CN118369117A

CN118369117A - Anti-TNFR 2 antibody pharmaceutical composition

Info

Publication number: CN118369117A
Application number: CN202280080852.9A
Authority: CN
Inventors: 李鑫鑫; 马飞; 胡建中; 赵晓峰; 唐任宏; 任晋生
Original assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Current assignee: Shandong Simcere Bio Pharmaceutical Co ltd
Priority date: 2021-12-28
Filing date: 2022-12-27
Publication date: 2024-07-19
Also published as: WO2023125483A1; TW202333788A

Abstract

A TNFR2 antibody pharmaceutical composition is provided, comprising a TNFR2 antibody, or antigen-binding fragment thereof, a buffer, a protein stabilizer, and a surfactant. The pharmaceutical composition can maintain the stability of the TNFR2 antibody during long-term storage and transportation of the product, thereby ensuring the stability, safety and effectiveness of the medicine.

Description

Anti-TNFR 2 antibody pharmaceutical composition

The present application claims priority from the chinese patent office, application number 202111624712.7, entitled "an anti-TNFR 2 antibody pharmaceutical composition" filed on day 28, 12, 2021, the entire contents of which are incorporated herein by reference.

Technical Field

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

Background

Formulations of macromolecular protein drugs (e.g., antibodies, etc.) are typically injectable formulations, suitable for parenteral administration, and typically include subcutaneous, intravenous, intramuscular, and the like. Because the protein is relatively unstable in the solution, particles, aggregation and the like are very easy to form, and the stability becomes a difficult problem for developing macromolecular protein medicines. Therefore, the development of liquid formulations of macromolecular protein drugs (such as antibodies) is first required to solve the problem of protein stability.

Disclosure of Invention

In view of this, the present invention provides an anti-TNFR 2 antibody pharmaceutical composition.

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

In a first aspect, the present disclosure provides a pharmaceutical composition comprising an anti-TNFR 2 antibody, or antigen-binding fragment thereof, a buffer, a protein stabilizer, and a surfactant.

In one embodiment, the anti-TNFR 2 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 comprises:

a. selected from SEQ ID NOs: 19-23,

B. Selected from the group consisting of SEQ ID NOs: 19-23, or a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences,

C. Selected from the group consisting of SEQ ID NOs: 19-23 VH sequences having 1,2,3,4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or substitutions compared to any of the sequences;

(2) The light chain variable region comprises:

a. Selected from SEQ ID NOs: 24-28,

B. Selected from the group consisting of SEQ ID NOs: 24-28, or a VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences,

C. Selected from the group consisting of SEQ ID NOs: 24-28 VL sequences having 1,2,3,4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or substitutions compared to any of the sequences;

alternatively, the amino acid insertions, deletions and/or substitutions occur in the FR region of the heavy chain variable region or/and the light chain variable region;

Alternatively, the substitution is a conservative amino acid substitution.

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

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

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

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

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

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

(6) Has a VH shown as SEQ ID NO.23 and a VL shown as SEQ ID NO. 28;

(7) A VH and VL combination having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequence combinations (1) to (6) above;

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

(9) A VH and VL combination having a CDR that is substantially identical to that of any one of (1) to (8) above, and an FR that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical to the sequence combination;

Alternatively, the substitution is a conservative amino acid substitution.

In a specific embodiment, the anti-TNFR 2 antibody or antigen-binding fragment thereof is selected from a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, fab ', F (ab') 2, fd, fv, scFv, or a diabody (diabody).

In another embodiment, the concentration of the anti-TNFR 2 antibody is from 1mg/mL to 100mg/mL, preferably 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, more preferably 30mg/mL.

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

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

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

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

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

In a specific embodiment, the concentration of the surfactant is 0.02 to 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).

In another embodiment, the pharmaceutical composition has a pH of 4.5 to 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 comprising:

a) anti-TNFR 2 antibody at a concentration of 1mg/mL to 100mg/mL, b) histidine-hydrochloride buffer at a concentration of 10-100mM, pH 4.5-7.5, c) sorbitol at a concentration of 1-10% (w/v), and d) polysorbate 80 at a concentration of 0.02-0.1% (w/w);

Preferably, the pharmaceutical composition comprises:

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

more preferably, the pharmaceutical composition comprises:

a) anti-TNFR 2 antibody at a concentration of 30mg/mL, b) histidine-hydrochloride buffer at a concentration of 20mM, 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 formulation.

In a third aspect, the present disclosure provides a method of preparing a pharmaceutical composition according to the first or second aspects, comprising the step of subjecting an anti-TNFR 2 antibody solution to buffer displacement, preferably a histidine-histidine hydrochloride buffer, preferably at a concentration of 20mM, and preferably at a pH of 5.5.

In a fourth aspect, the present disclosure provides a pharmaceutical composition of an anti-TNFR 2 antibody, or antigen-binding fragment thereof, prepared by the method of the third aspect.

In a fifth aspect, the present disclosure provides a pharmaceutical composition according to the first to second aspects, a product prepared by the method according to the third aspect, or a use of the pharmaceutical composition according to the fourth aspect in the preparation of a medicament for the treatment and/or prevention of an immune abnormality related disease; preferably, the immune abnormality related disorder is a Treg cell and/or MDSC function related disorder; preferably, the disease is cancer or an autoimmune disease; more preferably, the cancer is selected from ovarian cancer, advanced epidermoid T cell lymphoma, metastatic colorectal cancer stage III/IV, 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 therapies, such as metastatic melanoma; more preferably, the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis spectrum diseases, systemic lupus erythematosus, myasthenia gravis, igG 4-related diseases.

In a sixth aspect, the present disclosure provides a method of treating and/or preventing an immune abnormality related disorder, the method comprising administering to a subject an effective amount of a pharmaceutical composition according to the first to second aspects, a product prepared by a method according to the third aspect, or a pharmaceutical composition according to the fourth aspect; preferably, the immune abnormality related disorder is a Treg cell and/or MDSC function related disorder; preferably, the disease is cancer or an autoimmune disease; more preferably, the cancer is selected from ovarian cancer, advanced epidermoid T cell lymphoma, metastatic colorectal cancer stage III/IV, 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 therapies, such as metastatic melanoma; more preferably, the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis spectrum diseases, systemic lupus erythematosus, myasthenia gravis, igG 4-related diseases.

The beneficial effects are that: the TNFR2 antibody preparation can maintain the stability of an anti-TNFR 2 antibody in the long-term storage and transportation process of products, thereby ensuring the stability, safety and effectiveness of medicines.

Drawings

FIG. 1 shows a trend graph of a summary of CEX-HPLC results examined at 2-8deg.C and 25deg.C for sample placement in prescription validation stability;

FIG. 2 shows a trend graph of a summary of SE-HPLC results for sample placement at 2-8deg.C and 25deg.C for prescription validation stability;

FIG. 3 shows a trend graph summarizing the results of investigation of CE-SDS (NR) at 2-8deg.C and 25deg.C for sample placement in prescription validation stability;

FIG. 4 shows a trend graph of a summary of results of investigation of CE-SDS (R) at 2-8deg.C and 25deg.C for sample placement in order to confirm stability.

Detailed Description

The invention discloses an anti-TNFR 2 antibody pharmaceutical composition, and a person skilled in the art can properly improve the technological parameters by referring to the content of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the 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 invention is further illustrated by the following examples:

term interpretation:

Unless otherwise indicated, terms used herein have meanings commonly understood by one of ordinary skill in the art. For a term explicitly defined herein, the meaning of that term controls the definition.

As used herein, the term "TNFR2" refers to tumor necrosis factor receptor 2, also known as tumor necrosis factor receptor superfamily member 1B (TNFRSF 1B) or CD120B, which is a membrane receptor that binds tumor necrosis factor-alpha (tnfα). The TNFR2 is preferably human TNFR2.

As used herein, the terms "anti-tumor necrosis factor receptor 2 antibody", "anti-TNFR 2 antibody", "anti-TNFR 2 antibody portion", and/or "anti-TNFR 2 antibody fragment", etc., refer to any protein or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule capable of specifically binding TNFR2 (e.g., 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 or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof). TNFR2 antibodies also include antibody-like protein scaffolds (e.g., tenth fibronectin type III domain (10 Fn 3)) that 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 that of the IgG heavy chain variable region, and by replacing residues of the BC, DE and FG loops of 10Fn3 with residues of the CDR-H1, CDR-H2 or CDR-H3 regions 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 with an antigen of interest, including polyclonal, monoclonal, genetically engineered, and other modified forms of the antibody (including, but not limited to, chimeric antibodies, humanized antibodies, fully human antibodies, heteroconjugate antibodies (e.g., bispecific, trispecific, and tetraspecific antibodies, diabodies, triabodies, and tetrabodies), antibody conjugates, and antigen-binding fragments of antibodies (including, e.g., fab ', F (Ab') 2, fab, fv, rIgG, and scFv fragments). Furthermore, unless otherwise indicated, 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 (cleared more rapidly from the animal circulation) and thus lack Fc-mediated effector function (effector function) (see Wahl et al, J.Nucl. Med.24:316,1983; the contents of which are incorporated herein by reference).

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 may be performed by a fragment of a full-length antibody. The antibody fragment may be a Fab, F (ab') 2, scFv, SMIP, diabody, triabody, affibody (affibody), nanobody, aptamer, or domain antibody. Examples of binding fragments that encompass the term "antigen-binding fragment" of an antibody include, but are not limited to: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHl domains; (ii) A F (ab) 2 fragment, a bivalent fragment comprising two Fab fragments linked at a hinge region by a disulfide bond; (iii) an Fd fragment consisting of VH and CHl domains; (iv) Fv fragments consisting of the VL and VH domains of the antibody single arm; (V) a dAb comprising VH and VL domains; (vi) dAb fragments consisting of VH domains (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) a combination of two or more isolated CDRs, which may optionally be connected by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, these two domains can be joined, using recombinant methods, by a linker that enables them to be made into a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as 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 whole antibodies. Antigen binding fragments may 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 a hypervariable region found in both the light and heavy chain variable domains. The more conserved portions of the variable domains are called the Framework Regions (FR). As 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 may be considered heterozygous hypervariable positions, as these positions may be considered to be within the hypervariable region under one set of criteria (e.g. IMGT or KABAT) and outside the hypervariable region under a different set of criteria (e.g. KABAT or IMGT). One or more of these locations may also be found in the extended hypervariable region. The invention includes antibodies comprising modifications in these heterozygous hypervariable positions. The variable domains of the natural heavy and light chains each comprise four framework regions, principally in a lamellar configuration, which are linked by three CDRs (CDR 1, CDR2 and CDR 3) that form loops connecting the lamellar structure and in some cases form part of the lamellar structure. The CDRs in each chain are held closely together by the FR regions in sequence FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and with CDRs from other antibody chains contribute to the formation of the antigen binding site of the antibody (see Kabat et al Sequences of Protein sofImmunological Interest, national Institute of Health, bethesda, md.1987; incorporated herein by reference). For example, herein, CDR1-VH, CDR2-VH and CDR3-VH refer to the first CDR, the second CDR and the third CDR, respectively, of a heavy chain variable region (VH), which three CDRs constitute the CDR combination (VHCDR combination) of the heavy chain (or variable region thereof); CDR1-VL, CDR2-VL and CDR3-VL refer to the first CDR, second CDR and third CDR, respectively, of the light chain variable region (VL) and these three CDRs constitute the CDR combinations (VLCDR combinations) of the light chain (or variable regions thereof).

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 Fv, scFv, or Fab). The term "VL" refers to the variable region of an immunoglobulin light chain (including the light chain of Fv, scFv, dsFv or Fab).

The term "heavy chain constant region" herein refers to the carboxy-terminal portion of an antibody heavy chain that does not directly participate in binding of the antibody to an antigen, but exhibits effector functions, such as interactions with Fc receptors, that have more conserved amino acid sequences relative to the variable domains of the antibody. The "heavy chain constant region" comprises at least: a CH1 domain, a hinge region, a CH2 domain, a CH3 domain, or a variant or fragment thereof. "heavy chain constant regions" include "full length heavy chain constant regions" having a structure substantially similar to that of a natural antibody constant region and "heavy chain constant region fragments" including only a portion of the "full length heavy chain constant region. Illustratively, a typical "full length antibody heavy chain constant region" consists of a CH1 domain-hinge region-CH 2 domain-CH 3 domain; when the antibody is IgE, it further comprises a CH4 domain; when an antibody is a heavy chain antibody, then it does not include a CH1 domain. Exemplary, a typical "heavy chain constant region fragment" may be selected from a CH1, fc, or CH3 domain.

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

As used herein, the terms "percent (%) sequence identity" and "percent (%) sequence identity" are interchangeable, and refer to the percentage of amino acid (or nucleotide) residues of a candidate sequence that are identical to amino acid (or nucleotide) residues of a reference sequence after aligning the sequences and introducing gaps (if desired) for maximum percent sequence identity (e.g., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for alignment purposes). For the purpose of determining percent sequence identity, the alignment may be accomplished in a variety of ways well known to those skilled in the art, for example using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAIi) software. One skilled in the art can determine appropriate parameters for measuring the alignment, including any algorithm that requires maximum alignment over the full length of the sequences being compared. For example, a reference sequence for comparison to a candidate sequence may show that the candidate sequence exhibits from 50% to 100% sequence identity over the entire length of the candidate sequence or over selected portions of consecutive amino acid (or nucleotide) residues of the candidate sequence. 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 belonging to the same class or having similar characteristics (e.g., charge, side chain size, hydrophobicity, hydrophilicity, backbone conformation, and rigidity). Illustratively, the amino acids within each of the following groups belong to conserved amino acid residues with each other, and the substitutions of amino acid residues within a group belong to conservative amino acid substitutions:

(1) Acidic amino acid: asp (D) and Glu (E);

(2) Basic amino acid: 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) Nonpolar uncharged amino acids: cys (C), met (M), and Pro (P);

(6) Aromatic amino acid: 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 that are specifically recognized, for example, by antibodies or antigen binding fragments thereof. 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 100nM (e.g., between 1pM 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 of greater than 100nM (e.g., greater than 500nM, 1 μm, 100 μm, 500 μm, or 1 mM) for the particular antigen or epitope thereof. Antibodies that specifically immunoreact with a particular protein or carbohydrate may be selected using a variety of immunoassay formats. For example, solid phase ELISA immunoassays are routinely used to select antibodies that specifically immunoreact with a protein or carbohydrate. See, harlow & Lane, antibodies, ALaboratory Manual, cold Spring Harbor Press, newYork (1988), harlow & Lane, using Antibodies, A Laboratory Manual, cold Spring Harbor Press, newYork (1999), which describe immunoassay formats and conditions that can be used to determine specific immunoreactivity.

As used herein, "pharmaceutical composition" means a mixture containing one or more of the compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and 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 the administration to organisms, and facilitate the absorption of the active ingredients so as to exert biological activity. As used herein, the terms "pharmaceutical composition" and "formulation" are not intended to be mutually exclusive.

The invention is further illustrated by the following examples:

EXAMPLE 1 humanized and expression purification of anti-TNFR 2 monoclonal antibodies

1.1 Humanization of anti-TNFR 2 monoclonal antibodies

The antibody is humanized by "CDRs grafting" method, i.e., the humanized antibody with highest homology is selected based on the sequence to provide antibody Framework Regions (FRs), and the antigen binding fragment Complementarity Determining Regions (CDRs) of the target antibody based on Kabat naming method are grafted to the humanized antibody variable region sequence of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in the former order. Wherein, antigen binding fragment Complementarity Determining Regions (CDRs) of the TNFR2 antibody Kabat naming method are shown 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-TNFR 2 antibody.

TABLE 1 KABAT analysis of candidate antibody CDRs

TABLE 2 VH and VL sequences of humanized anti-TNFR 2 antibodies

TABLE 3 pairing of VH and VL sequences of humanized anti-TNFR 2 antibodies

Antibody numbering	VH	VL
#1-1	VH1(SEQ ID NO.19)	VL1(SEQ ID NO.24)
#1-2	VH1(SEQ ID NO.19)	VL2(SEQ ID NO.25)
#2-1	VH2(SEQ ID NO.20)	VL3(SEQ ID NO.26)
#2-2	VH3(SEQ ID NO.21)	VL3(SEQ ID NO.26)
#3-1	VH4(SEQ ID NO.22)	VL4(SEQ ID NO.27)
#3-2	VH5(SEQ ID NO.23)	VL5(SEQ ID NO.28)

1.2 Expression of humanized anti-TNFR 2 antibodies

The humanized anti-TNFR 2 antibody is expressed by using CHO as a host cell, and is cultured in DYNAMIS AGT culture medium (manufacturer: gibco, product number: A26615-01) for no more than 15 days, wherein the reactor control parameter is pH 6.8-7.6, dissolved Oxygen (DO) is 20% -80%, the rotating speed is 79-81 RPM, and the initial culture temperature is 36.0-37.0 ℃; when culturing to day 8, the culture temperature was lowered to 32.0.+ -. 0.5 ℃ until harvest.

1.3 Purification of humanized anti-TNFR 2 antibodies

Purification of humanized anti-TNFR 2 antibodies were purified sequentially using multi-step chromatography, concentration and filtration unit procedures. The supernatant was captured by Protein A affinity chromatography (Mabselect PrismA). The captured antibody solution is treated with a low pH incubation to inactivate potential viruses. After neutralization of the antibody solution, the precipitate was removed by depth filtration. Then anion exchange chromatography (Capto Q) is performed sequentially to remove HCD, HCP, shed Protein a, etc., and cation exchange chromatography (Capto S Impact) is performed to remove HCP and aggregates, etc. Filtering with nanofiltration membrane to remove potential endogenous and exogenous viruses. And concentrating the antibody solution by using an ultrafiltration membrane package, and replacing the solution by using a buffer solution, thereby completing purification and obtaining the humanized anti-TNFR 2 antibody protein stock solution.

Example 2 buffer System screening

2.1 Buffer System screening protocol

The experiment covers 4 buffer salts (acetate, citrate, histidine and phosphate) with pH values from 4.5 to 7.5, a total of 12 formulation buffer systems were set up for screening (see table 4). Carrying out ultrafiltration concentration liquid exchange treatment on the protein stock solution obtained in the example 1, wherein the stock solution is replaced into 12 corresponding preparation buffer systems: adding the protein stock solution into corresponding ultrafiltration tubes in 12 groups, carrying out ultrafiltration concentration by using a centrifuge, intercepting protein, enabling buffer solution of the protein stock solution to flow through an ultrafiltration membrane to achieve the purpose of protein concentration, then respectively adding corresponding 12 preparation buffer solutions, diluting the buffer solution of the protein stock solution, continuing ultrafiltration concentration, continuously adding corresponding 12 preparation buffer solutions after concentration is finished, continuing ultrafiltration concentration, and repeating the operation until the liquid exchange is finished. The humanized TNFR2 antibody #2-1 described in example 1 was used in each of the above formulations at a protein concentration of 30mg/mL. The stability of the protein in 12 buffer systems is comprehensively examined and compared by taking appearance, pH value, concentration, purity (size exclusion chromatography (SE-HPLC), non-reduced sodium dodecyl sulfate capillary electrophoresis (CE-SDS (NR)), charge isomerism (ion exchange chromatography (CEX-HPLC)), dynamic Light Scattering (DLS) and thermal stability (differential scanning fluorescence method (DSF)) as indexes through a high-temperature acceleration test at 40 ℃ so as to screen out the optimal buffer systems.

Table 4 formulation buffer system screening protocol

Remarks: x=appearance, pH, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS (NR), DLS, DSF (T0)

T0=survey start point; w = week; DSF (T0) means that the DSF parameter is only examined at T0.

2.2 Buffer System screening results

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

TABLE 5 screening of formulation buffer System results of accelerated test at 40℃

Remarks: na=inapplicable.

TABLE 6 screening of buffer System for formulations results of accelerated test at 40℃

Remarks: na=inapplicable.

2.3 Buffer System screening conclusions

In 12 buffer systems, the appearance is yellowish when being inspected for 4 weeks at 40 ℃, the pH and the concentration of the microemulsion liquid are within target values, and no obvious change exists;

Thermal stability assay, melting temperature (Tm) of protein: f4 prescriptions Tm1 at 68.46 ℃, significantly lower than other prescriptions, other prescriptions Tm1 all greater than 69 ℃, no significant difference;

DLS: particle size good and bad ordering (small to large ordering): f7, F8, F9> F3, F10, F11, F12> F1> F6, F2> F5, F4;

SE-HPLC: purity grade ordering 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;

Acid peaks are increased, and the good and bad orders F7, F1> F8, F2> F3, F4, F5, F6, F9> F10> F11> F12;

In summary, through the screening of the buffer system, the prescriptions are sorted as follows: f7> F8> F9, F3> F2> F1> F4, F5, F6, F10, F11, F12. The F7 prescription is comprehensively superior to other prescriptions in the aspects of purity, degradation, aggregation, charge heterosomes and the like in the accelerated experiment investigation at 40 ℃ and shows good stability. Therefore, the F7 prescription (20 mM histidine-histidine hydrochloride, pH 5.5) is initially selected as the optimal prescription and used as the buffer system of the product preparation for the next adjuvant screening study.

EXAMPLE 3 adjuvant screening

3.1 Adjuvant screening protocol

And (3) adding corresponding auxiliary materials including 7 prescriptions of sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose, mannitol and sorbitol into the optimal buffer system according to the screening result of the buffer system and the aim of developing the freeze-dried preparation, wherein the protein concentration is 30mg/mL. The stability of each prescription was examined by freeze thawing test (-20 ℃ to 25 ℃ for one round of freeze thawing, 3-5 rounds of continuous freeze thawing), 40 ℃ high temperature acceleration test, and 1 optimal formulation prescription was selected for the next round of surfactant screening by using appearance, pH, concentration, purity (size exclusion chromatography (SE-HPLC), non-reducing sodium dodecyl sulfate capillary electrophoresis size exclusion chromatography (CE-SDS (NR)), charge isomerism (ion exchange chromatography (CEX-HPLC)), dynamic Light Scattering (DLS), thermal stability (differential scanning fluorescence method (DSF)) as indicators.

Table 7 adjuvant screening protocol

Remarks: x=appearance, pH value, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS (NR), DLS, DSF (T0), osmotic pressure (T0)

T0=survey start point; w = week; DSF (T0) means that DSF parameters are only examined at T0; osmotic pressure (T0) means that the osmotic pressure parameter is only examined at T0.

3.2 Screening results of adjuvant

The main results of the adjuvant screening are summarized in tables 8-11 below.

Table 8 results of accelerated test at 40 ℃ for adjuvant screening

Remarks: na=inapplicable.

Table 9 results of accelerated test at 40 ℃ for adjuvant screening

Remarks: na=inapplicable.

TABLE 10 results of freeze-thaw test at-20℃to 25℃for adjuvant screening

Remarks: na=inapplicable.

Table 11 findings screening results of freeze-thaw test at-20 ℃ to 25 ℃

Remarks: na=inapplicable.

3.3 Findings of adjuvant screening

The concentration of the sample is 30mg/mL, 5 times of freeze thawing experiments at-20 ℃ to 25 ℃ and auxiliary material screening at 40 ℃ for 4 weeks are respectively carried out.

The appearance is yellowish, the pH and the concentration of the microemulsion are within the target values, and no obvious change exists;

Thermal stability test (DSF), melting temperature of proteins (Tm 1 and Tm 2): f7-4, F7-5> F7-2, F7-6, F7-7, no auxiliary material (F7) > F7-1, F7-3;

DLS: when the temperature is examined for 4 weeks at 40 ℃, the particle size of F7-1 is obviously increased relative to T0; the grain diameters of F7-2 and F7-6 are obviously increased relative to T0 after freeze thawing for 5 times;

SE-HPLC: the purity of F7-2, F7-3, F7-4, F7-5, F7-6, F7-7> F7-1 is examined for 4 weeks at 40 ℃; freeze thawing for 5 times, and the purities of F7-1, F7-3, F7-4, F7-5, F7-7> F7-2> F7-6;

CE-SDS (NR): the main peak purities of CE (NR) F7-7, F7-5, F7-6, F7-4, F7-2> F7-1, F7-3 were examined at 40℃for 4 weeks; no obvious change is caused after 5 times of freezing and thawing;

CEX-HPLC: the main peak purities F7-3> F7-1, F7-6, F7-7> F7-2, F7-4 and F7-5 were examined at 40℃for 4 weeks; acid peaks are increased, and the good and bad sequences F7-1, F7-3> F7-6, F7-7> F7-4, F7-5 and F7-2 are ordered; there was no obvious difference between the prescriptions for 5 times of freeze thawing.

In summary, the auxiliary material screening is superior to other prescriptions in terms of purity, degradation, aggregation, charge heterosomes and the like comprehensively in 5 times of freeze thawing investigation at-20 to 25 ℃ in the 40 ℃ accelerating experiment by evaluating each stability index, and the F7-7 prescription shows good stability. Thus, the F7-7 formulation (4.7% sorbitol (W/V), 20mM histidine-HCl, pH 5.5) was initially selected as the optimal formulation for the next surfactant screening study.

Example 4 surfactant screening

4.1 Surfactant screening protocol

And adding surfactants with different concentrations into an optimal buffer system and auxiliary materials, wherein the surfactants comprise 6 prescriptions which do not contain polysorbate 80, 0.02%, 0.04%, 0.06%, 0.08% and 0.1% of polysorbate 80 (W/W), the protein concentration is 30mg/mL, and the stability of each prescription is inspected by using an appearance, a pH value, the concentration, the purity (size exclusion chromatography (SE-HPLC), a non-reducing sodium dodecyl sulfate capillary electrophoresis size exclusion chromatography (CE-SDS (NR)), a charge isomerism (ion exchange chromatography (CEX-HPLC)), dynamic Light Scattering (DLS) and insoluble particles as indexes, screening out 1 optimal preparation prescription to carry out a prescription confirmation stability experiment, and confirming the final prescription, wherein the specific prescription composition and the inspection scheme are shown in table 12.

Table 12 surfactant screening protocol

Remarks: x=appearance, pH, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS (NR), DLS, insoluble microparticles (micro flow imaging Method (MFI), T0 and endpoint of investigation)

T0=survey start point; w = week; d = day.

4.2 Surfactant screening results

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

TABLE 13 surfactant screening results of 40 ℃ accelerated test

Remarks: na=inapplicable.

Table 14 results of surfactant screening shake test

Remarks: na=inapplicable.

4.3 Surfactant screening conclusion

The sample concentration was 30mg/mL and examined at 40℃for 4 weeks. The appearance is colorless micro-emulsion liquid; the pH and the concentration are within the target values, and no obvious change exists;

SE-HPLC: the prescriptions are good and bad, F7-7-3, F7-7-5> F7-7-2, F7-7-6> F7-7-4> F7-7-1;

CE-SDS (NR): the prescriptions are good and bad, F7-7-6, F7-7-2> F7-7-4, F7-7-3> F7-7-5, F7-7-1;

DLS: the particle size of each prescription is good or bad, F7-7-1, F7-7-6> F7-7-5> F7-7-2> F7-7-4> F7-7-3;

insoluble particles: the advantages and disadvantages of each prescription are that the granule with the F7-7-1 prescription of more than or equal to 10 mu m is always obviously more than other prescriptions; no obvious difference exists in other prescriptions;

CEX-HPLC: the F7-7-3 recipe is slightly better than the other recipes.

Shake at 25℃and 300rpm for 7 days. SE-HPLC, DLS, CEX-HPLC: the prescriptions have no obvious difference; insoluble particles: the particles with the prescription of F7-7-1 more than or equal to 10 mu m are always obviously more than other prescriptions; no significant difference was found between CE-SDS (NR) for the other formulas: the main peak contents F7-7-3, F7-7-6> F7-7-4> F7-7-1> F7-7-2 and F7-7-5.

In summary, the adjuvant screening was performed by evaluating the respective stability index: f7-7-3, F7-7-6> F7-7-2, F7-7-4> F7-7-5> F7-7-1, i.e., the optimal prescription is F7-7-3 or F7-7-6.

However, the polysorbate 80 content of the recipe F7-7-2, F7-7-4 is at the upper and lower edges of the polysorbate 80 content of the recipe F7-7-3, the polysorbate 80 content of the recipe F7-7-5 is at the edges of the polysorbate 80 content of the recipe F7-7-6, and both of the recipes F7-7-2, F7-7-4 are superior to the recipe F7-7-5.

Therefore, from the viewpoint of the operational space of production, it is more appropriate to select the F7-7-3 prescription (0.04% polysorbate 80 (W/W), 20mM histidine-histidine hydrochloride, 4.7% sorbitol, pH 5.5). Thus, the F7-7-3 prescription (0.04% polysorbate 80 (W/W), 20mM histidine-histidine hydrochloride, 4.7% sorbitol (W/V), pH 5.5) was the optimal prescription for this screening, and the next prescription confirmation stability study was prepared and the final confirmation of this prescription was performed.

EXAMPLE 5 prescription confirmation stability

5.1 Prescription confirmation stability protocol

The prescription confirmation stability and package material selection research experiment determines 1 prescription based on the screening results of a buffer system, auxiliary materials and a surfactant: 30mg/mLTNFR of monoclonal antibody (# 2-1), 20mM histidine-histidine hydrochloride, 4.7% sorbitol (W/V), 0.04% polysorbate 80 (W/W), pH5.5,5 mL/bottle, split charging into penicillin bottles, and respectively placing and inverting the bottles in the normal direction and the reverse direction by using a rubber plug and an aluminum plastic cover to form a closed packaging system, and performing real-time stability and accelerated stability investigation for 6 months, and performing stability confirmation study on the final prescription.

The specific examination protocol is shown in Table 15 below.

Table 15 prescription confirmation stability regimen

Remarks: x=appearance, pH, protein concentration, CEX-HPLC, SE-HPLC, CE-SDS (NR), CE-SDS (R), osmotic pressure (T0), active, insoluble microparticles (microfluidic imaging method, MFI)

T0=survey start point; m = month; osmotic pressure (T0) means that the osmotic pressure parameter is only examined at T0.

In the scheme, the activity detection method adopts Elisa and CELL ASSAY blocking methods, and is specifically as follows:

Elisa method: half maximal effect concentration of anti-TNFR 2 monoclonal antibody on 50ng of TNFR2 was measured using the binding of anti-TNFR 2 monoclonal antibody to human TNFR 2. Antigen (human TNFR 2-his) is pre-coated on a 96-well plate (Sino Biological, 10417-H08H), an anti-TNFR 2 monoclonal antibody is combined with the antigen on an ELISA plate, a plate is washed to remove various non-combined components, and then Peroxidase-conjugated Mouse anti Human IgG Fc gamma (Jackson Immuno, 209-035-098) is added, and the plate is washed to remove redundant ELISA antibodies after incubation; finally, HRP substrate TMB (Thermo, 34029) was added to produce a blue product with a shade proportional to the anti-TNFR 2 monoclonal antibody concentration. After termination of the stop solution, the blue product turned yellow, and the OD value was read by an ELISA reader.

CELL ASSAY blocking method: the humanized anti-TNFR 2 monoclonal antibody is a monoclonal antibody which specifically binds to Human TNFR2 and can inhibit the binding of Human TNFα and receptor Human TNFR 2. The method adopts a monoclonal cell strain CT26-2A03 which is obtained by self-screening and over-expresses TNFR2 as a cell for detection. The humanized anti-TNFR 2 monoclonal antibody is added, then biotin-labeled TNFα (TNFα -biotin) (AcroBiosystems, TNA-H82E 3) is added, ligand TNFα interacts with receptor TNFR2, then PE-labeled strepitavidin (bioleged, 405204) is added for incubation to form CHO-TNF2-TNFα -biotin-strepitavidin-PE complex, at this time, the cells are indirectly labeled with PE dye, and a significant positive signal can be seen through detection by an enzyme-labeled instrument. If the binding site of the humanized anti-TNFR 2 monoclonal antibody and the Human TNFR2 collides with the binding site of Human TNFα, the binding of Human TNFα and Human TNF2 is affected, the strength of PE signal is finally affected, and the decrease of PE signal is detected on an enzyme-labeled instrument. Using this principle, the function of humanized anti-TNFR 2 monoclonal antibodies to inhibit HumanTNFR's binding to HumanTNF. Alpha. At the cellular level can be examined.

5.2 Prescription confirmation stability results

The main results of the stability of the prescription verification are summarized in the following tables 16 to 17, and figures 1 to 4.

Table 16 prescription validation stability results

Table 17 prescription validation stability results

5.3 Prescription confirmation stability study conclusion

The sample is placed for 6 months at a low temperature of 2-8 ℃, the normal and the inverted samples have no obvious changes in the aspects of conventional detection indexes, insoluble particles, purity, charge heterosomes, activity and the like, and the normal and the inverted samples have no obvious differences.

The accelerated stability at 25 ℃ is examined for 6 months, the normal and the inverted samples are in the standard quality range in terms of conventional detection indexes, insoluble particles, purity, charge heterosomes, activity and the like, and no obvious difference exists between the normal and the inverted samples.

The accelerated stability at 40 ℃ is examined for 3 months, and the CE-SDS (NR) and CEX-HPLC in the purity indexes of the normal and inverted samples exceed the quality standard, and other purity indexes, conventional detection indexes, insoluble particles, charge heterosomes, activities and the like are all in the quality standard range, and no obvious difference exists between the normal and inverted samples.

In conclusion, in the process of low-temperature (2-8 ℃) real-time stability investigation, the conventional detection index, insoluble particles, purity, charge heterosomes, activity and the like of the sample are not changed remarkably, and in the 25 ℃ acceleration experiment, all indexes are in the quality standard range, so that the protein in the formula has good stability, and the aim of the invention is achieved. In the accelerated stability investigation experiment at the high temperature of 40 ℃, the purity and the charge heterosomes have a descending trend, which is consistent with the quality index trend of the prescription in the prescription screening stage. Under the high temperature condition, some key indexes show a descending trend, which also indicates that the product is sensitive to high temperature, and low temperature (2-8 ℃) storage and transportation of the product are recommended.

The anti-TNFR 2 antibody pharmaceutical composition provided by the invention is described in detail above. The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to facilitate an understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications fall within the scope of the claims of the invention.

Sequence listing

Claims

A pharmaceutical composition comprising an anti-TNFR 2 antibody, or antigen-binding fragment thereof, a buffer, a protein stabilizer, and a surfactant.
The pharmaceutical composition of claim 1, wherein 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.
The pharmaceutical composition of claim 1 or 2, wherein the anti-TNFR 2 antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region and a light chain variable region:

(1) The heavy chain variable region comprises:

a. selected from SEQ ID NOs: 19-23,

B. Selected from the group consisting of SEQ ID NOs: 19-23, or a VH sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequences,

C. Selected from the group consisting of SEQ ID NOs: 19-23 VH sequences having 1,2,3,4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or substitutions compared to any of the sequences;

(2) The light chain variable region comprises:

a. Selected from SEQ ID NOs: 24-28,

B. Selected from the group consisting of SEQ ID NOs: 24-28, or a VL sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any of the sequences,

C. Selected from the group consisting of SEQ ID NOs: 24-28 VL sequences having 1,2,3,4, 5, 6, 7, 8, 9, 10 or more amino acid insertions, deletions and/or substitutions compared to any of the sequences;

alternatively, the amino acid insertions, deletions and/or substitutions occur in the FR region of the heavy chain variable region or/and the light chain variable region;

Alternatively, the substitution is a conservative amino acid substitution.
A pharmaceutical composition according to claim 3, wherein the heavy chain variable region and the light chain variable region are selected from the group consisting of:

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

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

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

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

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

(6) Has a VH shown as SEQ ID NO.23 and a VL shown as SEQ ID NO. 28;

(7) A VH and VL combination having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of the sequence combinations (1) to (6) above;

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

(9) A VH and VL combination having a CDR that is substantially identical to that of any one of (1) to (8) above, and an FR that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identical to the sequence combination;

alternatively, the amino acid insertions, deletions and/or substitutions occur in the FR region of the heavy chain variable region or/and the light chain variable region;

Alternatively, the substitution is a conservative amino acid substitution.
The pharmaceutical composition of any one of claims 1-4, wherein the anti-TNFR 2 antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a polyclonal antibody, a natural antibody, an engineered antibody, a monospecific antibody, a multispecific antibody (e.g., bispecific antibody), a monovalent antibody, a multivalent antibody, a full-length antibody, an antibody fragment, fab ', F (ab') 2, fd, fv, scFv, or a diabody (diabody).
The pharmaceutical composition according to any one of claims 1-5, wherein the concentration of the anti-TNFR 2 antibody is 1mg/mL to 100mg/mL, preferably 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, more preferably 30mg/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, histidine-histidine hydrochloride buffer and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, preferably histidine-histidine hydrochloride buffer.
The pharmaceutical composition according to claim 7, wherein the buffer concentration is 10-100mM, preferably 10-60mM, more preferably 20mM.
Pharmaceutical composition according to any one of claims 1 to 8, wherein the protein stabilizing agent is selected from sodium chloride, glycine, arginine hydrochloride, sucrose, trehalose or sorbitol, preferably sorbitol.
Pharmaceutical composition according to claim 9, wherein the concentration of the protein stabilizing agent is 1-10% (w/v), preferably 2-8% (w/v), more preferably 4.7% (w/v).
The pharmaceutical composition according to any one of claims 1 to 10, wherein the surfactant is selected from polysorbate 80, poloxamer or glycerol fatty acid esters, 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 of the pharmaceutical composition is 4.5 to 7.5, preferably 4.5, 5, 5.5, 6, 6.5, 7, 7.5, more preferably 5.5.
The pharmaceutical composition according to any one of claims 1-13, comprising:

a) An anti-TNFR 2 antibody at a concentration of 1mg/mL to 100mg/mL,

B) Histidine-histidine hydrochloride buffer at a concentration of 10-100mM, pH 4.5-7.5,

C) Sorbitol at a concentration of 1-10% (w/v), and

D) Polysorbate 80 at a concentration of 0.02-0.1% (w/w);

Preferably, the pharmaceutical composition comprises:

a) An anti-TNFR 2 antibody at a concentration of 10mg/mL to 60mg/mL,

B) Histidine-histidine hydrochloride buffer solution with the concentration of 10-60mM and the pH value of 5.0-6.0,

C) Sorbitol at a concentration of 2-8% (w/v), and

D) Polysorbate 80 at a concentration of 0.04% (w/w);

more preferably, the pharmaceutical composition comprises:

a) An anti-TNFR 2 antibody at a concentration of 30mg/mL,

B) Histidine-histidine hydrochloride buffer at a concentration of 20mM, 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).
The pharmaceutical composition according to any one of claims 1-14, wherein the pharmaceutical composition is in the form of a liquid formulation.
A method for preparing a pharmaceutical composition according to any one of claims 1 to 15, comprising the step of subjecting said anti-TNFR 2 antibody solution to a buffer, preferably histidine-hcl histidine buffer, preferably at a concentration of 20mM, preferably at a pH of 5.5.
A pharmaceutical composition comprising an anti-TNFR 2 antibody, or antigen-binding fragment thereof, wherein the pharmaceutical composition is prepared by the method of claim 16.
Use of a pharmaceutical composition according to any one of claims 1-15, a product prepared by a method according to claim 16, or a pharmaceutical composition according to claim 17 for the preparation of a medicament for the treatment and/or prevention of an immune abnormality related disease;

Preferably, the immune abnormality related disorder is a Treg cell and/or MDSC function related disorder;

preferably, the disease is cancer or an autoimmune disease;

More preferably, the cancer is selected from ovarian cancer, advanced epidermoid T cell lymphoma, metastatic colorectal cancer stage III/IV, 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 therapies, such as metastatic melanoma;

More preferably, the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis spectrum diseases, systemic lupus erythematosus, myasthenia gravis, igG 4-related diseases.
A method of treating and/or preventing an immune abnormality related disorder, comprising administering to a subject an effective amount of the pharmaceutical composition of any one of claims 1-15, the product made by the method of claim 16, or the pharmaceutical composition of claim 17;

Preferably, the immune abnormality related disorder is a Treg cell and/or MDSC function related disorder;

preferably, the disease is cancer or an autoimmune disease;

More preferably, the cancer is selected from ovarian cancer, advanced epidermoid T cell lymphoma, metastatic colorectal cancer stage III/IV, 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 therapies, such as metastatic melanoma;

More preferably, the autoimmune disease may be selected from rheumatoid arthritis, multiple sclerosis, systemic sclerosis, neuromyelitis spectrum diseases, systemic lupus erythematosus, myasthenia gravis, igG 4-related diseases.