancer is selected from ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or response to CTLA-4 and Advanced solid tumors that are resistant to PD-1 therapy, such as metastatic melanoma; More preferably, the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis optica spectrum disease, systemic lupus erythematosus, myasthenia gravis, and IgG4-related diseases. A method for treating and/or preventing diseases related to immune abnormalities, characterized in that the method includes administering to a subject an effective amount of the pharmaceutical composition according to any one of claims 1 to 15, or the pharmaceutical composition prepared by the method of claim 16. The product, or the pharmaceutical composition described in claim 17; Preferably, the immune abnormality-related disease is a disease related to Treg cell and/or MDSC function; Preferably, the disease is cancer or an autoimmune disease; More preferably, the cancer is selected from the group consisting of ovarian cancer, advanced epidermal T-cell lymphoma, stage III/IV metastatic colorectal cancer, triple-negative breast cancer, pancreatic cancer, non-small cell lung cancer, and/or response to CTLA-4 and Advanced solid tumors that are resistant to PD-1 therapy, such as metastatic melanoma; More preferably, the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, multiple Sclerosis, systemic sclerosis, neuromyelitis optica spectrum disease, systemic lupus erythematosus, myasthenia gravis, IgG4-related diseases.

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