CN116688115A

CN116688115A - PD-L1/TGF-beta double-function fusion protein preparation and application thereof

Info

Publication number: CN116688115A
Application number: CN202310244201.5A
Authority: CN
Inventors: 梁羽; 颜晓丹; 刘吉; 杨利; 方言
Original assignee: Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
Current assignee: Shanghai Qilu Pharmaceutical Research and Development Centre Ltd
Priority date: 2022-03-18
Filing date: 2023-03-13
Publication date: 2023-09-05
Anticipated expiration: 2043-03-13
Also published as: CN116688115B

Abstract

The invention relates to a PD-L1/TGF-beta double-function fusion protein preparation and application thereof. The pharmaceutical formulation contains a PD-L1/TGF-beta bifunctional fusion protein, a stabilizer, a buffer and a surfactant. The PD-L1/TGF-beta double-function fusion protein pharmaceutical preparation is very stable, can still meet the pharmaceutical use requirements after long-time storage, strong light and high temperature, and has wide application prospect.

Description

PD-L1/TGF-beta double-function fusion protein preparation and application thereof

Technical Field

The invention belongs to the field of antibody and fusion protein preparations, and particularly relates to a PD-L1/TGF-beta dual-function fusion protein preparation and application thereof.

Technical Field

Programmed death receptor-1 (PD-1) is a transmembrane protein on T cells, and is mainly expressed on the surfaces of activated T lymphocytes, B lymphocytes and macrophages, and is a negative regulator of T cell proliferation. After being combined with the receptors PD-L1 and PD-L2, the polypeptide can inhibit the functions of T cells and induce apoptosis. The expression of PD-L1 of various tumor cells is up-regulated, and the tumor cells can interact with PD-1 on the surfaces of tumor-infiltrated lymphocytes, so that the killing of the lymphocytes on the tumors is inhibited, and the immune escape of the tumors is caused. The PD-1/PD-L1 signal path is blocked by using the anti-PD-1 and PD-L1 antibodies, so that the immune killing function of the T cells is partially recovered, and the anti-PD-1 and PD-L1 antibody has good clinical application value.

Transforming growth factor beta (Transforming Growth Factor-beta, TGF-beta) is a multifunctional cytokine, totaling 3 subtypes, namely TGF beta-1, TGF beta-2 and TGF beta-3. TGF- β plays an important role in cell proliferation, differentiation, apoptosis, epithelial-mesenchymal transition, and regulation of inflammatory responses through TGF- β/SMAD signaling pathways. In advanced cancers, a large number of tumor cells overexpress TGF- β, and high levels of TGF- β block immature T cell differentiation, resulting in immune escape of tumor cells, accelerating tumor progression. TGF-beta 1 also can directly prevent the differentiation of T cells and inhibit the function of T cells and NK cells to kill cancer cells.

Although anti-PD-1/PD-L1 antibody drugs have achieved significant effects in the treatment of many tumors, etc., their objective effective rate (ORR) ranges between 10% -35%. The reasons for lower ORR may be related to other tumor immune evasions beyond the PD-1/PD-L1 mediated immunosuppressive mechanisms, including intratumoral inhibitory microenvironments (tgfβ, IL-10, vegf, etc.). Therefore, the dual-function fusion protein medicine of the PD-L1 antibody and the TGF-beta receptor is developed, and simultaneously the PD-1/PD-L1 signal path and the TGF-beta signal path are blocked, so that the effect of killing tumor cells is cooperatively generated, and the response rate of immunotherapy is further improved.

Disclosure of Invention

The inventors first obtained a bifunctional fusion protein targeting PD-L1 and TGF-beta receptors. On the basis of obtaining the bifunctional fusion protein, a great deal of research and study is further carried out on the formulation prescription of the bifunctional fusion protein, and finally, a freeze-dried formulation which is most applicable to the bifunctional fusion protein and can stably store the bifunctional fusion protein is obtained, and the formulation can fully prevent aggregation, degradation, oxidation or denaturation and the like of the bifunctional fusion protein, so that the biological activity of the effective components of the bifunctional fusion protein is maintained, and the preparation is suitable for clinical use. Furthermore, on the basis of obtaining the double-function fusion protein preparation, the pharmaceutical function of the preparation is studied intensively, and the preparation is found to have good anti-tumor activity.

Detailed Description

1. Terminology

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Certain embodiments disclosed herein encompass a range of values, and certain aspects of the invention may be described by way of the range. Unless otherwise indicated, it should be understood that the numerical ranges or the manner in which the ranges are described are for the purpose of brevity and convenience only and should not be construed as a strict limitation on the scope of the invention. Accordingly, the description of a range format should be considered to specifically disclose all possible sub-ranges and all possible specific numerical points within the range as if such sub-ranges and numerical points had been explicitly written herein. The above principle applies equally regardless of the breadth of the values. When a range description is employed, the range includes the endpoints of the range.

The term "about" when referring to a measurable value such as an amount, temporal duration, or the like, is meant to include a change of + -20%, or in some cases + -10%, or in some cases + -5%, or in some cases + -1%, or in some cases + -0.1% of the specified value.

The amino acid three-letter codes and one-letter codes used herein are as described in J.biol. Chem,243, p3558 (1968).

The term "anti-human PD-L1" antibody refers to an antibody capable of recognizing, binding to a PD-L1 molecule from a human.

The term "antibody" as used herein typically refers to a Y-type tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have such a structure. Each light chain consists of one variable domain (VL) and one constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant region.

Five main classes of antibodies are known in the art: igA, igD, igE, igG and IgM, the corresponding heavy chain constant domains are referred to as α, δ, ε, γ and μ, respectively, igG and IgA can be further divided into different subclasses, e.g., igG can be divided into IgG1, igG2, igG3, igG4, igA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, termed kappa and lambda, based on the amino acid sequences of their constant domains.

In the case of IgG, igA and IgD antibodies, the constant region comprises three domains called CH1, CH2 and CH3 (IgM and IgE have the fourth domain CH 4). In the IgG, igA and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable length proline and cysteine rich segment. Each class of antibodies further comprises interchain and intrachain disulfide bonds formed by paired cysteine residues.

The term "variable region" or "variable domain" shows a significant change in amino acid composition from one antibody to another and is primarily responsible for antigen recognition and binding. The variable region corresponding to each light/heavy chain forms an antibody binding site such that the complete IgG antibody has two binding sites (i.e., it is bivalent). The variable region (VH) of the heavy chain and the variable region (VL) of the light chain each comprise three regions of extreme variability, known as hypervariable regions (HVRs), or more generally as Complementarity Determining Regions (CDRs), each of VH and VL having 4 framework regions FR, denoted FR1, FR2, FR3, FR4, respectively. Thus, CDR and FR sequences are typically found in the following sequences of the heavy chain variable domain (or light chain variable domain): FRl-HCDR 1 (LCDR 1) -FR2-HCDR2 (LCDR 2) -FR3-HCDR3 (LCDR 3) -FR4.

The term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which region comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions.

As used herein, the broad class of "antibodies" can include, for example, polyclonal antibodies (polyclonal antibodies), monoclonal antibodies (monoclonal antibodies), chimeric antibodies, humanized and primatized antibodies, CDR-grafted antibodies (CDR-grafted antibodies), human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

The term "monoclonal antibody" (or "mab") refers to an antibody that is produced by a single cell clone that is substantially homogeneous and directed against only a particular epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, transgenic animals, synthetic techniques, combinations thereof, or the like.

The division of the CDRs and FRs of the variable region of the monoclonal antibody of the present invention was determined according to the Kabat definition. While other naming and numbering systems, such as Chothia, IMGT or AHo, etc., are also known to those skilled in the art. Thus, humanized antibodies comprising any naming system derived CDR or CDRs, based on the monoclonal antibody sequences of the present invention, are clearly maintained within the scope of the present invention.

The term "antibody fragment" encompasses at least a portion of an intact antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of an antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment in an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or immunogenic determining portion thereof, or a fusion protein product further derived from such fragment, e.g., a single chain antibody, an extracellular binding region in a chimeric antigen receptor, and the like. Exemplary antibody fragments or antigen-binding fragments thereof include, but are not limited to: variable light chain fragments, variable heavy chain fragments, fab fragments, F (ab') ₂ Fragments, fd fragments, fv fragments, single domain antibodies, linear antibodies, single chain antibodies (scFv), bispecific or multispecific antibodies formed from antibody fragments, and the like.

The term "antigen" refers to a substance recognized and specifically bound by an antibody or antibody binding fragment, and in a broad sense, an antigen may include any immunogenic fragment or determinant of a selected target, including a single epitope, multiple epitopes, a single domain, multiple domains, an intact extracellular domain (ECD), or a protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, portions thereof and combinations thereof may all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, and the like. An "antigen" may also refer to a molecule that initiates an immune response. Any form of antigen or cell or preparation containing the antigen can be used to generate antibodies specific for an antigenic determinant. The antigen may be an isolated full-length protein, a cell surface protein (e.g., immunized with a cell expressing at least a portion of the antigen on its surface), or a soluble protein (e.g., immunized with only the ECD portion of the protein), or a protein construct (e.g., fc antigen). The antigen may be produced in a genetically modified cell. Any of the foregoing antigens may be used alone or in combination with one or more immunogenicity enhancing adjuvants known in the art. The DNA encoding the antigen may be genomic or non-genomic (e.g., cDNA) and may encode at least a portion of the ECD sufficient to elicit an immunogenic response. Any vector may be used to transform cells in which the antigen is expressed, including but not limited to adenoviral vectors, lentiviral vectors, plasmids, and non-viral vectors such as cationic lipids.

The term "affinity" or "binding affinity" refers to the strength of the sum of all non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The term "KD" refers to the dissociation constant of a particular antibody-antigen interaction. Binding affinity can be determined using various techniques known in the art, such as surface plasmon resonance, biolayer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, isothermal titration calorimetry, ELISA, analytical ultracentrifugation, flow cytometry, and the like.

The term "bifunctional fusion protein" refers to a fusion protein capable of binding two proteins of interest of different specificities. The bifunctional fusion proteins of the invention bind TGF-beta and PD-L1. The term "bifunctional fusion protein" may also include a prefix, e.g. "tgfβ/PD-L1" or "PD-L1/tgfβ", to indicate that it specifically binds to two target proteins. Each of the two binding domains of PD-L1 of the bifunctional fusion proteins described herein is a PD-L1 antigen-binding domain (or "antigen-binding site") of a parent anti-PD-L1 monoclonal antibody. Each of the two binding domains of tgfβ of the bifunctional fusion proteins described herein is a tgfβ binding domain derived from a tgfβ receptor 2 protein (tgfβr2), and is also referred to as a "trap" (or "tgfβ trap") distinguishing the binding domain as a ligand binding domain derived from a receptor, rather than an antigen binding domain derived from an antibody.

The term "biological activity" refers to the ability of an antibody to bind to an antigen and result in a measurable biological response that can be measured in vitro or in vivo.

The term "pharmaceutical formulation" or "formulation prescription" means such an article of manufacture: the form of its presence allows the biological activity of the active ingredient to be effective and does not contain other components toxic to the subject to which the formulation is to be administered.

The term "solution formulation" means a formulation that is liquid at a temperature of at least about 2 ℃ to about 8 ℃ at atmospheric pressure.

The term "lyophilizate" refers to a lyophilizate prepared by lyophilization techniques well known in the art. The lyophilizate should be reconstituted using an aqueous reconstitution composition prior to administration to a patient. This step allows the antibodies and other components in the lyophilizate to be redissolved to give a solution suitable for injection into a patient. The volume of aqueous material used for reconstitution determines the concentration of antibody in the resulting pharmaceutical composition. Reconstitution with a reconstituted aqueous material of less volume than before lyophilization provides a more concentrated composition than before lyophilization. The reconstitution coefficient (volume of formulation after lyophilization: volume of formulation before lyophilization) can be from 1:0.5 to 1:6. the lyophilizates of the invention can be reconstituted to give aqueous compositions having anti-PD-L1 and TGF-beta bispecific fusion protein concentrations of at least 50mg/mL, 100mg/mL, 150mg/mL, and the volumes of the reconstituted components selected accordingly. The reconstituted formulation may be diluted, if desired, prior to administration to a patient, to deliver the desired dose as appropriate.

Typical reconstitution ingredients for lyophilized formulations include sterile water or buffers, optionally with a preservative. If the lyophilizate includes a buffer, the reconstituted ingredients may further include a buffer (which may be the same or a different lyophilizate), or it may not include a buffer (e.g., water for injection, saline, or dextrose injection).

The term "aggregated" antibody or fusion protein refers to an antibody or fusion protein that has been found to aggregate with other antibody or fusion protein molecules, particularly after freezing and/or agitation.

The term "stable" formulation is a formulation in which the protein substantially retains its physical and/or chemical stability and/or biological activity after storage. Stored at refrigeration temperature (2-8 ℃) for 3 months, preferably 6, more preferably 1 year, even more preferably up to 2 years. Further, it shows stability after preservation at 25℃for a period of time including 1 month, 3 months, 6 months or 30 days at 40 ℃.

Typical acceptable criteria for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of the active ingredient is degraded as measured by SEC-HPLC. By visual analysis, the pharmaceutical formulation was a pale yellow, nearly colorless clear liquid or a colorless or clear to slightly milky, or pale yellow, nearly colorless clear liquid. The concentration, pH of the formulation has a variation of no more than + -10%. Typically no more than about 10% truncation, preferably no more than 5% truncation is observed. Usually no more than 10% of aggregates are formed, preferably no more than 5% of aggregates.

The term "buffer" or "buffer" means a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature. Preferred pharmaceutically acceptable buffers include, but are not limited to: histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer or mixtures thereof and the like. The buffer may be pH adjusted with acids or bases known in the art to a value in the range of 4.0-6.0, especially to a value in the range of 4.0-5.5, most especially to a pH of 4.5.

The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active pharmaceutical ingredient and/or formulation from chemical and/or physical degradation during manufacture, storage and use. Stabilizers include, but are not limited to, sugars, amino acids, polyols, cyclodextrins, and the like.

The term "surfactant" means a pharmaceutically acceptable excipient used to protect a protein formulation against physical stress (e.g., agitation and shear). Pharmaceutically acceptable surfactants include: polyoxyethylene sorbitan fatty acid esters (tween), polyoxyethylene alkyl ethers (e.g. under the trade mark Brij ^TM Those sold below) and polyoxyethylene-polyoxypropylene copolymers (poloxamers, pluronic). The polyoxyethylene sorbitan-fatty acid ester comprises polysorbate 20 (under the trade name tween 20 ^TM Lower sales) and polysorbate 80 (inTween 80 trademark ^TM Lower sales).

The term "effective amount" refers to the dose of a pharmaceutical formulation of an antibody or fragment of the invention that produces a desired effect in a treated patient after administration to the patient in a single or multiple dose. The effective amount can be readily determined by the attending physician as a person skilled in the art by considering a number of factors: such as race differences; body weight, age and health; specific diseases involved; severity of disease; response of individual patients; specific antibodies administered; mode of administration: the bioavailability characteristics of the administration formulation; a selected dosing regimen; and the use of any concomitant therapy.

The term "kit" includes an effective amount of one or more unit dosage forms of the pharmaceutical formulation or combination of the invention. In some embodiments, the kit may contain a sterile container of the therapeutic or prophylactic composition; such containers may be in the form of boxes, ampoules, bottles, vials, tubes, bags, blister packs or other suitable containers known in the art. Such containers may be made of plastic, glass, laminated paper, metal foil or other materials suitable for holding medicaments. In addition, the kit may include instructions for administering the pharmaceutical formulation or combination of the invention to an individual. The instructions generally comprise methods of treating or preventing a disease using the pharmaceutical formulations or combination of the invention.

The engineered antibodies or antigen-binding fragments thereof of the invention can be prepared and purified by conventional methods. For example, cDNA sequences encoding the heavy and light chains can be cloned and recombined into expression vectors. Recombinant immunoglobulin expression vectors can stably transfect CHO cells. As a more recommended prior art, mammalian expression systems can lead to glycosylation of the antibody, particularly at the highly conserved N-terminus of the Fc region. Stable clones were obtained by expressing antibodies that specifically bound to human antigens. Positive clones were expanded in serum-free medium of the bioreactor to produce antibodies. The antibody-secreting culture may be purified and collected using conventional techniques. The antibodies can be concentrated by filtration using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves, ion exchange.

The term "individual" or "subject" as used herein refers to any animal, such as a mammal or a pouched animal. Individuals of the invention include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus or other types of macaque), mice, pigs, horses, donkeys, cattle, sheep, rats, and any variety of poultry.

The term "tumor" as used herein refers to a disease characterized by pathological proliferation of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is generally uncontrolled and progressive, not inducing or inhibiting normal cell proliferation. Tumors may affect a variety of cells, tissues or organs including, but not limited to, organs selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tube, gall bladder, heart, intestine, kidney, liver, lung, lymph node, nervous tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vagina, or tissues or corresponding cells. Tumors include cancers, such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells). Tumors according to the present invention may include, but are not limited to, leukemias (e.g., acute leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphomas (hodgkin's disease, non-hodgkin's disease), primary macroglobulinemia, heavy chain diseases, solid tumors such as sarcomas and cancers (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endothelial sarcoma, lymphatic sarcoma, vascular sarcoma, lymphatic endothelial sarcoma, mesothelioma), ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, carcinoma, bronchus cancer, medullary carcinoma, renal cell carcinoma, liver cancer, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, wilms' cell tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngeal tube tumor, ependymoma, pineal tumor, angioblastoma, auditory neuroma, oligodendroglioma, neuroblastoma, meningioma, melanoma, neuroblastoma, retinoblastoma), esophageal cancer, gallbladder cancer, renal cancer, multiple myeloma. Preferably, the "tumor" includes but is not limited to: pancreatic cancer, liver cancer, lung cancer, stomach cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gall bladder cancer, renal cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.

The term "disease" or "condition" or "disorder" or the like as used herein refers to any change or disorder that impairs or interferes with the normal function of a cell, tissue or organ. For example, the "disease" includes but is not limited to: tumors, pathogen infection, autoimmune diseases, T cell dysfunctional diseases, or defects in the ability to tolerate immunity (e.g., transplant rejection).

The term "treatment" as used herein refers to a clinical intervention in an attempt to alter the course of a disease caused by an individual or a treated cell, either prophylactically or during a clinical pathology. Therapeutic effects include, but are not limited to, preventing occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the rate of progression of a disease, improving or alleviating a condition, alleviating or improving prognosis, and the like.

2. Summary of the invention

The present invention aims to provide a stable formulation of bifunctional fusion proteins suitable for binding of a specific anti-human PD-L1 antibody to the TGF-beta receptor and uses thereof.

The invention provides a pharmaceutical preparation of PD-L1/TGF-beta double-function fusion protein, which comprises the PD-L1/TGF-beta double-function fusion protein and buffer solution, wherein the PD-L1/TGF-beta double-function fusion protein is formed by connecting an IgG1 type anti-PD-L1 antibody with an N-terminal truncated form of an extracellular region of TGF-beta RII through each C-terminal end of an Fc segment of the anti-PD-L1 antibody by a flexible linker, and the light chain sequence of the PD-L1 antibody is shown as SEQ ID NO:1, the sequence of the whole heavy chain and the N-terminal truncated form of the extracellular region of TGF-beta RII is shown as SEQ ID NO: 2.

Preferably, the buffer is selected from acetate, succinate, tartrate and/or citrate buffers. Acetate and/or citrate are preferred.

Preferably, the acetate buffer is acetic acid-sodium acetate buffer; the citric acid buffer solution is citric acid-sodium citrate buffer solution.

Preferably, the concentration of the buffer is 5mM to 30mM, preferably 10mM to 25mM, most preferably about 25mM.

Preferably, the concentration of the bifunctional fusion protein is from 30mg/mL to 100mg/mL, preferably from 30mg/mL to 70mg/mL, and most preferably about 50mg/mL.

Preferably, the preparation further comprises a stabilizer, wherein the stabilizer comprises one or more of saccharides, polyalcohols, sodium chloride, arginine hydrochloride, glycine and methionine; preferred stabilizers are saccharides and/or polyols, most preferred are saccharides.

Preferably, the saccharide is selected from sucrose and trehalose; the polyalcohol is selected from mannitol, sorbitol and glycerol; the stabilizer is preferably sucrose and/or trehalose, most preferably sucrose.

Preferably, the sugar concentration is 50mg/mL to 100mg/mL, preferably 60mg/mL to 90mg/mL, most preferably about 70mg/mL.

Preferably, the formulation further comprises a surfactant, preferably the surfactant is polysorbate, more preferably the surfactant is polysorbate 20.

Preferably, the surfactant concentration is from 0.2mg/mL to 0.7mg/mL, preferably from 0.3mg/mL to 0.6mg/mL, more preferably about 0.5mg/mL.

Preferably, the formulation has a pH of 4.0 to 7.0, preferably a pH of 4.0 to 5.5, more preferably a pH of 4.2 to 4.8.

Preferably, the preparation comprises any two or three of sucrose, citric acid-sodium citrate and polysorbate 20.

The invention also provides a pharmaceutical preparation of the PD-L1/TGF-beta bifunctional fusion protein, which comprises 30-100mg/ml bifunctional fusion protein, 50-100mg/ml sucrose, 0.2-0.7mg/ml polysorbate 20,5-30mmol/L citric acid-sodium citrate buffer solution and pH value of 4-7.

The invention also provides a freeze-dried preparation containing the PD-L1/TGF-beta double-function fusion protein, which is obtained by freeze-drying the pharmaceutical preparation.

The invention also provides a freeze-dried preparation containing the PD-L1/TGF-beta double-function fusion protein, and the freeze-dried preparation can form the pharmaceutical preparation after being re-dissolved.

The invention also provides a product comprising a container containing a pharmaceutical formulation according to the invention, or a lyophilized formulation according to the invention.

The invention also provides a preparation for treating or inhibiting a disease or a disorder related to tumor cell proliferation or tumor cell metastasis, wherein the disease or disorder is preferably tumor or cancer selected from lung cancer, gastric cancer, melanoma, renal cancer, breast cancer, intestinal cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophagus cancer, pancreatic cancer and head and neck tumor.

The pharmaceutical preparation of the bifunctional fusion protein combined by the anti-human PD-L1 antibody and the TGF-beta receptor has the following beneficial effects: the preparation is very stable, can meet the pharmaceutical use requirement after long-time storage, strong light and high temperature, and has wide application prospect.

Drawings

Fig. 1: schematic diagram of the structure of the bifunctional fusion protein.

Fig. 2: isoelectric point of the bifunctional fusion protein is shown schematically.

Fig. 3: physical stability of bifunctional fusion proteins at different pH conditions.

Fig. 4: chemical stability of bifunctional fusion proteins at different pH conditions.

Fig. 5: the pH of the bifunctional fusion protein was prescribed for different protectants.

Fig. 6: stability in formulations of different protectants for bifunctional fusion proteins.

Fig. 7: stability of bifunctional fusion proteins in different sucrose protectant formulations.

Fig. 8: stability of bifunctional fusion proteins in different buffer salts.

Fig. 9: the bifunctional fusion proteins are pH in different buffer salts.

Fig. 10: SEC stability under conditions of bifunctional fusion protein liquid formulations.

Fig. 11: non-reducing CE-SDS stability under conditions of liquid formulations of bifunctional fusion proteins.

Fig. 12: iCIEF stability under conditions of liquid formulations of bifunctional fusion proteins.

Fig. 13: SEC stability of the bifunctional fusion protein lyophilized formulation over long periods of time and under accelerated conditions.

Fig. 14: non-reducing CE-SDS stability of the bifunctional fusion protein lyophilized formulation over long periods of time and under accelerated conditions.

Fig. 15: the lyophilized preparation of the bifunctional fusion protein has iCIEF stability under long-term and accelerated conditions.

Fig. 16: stability of the bifunctional fusion protein lyophilized formulation at high temperature.

Fig. 17: stability of the bifunctional fusion protein lyophilized formulation under light conditions.

Detailed Description

The invention is further illustrated by the following examples. Changes in the concentration of formulation components or the addition of other substances on the basis of the present invention, without having a significant effect on the stability of the bifunctional fusion protein, are still considered to be part of the present invention.

Charge heterogeneity (iCIEF)

The charge heterogeneity and isoelectric point of the sample are detected by adopting a full-column imaging capillary isoelectric focusing electrophoresis method. The capillary is 100 mu m inner diameter FC coating fused quartz capillary, and the effective separation length is 5cm; the final concentration of the sample is 2.0 percent of GE pharmic acid 3-10, 2.0 percent of GE pharmic acid 5-8, 0.35 percent of hydroxypropyl methylcellulose, 4mol/L urea and 2mg/ml of sample are respectively added during sample treatment; the focus separation voltage and time are 1.5kV-1min,3kV-9min. The pI value of the target peak was calculated as the marker pI value.

Size Exclusion Chromatography (SEC)

Size exclusion chromatography was used to quantify polymers, monomers and fragments. This assay utilizes a TOSOH TSK gel G3000SWXL (7.8X105 mm,5 μm) column and is run on a Waters e2695-2489 HPLC or Arc HPLC system. The mobile phase was 50mM phosphate, 300mM sodium chloride buffer, pH6.8. The sample loading was 50. Mu.g of protein, the protein was isocratically eluted at a flow rate of 0.5mL/min for 30min, and the absorbance of the eluate was measured at 280 nm. Integration was performed using the Empower 3 software.

Capillary electrophoresis (CE-SDS)

The main peak and (lc+hc) purity were determined by non-reducing CE-SDS (nrCE) and reducing CE-SDS (rCE), respectively, and this determination was performed on a BECKMAN COULTER PA800 plus capillary electrophoresis system with 50 μm i.d. non-coated quartz capillaries with an effective separation length of 20cm (full length 30.2 cm), PDA220nm bandwidth of 10 nm.

The invention is illustrated by the following specific examples, which are to be understood as merely illustrative of the invention and not limiting the scope thereof.

Example 1 structural and sequence information of PD-L1/TGF-beta bifunctional fusion proteins

The PD-L1/TGF beta fusion protein is formed by connecting an anti-PD-L1 monoclonal antibody of an IgG1 type with TGF-beta RII extracellular through a flexible linker through the C-terminal of an Fc segment. The structure of which is shown in figure 1.

Wherein, the light chain sequence of the PD-L1 antibody is shown as SEQ ID NO:1, the sequence of the heavy chain and the TGF-beta RII receptor is shown as SEQ ID NO: 2.

EXAMPLE 2 preparation of PD-L1/TGF-beta bifunctional fusion protein

The PD-L1/TGF-beta bifunctional fusion protein is a bifunctional fusion protein expressed by CHO cells. The cell fermentation harvest liquid is purified after the treatment mode of disc centrifugation, deep filtration and sterilization filtration. The purification step comprises removing protein aggregate, fragments and impurities such as HCP, DNA and ProA through affinity chromatography, low pH virus inactivation, cationic chromatography and UF/DF, and finally obtaining the fusion protein stock solution.

Example 3 optimum pH Range screening

Since the isoelectric point of the fusion protein ranges from about 5.7 to about 6.9, see FIG. 2. Taking a protein stock solution, dividing the solution into two parts, respectively adjusting the two parts to different pH values by using 1MHCl and 1MNaOH, and detecting the turbidity of samples with different pH values according to a Chinese pharmacopoeia 0902 clarity inspection method and a second turbidity method turbidity meter method. The test results are shown in FIG. 3: the protein solution is easy to have extremely turbid isoelectric precipitation phenomenon in the isoelectric range. Thus, a solution with good clarity (limited by about 24NTU as a turbidity standard solution No. 3) is desired, and the pH range is required to be equal to or less than pH4.5.

From the above experiments, a batch of samples having pH4.0 to pH8.5 were prepared separately, and the chemical stability was examined to analyze the SEC and non-reducing CE-SDS purities. The test results are shown in FIG. 4: in solutions of different pH values, the protein showed a tendency to increase significantly with increasing pH value, the optimal pH value being pH4.5 in SEC results, too low pH aggregates also showed a slight increase. In the non-reducing CE-SDS results, the fragment content of each pH sample was not significantly different, and it can be seen that the impurity ratio was slightly higher in the low pH (3.93) and high pH (7.91-8.51) value solutions, while there was no significant difference between the other samples. Thus, combining the results of the two tests, a preliminary determination of pH4.5 was made as the basis for subsequent prescription development.

EXAMPLE 4 screening of protectant species and amounts

After pH screening, screening of protective agent species was first performed, prescription design: based on 50mg/ml of bifunctional fusion protein and 800.5mg/ml of polysorbate, 25mM acetic acid-sodium acetate, pH4.5 was used as a buffer system, and various protection was adopted for investigation, and the formulation recipe is shown in Table 1 below:

table 1 formulation prescription of different protective Agents for bifunctional fusion proteins

The above prescription pH test results show that each sample was shifted to a different degree of pH (shift degree greater than 0.1) as shown in FIG. 5, which may be caused by insufficient buffering capacity of acetate buffer salt under the formulation conditions. The high temperature stability of 40 ℃ is examined on the prescription samples, and the result shows that the high temperature of 40 ℃ is examined for 28 days, and as shown in figure 6, the aggregate content of each prescription sample is in an ascending trend under the same dosage condition of the protective agent. Among the mannitol and glycine protectant samples, the aggregate was the most variable and consistent with the trend of the no-added protection control sample, indicating that mannitol and glycine did not act to stabilize the protein monomer. The aggregate content of the sucrose, trehalose and sorbitol protectant samples is the least variable, and the aggregate content is the lowest, wherein the sucrose protectant sample is the most preferred protectant. Further examination of sucrose protectant usage was performed, and the results are shown in fig. 7, in which SEC purity tended to increase monotonically as sucrose usage increased. The isotonic range of the injection is 285-315 mOsm/kg, the osmotic pressure of the solution with a blank prescription without protein is shown in table 3, and the osmotic pressure is slightly increased after the bifunctional fusion protein is added, so that the highest sucrose concentration is selected by referring to the isotonic condition, and the 7% sucrose concentration is determined to be the optimal dosage.

Table 2 formulation prescription for investigation of different protective agent concentrations of bifunctional fusion proteins

Table 3 dual function fusion prescription solution osmolarity assay

Buffer solution	Sucrose concentration	Osmotic pressure (mOsm/kg)
			25mM citrate, pH4.5	50mg/ml	218
25mM citrate, pH4.5	60mg/ml	252
			25mM citrate, pH4.5	70mg/ml	282
25mM citrate, pH4.5	80mg/ml	314

EXAMPLE 5 buffer salt species screening

Buffer salt species screening was performed based on the determination of the optimum pH conditions. The most suitable pH condition is pH4.5, and the buffer salt is selected from acetic acid-sodium acetate, succinic acid-sodium succinate, tartaric acid-NaOH and citric acid-sodium citrate. The study recipe is shown in Table 4 below, with sucrose as the stabilizer and polysorbate 80 at 0.5mg/mL as the surfactant, and protein concentration at 50mg/mL.

Table 4 formulation recipe for bifunctional fusion of different buffers

The above prescription samples were examined at 40℃and the results are shown in FIG. 8, in which the stability of aggregates was better in a citric acid and acetic acid buffer system, which was the best. Since the pH shift was large in the above screening, pH detection was further performed on samples with different buffer salt concentrations, and as shown in fig. 9, different levels of pH shift were present between samples with different buffer salt systems at pH4.5, wherein the pH shift was large (0.24-0.31) for the acetic acid, succinic acid, and tartaric acid buffer systems, indicating that the buffering capacity was poor under the formulation conditions. The pH deviation of the citric acid slow system sample can be controlled within 0.1, which proves that the citric acid slow system sample has better buffering capacity under the preparation condition. And combining the experimental results to determine the citric acid buffer system as a preferred buffer system.

Example 6 surfactant type and amount screening

And (3) screening the types and the dosage of the surfactant on the basis of determining the optimal pH condition, the type of the buffer salt and the type of the protective agent. The types of polysorbate 80 and polysorbate 20 surfactants were examined on the basis of 25mM citric acid-sodium citrate, pH4.5 and 50mg/ml sucrose, the concentration ranges were examined to be 0.1-0.5 mg/ml, and the prescription design is shown in Table 5 below.

Table 5 formulation recipe for bifunctional fusion of different buffers

The samples were analyzed for pH, SEC, non-reducing CE-SDS, etc., and the experimental results are shown in Table 6 below:

TABLE 6 results of tests on different surfactant types and amounts of bifunctional fusion proteins

The above results indicate that there is no significant difference in SEC and non-reducing CE-SDS purity for each formulation among the different surfactants. However, in the MFI detection insoluble particle measurement result, insoluble particles (particles of 10 μm or more) of the polysorbate 20 recipe as a whole showed a smaller number of insoluble particles than the polysorbate 80 recipe. No significant differences were seen between the individual amounts of polysorbate 20. However, considering that macromolecular biological agents are easier to form unstable particles in the processes of stock solution preparation, purification, storage and the like, the dosage of the active agent with higher concentration is selected to ensure the stability of the bifunctional fusion protein.

EXAMPLE 7 stability

According to the experimental result, the optimal preparation composition is determined to be 50mg/ml of bifunctional fusion protein, 70mg/ml of sucrose, 0.5mg/ml of polysorbate 20, 25mmol/L of citric acid-sodium citrate buffer solution, and the pH value is 4.5. Stability studies were performed on the most preferred formulations described above. Comprising the following steps: forced condition test (high temperature test, strong light irradiation test), acceleration test, long term test. The sample placement mode in the stability study is positive; the liquid and the freeze-dried samples are inspected respectively for a long time, at an accelerated speed and at a high temperature, the liquid samples are inspected by freeze thawing, the freeze-dried samples are inspected by illumination, and the illumination test samples are removed from the outer package and the bottle label. The various conditions examined are set forth in Table 7 below:

table 7 conditions for examining stability of bifunctional fusion proteins

The lyophilization cycles used are described in table 8 below:

table 8 dual function fusion lyophilization cycle parameters

Stage(s)	Shelf temperature	Temperature change speed (DEG C/min)	Maintenance time (min)	Vacuum degree (mbar)
					Prefreezing	-45℃	1	120	/
Primary drying	-15℃	1	1800-3000	0.133
					Secondary drying	30℃	0.2	120-240	0.133
Plug adding	30℃	/	/	690-790

Stability studies show that the SEC, non-reduced CE-SDS and iCIEF purities are stable in the liquid state for 18 months under long-term (-40 ℃) and accelerating conditions (2-8 ℃), and the SEC, non-reduced CE-SDS and iCIEF purities are in a decreasing trend under high-temperature conditions (25 ℃), wherein the non-reduced CE-SDS and iCIEF are obviously decreased, as shown in FIGS. 10-12. The stability under the low temperature condition is better under the liquid state. The purities of SEC, non-reducing CE-SDS and iCIEF were stable in the lyophilized state for long term (2-8deg.C), accelerated (25deg.C), high temperature (40deg.C) and light conditions, wherein the product was stable for 18 months under long term conditions, see FIGS. 13-17. The above results prove that the bifunctional fusion protein is relatively stable under the proposed conditions (2-8 ℃). For better stability, the lyophilized preparation should be used as the main form.

EXAMPLE 8 evaluation of Activity of PD-L1/TGF-beta bifunctional fusion protein

Based on the above results, the bifunctional fusion protein of the optimal formulation in example 7 was evaluated for binding activity. The binding capacity of the product to TGF-beta and PD-L1 proteins was determined by ELISA. Human TGF-beta 1 protein is coated on a 96-well plate, then the gradient diluted product is added, and then biotin-labeled PD-L1 protein with fixed concentration is added. One end of the product is combined with TGF-beta 1 protein, the other end is combined with biotin-marked PD-L1 protein, and the horseradish peroxidase-marked streptavidin is used for color development, so that the combination ability of the product with TGF-beta 1 and PD-L1 proteins is detected. The test data were analyzed by four-parameter fitting using SoftMax Pro, plotted and calculated to obtain EC50 values, the results are shown in table 9. The result shows that the PD-L1/TGF beta double-function fusion protein has good binding activity with TGF-beta and PD-L1 proteins.

TABLE 9 PD-L1/TGF-beta bifunctional fusion protein binding Activity results

Sample name	EC50 value (ng/mL)
		PD-L1/TGF beta double-function fusion protein	0.0303

Claims

1. A pharmaceutical formulation of a PD-L1/TGF- β bifunctional fusion protein comprising a PD-L1/TGF- β bifunctional fusion protein and a buffer, said PD-L1/TGF- β bifunctional fusion protein being formed by an anti-PD-L1 antibody of IgG1 type linked by each C-terminus of its Fc segment to an N-terminal truncated form of the extracellular region of TGF- βrii by a flexible linker, wherein the light chain sequence of the PD-L1 antibody is as set forth in SEQ ID NO:1, the sequence of the whole heavy chain and the N-terminal truncated form of the extracellular region of TGF-beta RII is shown as SEQ ID NO: 2.

2. The pharmaceutical formulation of claim 1, wherein: the buffer is selected from acetate, succinate, tartrate and/or citrate buffers, preferably acetate and/or citrate buffers. .

3. The pharmaceutical formulation of claim 2, wherein: wherein the acetate buffer is acetic acid-sodium acetate buffer; the citric acid buffer solution is citric acid-sodium citrate buffer solution.

4. A formulation according to any one of claims 1 to 3, wherein the buffer is at a concentration of 5mM to 30mM, preferably 10mM to 25mM, most preferably about 25mM.

5. The formulation of any one of claims 1-4, wherein the concentration of the bifunctional fusion protein is between 30mg/mL and 100mg/mL, preferably between 30mg/mL and 70mg/mL, most preferably about 50mg/mL.

6. The formulation of any one of claims 1-5, further comprising a stabilizer, wherein the stabilizer comprises one or more of a saccharide, a polyol, sodium chloride, arginine hydrochloride, glycine, methionine; preferred stabilizers are saccharides and/or polyols, most preferred are saccharides.

7. The pharmaceutical formulation of claim 6, wherein the saccharide is selected from the group consisting of sucrose and trehalose; the polyalcohol is selected from mannitol, sorbitol and glycerol; the stabilizer is preferably sucrose and/or trehalose, most preferably sucrose.

8. The pharmaceutical formulation of any one of claims 6-7, wherein the concentration of the sugar is 50mg/mL to 100mg/mL, preferably 60mg/mL to 90mg/mL, most preferably about 70mg/mL.

9. A pharmaceutical formulation according to any one of claims 1 to 8, wherein the formulation further comprises a surfactant, preferably the surfactant is polysorbate, more preferably the surfactant is polysorbate 20.

10. The pharmaceutical formulation of claim 9, wherein the surfactant concentration is 0.2mg/mL to 0.7mg/mL, preferably 0.3mg/mL to 0.6mg/mL, more preferably about 0.5mg/mL.

11. Pharmaceutical formulation according to any one of claims 1-10, wherein the pH of the formulation is from 4.0 to 7.0, preferably from 4.0 to 5.5, more preferably from 4.2 to 4.8.

12. The pharmaceutical formulation according to any one of claims 1 to 11, wherein the formulation comprises any two or three of sucrose, citric acid-sodium citrate, polysorbate 20.

13. A pharmaceutical formulation according to any one of claims 1 to 12, wherein: it contains 30-100mg/ml bifunctional fusion protein, 50-100mg/ml sucrose, 0.2-0.7mg/ml polysorbate 20,5-30mmol/L citric acid-sodium citrate buffer solution, and pH value is 4-7.

14. A lyophilized formulation comprising a PD-L1/TGF- β bifunctional fusion protein, said lyophilized formulation being obtained by lyophilizing the pharmaceutical formulation of any one of claims 1-13.

15. A lyophilized formulation comprising a PD-L1/TGF- β bifunctional fusion protein, which upon reconstitution forms a pharmaceutical formulation according to any one of claims 1-13.

16. A product comprising a container containing the pharmaceutical formulation of any one of claims 1-13, or the lyophilized formulation of any one of claims 14-15.

17. The formulation according to any one of claims 1 to 15 for use in the preparation of a medicament for the treatment or inhibition of a disease or condition associated with tumor cell proliferation or tumor cell metastasis, preferably a tumor or cancer selected from lung cancer, gastric cancer, melanoma, renal cancer, breast cancer, intestinal cancer, liver cancer, ovarian cancer, cervical cancer, bladder cancer, esophageal cancer, pancreatic cancer and head and neck tumor.