Anti-PD-1 and mutant IL-2 immune complex preparations

Abstract

The present invention relates to a formulation of an immune complex ("αPD-1 / IL2m") comprising a recombinant anti-programmed cell death receptor 1 (PD-1) antibody and mutant interleukin 2 (IL2), particularly a stable liquid formulation, a method for preparing the formulation, and the use of the formulation in the treatment and / or prevention of disease.

Description

[Technical Field]

[0001] The present invention relates to the field of immune complex formulations. Specifically, the present invention relates to immune complex formulations (hereinafter also referred to as "αPD-1 / IL2m") comprising recombinant anti-programmed cell death receptor 1 (PD-1) antibody and mutant interleukin 2 (IL2), particularly stable liquid formulations, methods for preparing said formulations, and the use of said formulations in the treatment and / or prevention of disease. [Background technology]

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a multifunctional cytokine primarily produced by activated T cells, particularly CD4+ helper T cells. By binding to IL-2 receptors on various cells, IL-2 mediates diverse effects in the immune response. IL-2 has immune-stimulating effects and can stimulate the proliferation and differentiation of T cells and natural killer (NK) cells. Therefore, IL-2 is approved as an immunotherapy agent in the treatment of cancer and chronic viral infections. On the other hand, IL-2 promotes the maintenance of immunosuppressive CD4+CD25+ regulatory T cells (i.e., Treg cells) (Fontenot et al., Nature Immunol 6, 1142-51 (2005); D'Cruz and Klein, Nature Immunol 6, 1152-59 (2005); Maloy and Powrie, Nature Immunol 6, 1171-72 (2005)), and can also induce immunosuppression by activated Treg cells in patients.

[0003] Programmed cell death protein-1 (PD-1) is an important immune checkpoint protein, a 55 kDa type I transmembrane protein that is primarily induced on the surface of activated T cells, but is also expressed on B cells, NK cells, monocytes, and DC cells (DCs). Programmed cell death protein ligand 1 (PD-L1) and programmed cell death protein ligand 2 (PD-L2) have been identified as cell surface glycoprotein ligands for PD-1. PD-1 ligands are highly expressed in many cancer cells. When PD-1 binds to its ligand, it triggers T cell apoptosis, immune anergesia, T cell "exhaustion," and IL-10 secretion, thus inhibiting the PD1 pathway can restore T cell function in cancer patients (Sheridan, Nature Biotechnology 30(2012), 729-730). Monoclonal antibodies targeting PD-1 include nivolumab from Bristol-Myers Squibb (BMS) and pembrolizumab from Merck. Nivolumab (trade name OPDIVO®) is a fully humanized IgG4 antibody molecule, and pembrolizumab (trade name KEYTRUDA®) is a humanized IgG4 antibody molecule. Anti-PD-1 monoclonal antibodies bind to PD-1 on T lymphocytes, then inhibit the binding of PD-1 to its ligands, PD-L1 and PD-L2, thereby promoting T lymphocyte activation, proliferation, and the production of immune-activating cytokines, and can release the inhibition of PD-1 in immune surveillance of T lymphocytes with antitumor activity.

[0004] In light of the importance of IL-2 and PD-1 in regulating the immune response, the inventors have diligently conducted research and developed an anti-PD-1 / IL-2 mutant protein immune complex that targets PD-1 and IL-2 receptors (see Patent Application No. PCT / CN2022 / 120265; for reference, the full text of that application is incorporated into this application). Because the immune complex enhances antitumor effects and reduces side effects, it is an excellent candidate drug for the clinical treatment of tumors.

[0005] For clinical pharmaceuticals, drug stability is one of the most important indicators of efficacy and safety. Obtaining a formulation that provides good drug stability is a crucial condition for maintaining the safety and efficacy of the drug throughout its shelf life. Therefore, to meet the requirements for drug stability, those skilled in the art still need to develop formulations suitable for anti-PD-1 / IL-2 mutant protein immune complexes. [Overview of the project]

[0006] To address the aforementioned challenges, the inventors designed a formulation screening experiment for recombinant anti-PD-1 / IL-2 mutant protein immune complexes. High-temperature experiments were used to screen buffer pH, buffering system, and additives. Formulations were evaluated and confirmed by measuring indicators such as appearance, visible foreign matter, protein content, pH, purity, charge variants, polysorbate-80 content, and biological activity. Ultimately, research and development yielded the liquid formulation of the present invention. The formulation of the present invention not only guarantees product quality throughout its shelf life but can also be prepared by a stable and easily implementable sterile preparation process.

[0007] Accordingly, in a first embodiment, the present invention provides a liquid formulation containing an αPD-1 / IL2m immune complex. In some embodiments, the liquid formulation of the present invention is an injectable formulation, preferably an injectable solution for intravenous infusion.

[0008] In some embodiments, the liquid formulation of the present invention is (i) αPD-1 / IL2m immune complex, (ii) Buffering agent, (iii) Stabilizers, and (iv) containing a surfactant, Here, the pH of the liquid formulation is approximately 5.0 to approximately 6.5, for example, approximately 5.0 to approximately 5.5, and preferably approximately 5.0.

[0009] In some embodiments, the αPD-1 / IL2m immune complex according to the present invention comprises (a) an anti-PD-1 half-antibody and (b) a mutant IL2 fusion polypeptide. Here, the anti-PD-1 semiantibody includes a Fab fragment fused to the Fc region, and also includes three heavy chain CDR sequences contained in the heavy chain variable region sequence shown in SEQ ID NO: 8, and three light chain CDR sequences contained in the light chain variable region sequence shown in SEQ ID NO: 15, Here, the mutant IL2 fusion polypeptide comprises a mutant IL-2 polypeptide fused to the Fc region, and the mutant IL-2 polypeptide comprises (i) N88R + S130R, (ii) a B'C' loop sequence AGDASIH or AQSKNFH, and optionally (iii) T3A.

[0010] In some embodiments, the liquid formulation is the immune complex in a concentration of about 0.5 mg / ml to about 150 mg / ml, and preferably the concentration of the immune complex is about 0.5 mg / ml to about 5 mg / ml.

[0011] In some embodiments, the buffer is selected from histidine buffers, glutamate buffers, phosphate buffers, acetate buffers, citrate buffers, trishydroxymethylaminomethane (Tris) buffers, and combinations thereof. Preferably, the liquid formulation according to the present invention contains a histidine buffer. In some embodiments, the (total) concentration of the buffer in the liquid formulation according to the present invention is about 0.5 mM to about 200 mM, about 5 mM to about 50 mM, or about 5 mM to about 20 mM.

[0012] In some embodiments, the stabilizer is selected from sugars (such as sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, and dextran), amino acids (methionine, arginine, and their salts), or combinations thereof. Preferably, the stabilizer includes sucrose, methionine, or combinations thereof. In some embodiments, the (total) concentration of the stabilizer is about 1 mM to about 1000 mM, or about 50 mM to about 500 mM.

[0013] In some embodiments, the surfactant is selected from polysorbate surfactants such as polysorbate 80 and polysorbate 20, for example, preferably the surfactant is polysorbate 80. In some embodiments, the (total) concentration of the surfactant is about 0.01 mg / ml to about 10 mg / ml, about 0.05 mg / ml to about 5 mg / ml, about 0.1 mg / ml to about 5 mg / ml, or about 0.1 mg / ml to about 1 mg / ml.

[0014] In some embodiments, the liquid formulation of the present invention is (i) αPD-1 / IL2m immune complex, (ii) Histidine buffer, (iii) Sucrose and methionine, (iv) Polysorbate-80, and optional (v) EDTA, Here, the pH of the liquid formulation is approximately 5.0 to approximately 6.5, for example, approximately 5.0 to approximately 5.5, and preferably approximately 5.0.

[0015] In some embodiments, the liquid formulation of the present invention is (i) αPD-1 / IL2m immune complex, (ii) Histidine and histidine hydrochloride, (iii) Sucrose and methionine, (iv) Polysorbate-80, (v) EDTA, which is optional. Here, the pH of the liquid formulation is approximately 5.0 to approximately 6.5, for example, approximately 5.0 to approximately 5.5, and preferably approximately 5.0.

[0016] Preferably, the liquid formulation of the present invention is (i) αPD-1 / IL2m immune complex in a concentration of approximately 1 mg / ml to approximately 10 mg / ml (ii) Histidine buffer of approximately 10 mM to approximately 30 mM, (iii) sucrose at about 70 mg / ml to about 90 mg / ml, or a combination of sucrose at about 50 mg / ml to about 80 mg / ml and methionine at about 40 mM to about 60 mM, and (iv) containing about 0.2 mg / ml to about 0.4 mg / ml of polysorbate 80, where the pH of the liquid formulation is about 5.0 - 5.5, preferably about pH 5.0, alternatively, the liquid formulation (i) about 1.0 mg / ml of αPD - 1 / IL2m immune complex, (ii) about 10 mM of histidine buffer, (iii) about 80 mg / ml of sucrose, or a combination of about 70 mg / ml of sucrose and about 50 mM of methionine, and (iv) containing about 0.3 mg / ml of polysorbate 80, where the pH of the liquid formulation is about 5.0 - 5.5, preferably about pH 5.0. In any of the above embodiments, preferably, the histidine buffer is a buffer system containing histidine and its salts, particularly a histidine buffer system containing histidine and histidine hydrochloride.

[0017] In a more preferred embodiment, the liquid formulation of the present invention (i) about 1.0 mg / ml of αPD - 1 / IL2m immune complex, (ii) about 0.12 mg / ml of histidine and about 1.94 mg / ml of histidine hydrochloride, (iii) about 70 mg / ml of sucrose and about 7.49 mg / ml of methionine, and (iv) containing about 0.3 mg / ml of polysorbate 80, where the pH of the liquid formulation is about 5.0 - 5.5, preferably about pH 5.0.

[0018] In a second embodiment, the present invention provides a solid formulation obtained by solidifying a liquid formulation of the present invention. The solidification process is carried out, for example, by crystallization, spray drying, or freeze-drying. In a particular preferred embodiment, the solid formulation is, for example, in the form of a freeze-dried powder formulation for injection. The solid formulation can be reconstituted with a suitable solvent at the time of use to form a reconstituted formulation of the present invention. The reconstituted formulation is also one of the liquid formulations of the present invention. In a particular embodiment, the suitable solvent is selected from water for injection, organic solvents for injection (including, but not limited to, oils for injection, ethanol, propylene glycol, etc.), or a combination thereof.

[0019] In a third aspect, the present invention provides a pharmaceutical container containing a liquid formulation of the present invention. In some embodiments, the container is a formulation container for injection.

[0020] In a fourth aspect, the present invention provides a delivery device comprising the liquid or solid formulation of the present invention. In certain embodiments, the delivery device of the present invention is provided in the form of a pre-filled syringe comprising the liquid or solid formulation of the present invention for use with intravenous, subcutaneous, intradermal or intramuscular injection or intravenous infusion.

[0021] In a fifth aspect, the present invention provides a method for delivering αPD-1 / IL2m immune complexes to a subject, such as a mammal, comprising the procedure of administering a liquid or solid formulation of the present invention to the subject by a delivery device using a prefilled syringe.

[0022] In a sixth aspect, the present invention provides the use of a liquid or solid formulation of the present invention for the treatment or prevention of cancer in a subject, or for preparing a drug or delivery device (e.g., a pre-filled syringe) for the treatment or prevention of cancer in a subject. Preferably, the cancer is a solid tumor or hematological malignancy, e.g., a gastrointestinal tumor or melanoma, e.g., colorectal cancer or colon cancer, e.g., the cancer is PD-1 antibody-resistant cancer.

[0023] Other embodiments of the present invention will become apparent by referring to the following detailed description. [Brief explanation of the drawing]

[0024] The preferred embodiments of the present invention, described in detail below, will be better understood when read in conjunction with the drawings. For the purpose of illustrating the present invention, the drawings show preferred embodiments at present. However, it should be understood that the present invention is not limited to the exact arrangement and means of the embodiments shown in the drawings.

[0025] [Figure 1] Figure 1 shows the trend of changes in the formulation evaluation results. (A) is the trend of changes in protein content (UV method), (B) is the trend of changes in purity (SEC-HPLC method), (C) is the trend of changes in purity (nrCE-SDS method), (D) is the trend of changes in charge variant-acidic component (iCIEF method), (E) is the trend of changes in charge variant-major component (iCIEF method), and (F) is the trend of changes in charge variant-basic component (iCIEF method).

[0026] [Figure 2-1] Figure 2 shows the trend of changes in the formulation confirmation study results. (A) to (E) are the stability study results at 40°C, and (F) to (J) are the stability study results at 25°C. (A) and (F) are the trend of changes in purity (SEC-HPLC method), (B) and (G) are the trend of changes in purity (nrCE-SDS method), (C) and (H) are the trend of changes in charge variant-acidic component (iCIEF method), (D) and (I) are the trend of changes in charge variant-main component (iCIEF method), and (E) and (J) are the trend of changes in charge variant-basic component (iCIEF method). [Figure 2-2]Figure 2 shows the trend of changes in the formulation confirmation study results. (A) to (E) are the stability study results at 40°C, and (F) to (J) are the stability study results at 25°C. (A) and (F) are the trend of changes in purity (SEC-HPLC method), (B) and (G) are the trend of changes in purity (nrCE-SDS method), (C) and (H) are the trend of changes in charge variant-acidic component (iCIEF method), (D) and (I) are the trend of changes in charge variant-main component (iCIEF method), and (E) and (J) are the trend of changes in charge variant-basic component (iCIEF method).

[0027] [Figure 3A] Figure 3 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse MC38 tumors. (B) shows the survival curve reflecting the antitumor effect of 2149 against mouse MC38 tumors. (C) shows the effect of 2149 on mouse body weight. [Figure 3B] Figure 3 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse MC38 tumors. (B) shows the survival curve reflecting the antitumor effect of 2149 against mouse MC38 tumors. (C) shows the effect of 2149 on mouse body weight. [Figure 3C] Figure 3 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse MC38 tumors. (B) shows the survival curve reflecting the antitumor effect of 2149 against mouse MC38 tumors. (C) shows the effect of 2149 on mouse body weight.

[0028] [Figure 4A]Figure 4 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (B) shows the individual mouse tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (C) shows the survival curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (D) shows the effect of 2149 on mouse body weight. [Figure 4B] Figure 4 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (B) shows the individual mouse tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (C) shows the survival curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (D) shows the effect of 2149 on mouse body weight. [Figure 4C] Figure 4 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (B) shows the individual mouse tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (C) shows the survival curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (D) shows the effect of 2149 on mouse body weight. [Figure 4D] Figure 4 shows the in vivo efficacy of the immune complex 2149 protein. (A) shows the tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (B) shows the individual mouse tumor volume change curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (C) shows the survival curve reflecting the antitumor effect of 2149 against mouse B16F10 tumors. (D) shows the effect of 2149 on mouse body weight.

[0029] [Figure 5A]Figure 5 shows a comparison of the in vivo efficacy of the control molecule 2061 and the immune complex protein 2149. (A) shows the mouse individual tumor volume change curve reflecting the antitumor effects of 2061 and 2149 against mouse B16F10 tumors. (B) shows the survival curve reflecting the antitumor effects of 2061 and 2149 against mouse B16F10 tumors. (C) shows the effect of 2061 and 2149 on mouse body weight. [Figure 5B] Figure 5 shows a comparison of the in vivo efficacy of the control molecule 2061 and the immune complex protein 2149. (A) shows the mouse individual tumor volume change curve reflecting the antitumor effects of 2061 and 2149 against mouse B16F10 tumors. (B) shows the survival curve reflecting the antitumor effects of 2061 and 2149 against mouse B16F10 tumors. (C) shows the effect of 2061 and 2149 on mouse body weight. [Figure 5C] Figure 5 shows a comparison of the in vivo efficacy of the control molecule 2061 and the immune complex protein 2149. (A) shows the mouse individual tumor volume change curve reflecting the antitumor effects of 2061 and 2149 against mouse B16F10 tumors. (B) shows the survival curve reflecting the antitumor effects of 2061 and 2149 against mouse B16F10 tumors. (C) shows the effect of 2061 and 2149 on mouse body weight.

[0030] [Figure 6] Figure 6 shows a schematic diagram of the αPD-1 / IL2m immune complex of the present invention. [Figure 7] Figure 7 shows a schematic diagram of sequence alignment. Detailed description of the invention

[0031] Before describing the present invention in detail, it should be understood that the present invention is not limited to the specific methods and experimental conditions described herein, as these can be modified. Furthermore, the terms used herein are solely for the purpose of describing specific embodiments and are not intended to be limiting.

[0032] Unless otherwise defined, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art. For the purposes of the present invention, the following terms have been defined.

[0033] When used in conjunction with a number, the term "approximately" means to encompass a range of numbers that have a lower limit of 5% lower and an upper limit of 5% higher than the specified number.

[0034] When the term "and / or" is used in relation to two or more options, it is understood to mean either one of the options or two or more of the options.

[0035] In this specification, the terms “inclusion” or “contains” are intended to include the elements, integers, or procedures described, but not to exclude other elements, integers, or procedures. In this specification, when the terms “inclusion” or “contains” are used, unless otherwise specified, they also include combinations of the elements, integers, or procedures described. For example, when an antibody variable region “contains” a specific sequence is mentioned, it means including the antibody variable region consisting of that sequence.

[0036] In this specification, the term "antibody" refers to a polypeptide comprising at least a light chain and a heavy chain immunoglobulin variable region, wherein the immunoglobulin variable region specifically recognizes and binds to an antigen. The term includes, but is not limited to, various antibody structures such as monoclonal antibodies, chimeric antibodies or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the expected antigen-binding activity.

[0037] In this specification, "whole antibody" (used interchangeably with "full-length antibody," "complete antibody," and "intact antibody") refers to a protein molecule containing at least two heavy chains (H) and two light chains (L) linked together by disulfide bonds. Each heavy chain consists of a variable heavy chain region (abbreviated as VH in this specification) and a constant heavy chain region. The constant heavy chain region consists of three domains: CH1, CH2, and CH3. Thus, a single antibody heavy chain generally consists of a variable heavy chain region, and constant heavy chain domains CH1, CH2, and CH3, from the N-terminus to the C-terminus. In this specification, the C-terminal region of the heavy chain, located at the C-terminus of the CH1 domain and containing the CH2 and CH3 domains, is also called the Fc region or Fc fragment. Each light chain consists of a variable light chain region (abbreviated as VL in this specification) and a constant light chain region. The constant light chain region consists of one domain CL. Therefore, a single antibody light chain generally contains a variable light chain region and a constant light chain domain (CL) from the N-terminus to the C-terminus. The variable regions of the antibody's heavy chain and light chain pair up to form an antigen-binding structure domain responsible for binding to the antigen. The constant regions of the heavy and light chains do not directly participate in antigen binding, but they exhibit various effector functions.

[0038] In this specification, the term “anti-PD-1 semi-antibody” refers to a monovalent antigen-binding polypeptide containing one heavy chain and one light chain of a complete anti-PD-1 antibody. In such semi-antibodies, the VH-CH1 region of the heavy chain is generally paired with the VL-CL region of the light chain to form a Fab fragment that specifically binds to the PD-1 antigen, and is covalently bound to the Fc region of the heavy chain at the C-terminus of the CH1 domain via an immunoglobulin hinge region. Thus, an anti-PD-1 semi-antibody is a polypeptide containing a Fab fragment that fuses to the Fc region and specifically binds to PD-1.

[0039] In this specification, the term “mutant IL-2 fusion polypeptide” refers to a fusion polypeptide comprising a mutant IL-2 polypeptide, wherein the mutant IL-2 polypeptide is fused to an Fc region at its C-terminus. Typically, the fusion polypeptide comprises the mutant IL-2 polypeptide, an optional linker sequence, a hinge region, and an Fc region, from the N-terminus to the C-terminus.

[0040] In this specification, the term αPD-1 / IL2m immune complex refers to an immune complex comprising, substantially consisting of, (i) an anti-PD-1 semiantibody and (ii) a mutant IL-2 fusion polypeptide. An exemplary structure of an αPD-1 / IL2m immune complex is shown in Figure 6. As shown in Figure 6, in the αPD-1 / IL2m immune complex, the VH-CH1 region of the anti-PD-1 heavy chain pairs with the VL-CL region of the light chain to form a Fab fragment that specifically binds to PD-1, and the Fc region of the mutant IL-2 fusion polypeptide pairs with the Fc region of the anti-PD-1 heavy chain to dimerize. It should be understood that the antibody constant regions contained in the immune complex of the present invention can be assigned to any class or subclass based on their sequence and may be native sequence constant regions or mutant sequence constant regions.

[0041] In this specification, the class and subclass of an antibody constant region (e.g., the Fc region) are determined by the sequence of the constant region. Generally, antibody light chain constant regions can be classified into two types (called kappa (κ) and lambda (λ)) based on their amino acid sequence. Antibody heavy chain constant regions can be classified into five distinct classes based on their amino acid sequence: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA2. Therefore, when an immune complex is said to have an Fc region of the IgG class, it means that the immune complex has an Fc region that belongs to the IgG class based on its amino acid sequence. Similarly, when an immune complex is said to have a kappa light chain constant region, it means that the immune complex has a light chain constant region that belongs to the kappa class based on its amino acid sequence.

[0042] In this specification, the term “natural sequence Fc region” includes various immunoglobulin Fc region sequences, e.g., various Ig subclasses and their allotype Fc region sequences (Gestur Vidarsson et al., IgG subclasses and allotypes: from structure to effector functions, 20 October 2014, doi: 10.3389 / fimmu.2014.00520). In some embodiments, the human IgG heavy chain Fc region has an amino acid sequence extending from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. In some further embodiments, the human IgG heavy chain Fc region has a natural immunoglobulin hinge sequence or partial hinge sequence at the N-terminus, such as a sequence from E216 to T225 or a sequence from D221 to T225 according to EU numbering.

[0043] In this specification, the term “mutant sequence Fc region” refers to an Fc region polypeptide that includes modifications to the native sequence Fc region polypeptide. Such modifications may involve the addition, deletion, or substitution of amino acid residues. Substitutions may include native and non-native amino acids. The purpose of the modifications may be to alter the binding of the Fc region to its receptor and the effector function induced thereby.

[0044] In this specification, the terms “complementarity-determining region,” “CDR region,” “CDR,” or “hypervariable region” can be used interchangeably and refer to a region in the antibody variable domain whose sequence is hypervariable and which contains structurally determined loops ("hypervariable loops") and / or antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigen epitopes. In this specification, CDRs of the antibody heavy chain and light chain are generally referred to as CDR1, CDR2, and CDR3, and are numbered from the N-terminus. CDRs located in the antibody heavy chain variable domain are also called HCDR1, HCDR2, and HCDR3, and CDRs located in the antibody light chain variable domain are called LCDR1, LCDR2, and LCDR3. In the amino acid sequence of a given light chain variable region or heavy chain variable region, its CDR sequence can be determined using various methods known in the art. Various methods known in the art that can be used to determine the CDR sequence in a given VH or VL amino acid sequence include the Chothia definition (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), the Kabat definition (Kabat et al., Sequences of Proteins of Immunological Interest, 4th edition, US Department of Health and Human Services, National Institutes of Health (1987)), the AbM definition (University of Bath), the Contact definition (University College London), the IMGT definition (http: / / imgt.cines.fr), and the North CDR definition based on affinity propagation clustering using numerous crystal structures.Furthermore, the CDR can also be determined based on having the same Kabat numbering positions as the reference CDR sequence. Unless otherwise stated, when residue positions in the antibody heavy / light chain variable region are referred to in this invention, they refer to numbering positions based on the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

[0045] In this specification, the term “sequence identity” refers to the degree to which sequences are homologous nucleotide-wise or amino acid-wise across the entire comparison window. “Percentage of sequence identity” can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions in the two sequences where the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, Met) is present, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to calculate the percentage of sequence identity. Optimal alignment for determining the percentage of sequence identity can be achieved in various ways known in the art using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm necessary to achieve maximum alignment within the full-length sequence range or target sequence region to be aligned.

[0046] In this specification, with respect to antibody sequences, the amino acid sequence identity percentage is determined after optimal alignment of the candidate antibody sequence with a given antibody sequence, and, in a preferred embodiment, after optimal alignment according to the Kabat numbering rules. In this specification, when no comparison window (i.e., the target antibody region to be compared) is specified, it can be applied to comparisons over the full length of a given antibody sequence.

[0047] In this specification, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the biological function of a protein / polypeptide containing an amino acid sequence. For example, conservative substitutions can be introduced by standard methods known in the art, such as site-directed mutagenesis or PCR-mediated mutagenesis. A typical conservative amino acid substitution refers to the substitution of one amino acid with another amino acid having similar chemical properties (e.g., charge or hydrophobicity). A table of conservative substitutions of functionally similar amino acids is well known in the art.

[0048] In this specification, the term “immune complex preparation” refers to a preparation in which the biological activity of an immune complex as the active ingredient is effectively exerted and which does not contain other ingredients that are unacceptably toxic to the subject to which it is administered. Such preparations are generally sterile. Preparations generally contain pharmaceutically acceptable excipients. A “pharmaceutically acceptable” excipient is a drug that can be appropriately administered to a test mammal so as to deliver an effective dose of the active ingredient used in the preparation to the test mammal. The concentration of the excipient is suitable for the method of administration and, for example, is acceptable for injection.

[0049] In this specification, a “stable” immune complex formulation means that the immune complex in the formulation can maintain an acceptable degree of physical and / or chemical stability when stored under specific conditions. While the immune complex contained in an immune complex formulation may not retain 100% of its chemical structure after storage for a period of time, an immune complex formulation is generally considered “stable” if approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the structure or function of the immune complex is maintained after storage for a period of time. In some embodiments, the immune complexes of the present invention retain substantially their physical and chemical stability after storage.

[0050] Various analytical techniques for measuring protein stability are known in the art; see, for example, Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993). Stability can be measured at a selected temperature and a selected storage time. For example, the storage period can be selected based on the expected storage period of the formulation. Alternatively, accelerated stability testing or high-temperature stress testing may be used. In some embodiments, stability testing is carried out by various stress tests on the formulation. These tests can reproduce not only the extreme conditions that the formulation may encounter during manufacturing, storage, or transport, but also conditions that may accelerate the instability of immune complexes in the formulation during periods other than manufacturing, storage, or transport. For example, the prepared immune complex formulation can be filled into a glass vial and the stability of the immune complexes under high-temperature stress can be tested.

[0051] If, after storage for a certain period, the formulation does not exhibit aggregation, precipitation, turbidity, and / or denaturation, or exhibits little to no aggregation, precipitation, turbidity, and / or denaturation, the immune complexes can be considered to "maintain physical stability" within the formulation. Aggregation of immune complexes in a formulation can lead to an enhancement of the patient's immune response and potentially lead to safety issues. Therefore, it is necessary to minimize or prevent the aggregation of immune complexes in the formulation. Light scattering can be used to measure visible aggregates in the formulation. SEC can be used to measure soluble aggregates in the formulation. The stability of a formulation can also be indicated by visual inspection of the appearance, color, and / or clarity of the formulation, detection of turbidity of the formulation by the OD350nm method, or measurement of the purity of the formulation by the non-reducing CE-SDS method. In certain embodiments, the stability of a formulation can be measured by measuring the percentage of immune complex protein monomers in the formulation after storage for a specific period at a specific temperature, with a higher percentage of immune complex protein monomers indicating greater formulation stability.

[0052] In this specification, "immune complex protein monomer" (or "immune complex monomer" or "monomer") in relation to the stability of the formulation refers to a complete, functional immune complex protein molecule. An immune complex protein monomer differs from a protein aggregate composed of multiple such protein monomers, and also differs from an immune complex protein fragment, such as a semi-antibody portion or an IL-2 mutant fusion protein portion, which are part of the immune complex protein. SEC-HPLC analysis can distinguish between the monomer form and the aggregate form of the immune complex protein. Furthermore, non-reducing CE-SDS analysis can also distinguish between the monomer form and the fragment form of the immune complex protein.

[0053] "Acceptable" physical stability can be expressed as the detection of at least about 92% of immune complex protein monomers in the formulation after storage at a specific temperature for a specific period of time. In some embodiments, after storage at a specific temperature for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months or longer, acceptable physical stability is indicated by the retention of at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of immune complex protein monomers in the formulation. When evaluating physical stability, the specific storage temperature for the drug formulation can be any temperature between approximately -80°C and approximately 45°C, for example, approximately -80°C, approximately -30°C, approximately -20°C, approximately 0°C, approximately 4°C-8°C, approximately 5°C, approximately 25°C, approximately 35°C, approximately 37°C, approximately 40°C, approximately 42°C, or approximately 45°C. For example, if at least approximately 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of immune complex protein monomers are detected after storage at approximately 40°C ± 2°C for two or four weeks, the drug formulation is considered stable. A drug formulation is considered stable if, after storage at approximately 25°C ± 2°C for 4 weeks, 2 months, or 3 months, at least approximately 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of immune complex protein monomers are detected. A drug formulation is considered stable if, after storage at approximately 5°C ± 3°C for 3 months, at least approximately 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% of immune complex protein monomers are detected.

[0054] If the immune complex proteins in a formulation do not show significant chemical changes after storage for a predetermined period, the immune complex proteins are considered to have "maintained chemical stability" in the formulation. Most chemical instability is due to the formation of covalently modified forms of immune complex proteins (e.g., charge variants of immune complex proteins). For example, basic variants are formed by aspartate isomerization, N-terminal and C-terminal modifications, and acidic variants are formed by deamidation, sialylation, and glycation. Chemical stability can be evaluated by detecting and / or quantifying the form of chemical change in the immune complex proteins. For example, charge variants of immune complex proteins in a formulation can be detected by imaging capillary isoelectric focusing (iCIEF). In certain embodiments, the stability of a formulation can be measured by measuring the percentage change in charge variants of immune complex proteins in the formulation after storage at a specific temperature for a specific period; a smaller change indicates higher formulation stability.

[0055] "Acceptable" chemical stability can be expressed as a percentage change of 50% or less in immune complex protein charge variants (e.g., the main component, acidic component, or basic component) in the formulation after storage at a specific temperature for a specific period of time, for example, 30% or less, or 20% or less. In some embodiments, after storage at a specific temperature for at least two weeks, at least 28 days, at least one month, at least two months, at least three months, at least four months, at least five months, at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least eighteen months, at least twenty-four months or longer, an acceptable degree of chemical stability can be expressed by the percentage change in charge variant of the main component being less than or equal to about 50%, 40%, 30%, 20%, or 15%. When evaluating chemical stability, the temperature of the stored drug formulation can be any temperature between about -80°C and about 45°C, for example, about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C. For example, a drug formulation is considered stable if, after 24 months of storage at 5°C, the percentage change in charge variant of the main component is less than approximately 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. A drug formulation is considered stable if, after 2 months of storage at 25°C, the percentage change in charge variant of the main component is less than approximately 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. A drug formulation is considered stable if, after storage at 40°C for one month, the percentage change in charge variant of the main component is approximately 50%, 40%, 30%, 20%, 10%, 5%, or less than 4%.

[0056] The term "freeze-dried formulation" refers to a composition obtained or obtained by freeze-drying a liquid formulation. Preferably, it is a solid composition having a water content of less than 5%, more preferably less than 3%.

[0057] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (e.g., a lyophilized formulation) in a physiologically acceptable solution.

[0058] In this specification, the term "room temperature" refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, and particularly preferably 25°C.

[0059] The term "stress conditions" refers to chemically and / or physically unfavorable environments for immune complexes that can cause unacceptable destabilization of immune complex proteins. The term "high-temperature stress" refers to storing immune complex preparations at room temperature or even higher temperatures (e.g., 40°C ± 2°C) for a certain period of time. The stability of immune complex preparations can be confirmed by room-temperature accelerated testing or high-temperature stress forced testing.

[0060] In this specification, the term “extra-gastrointestinal administration” refers to methods of administration other than enteral and topical administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intra-articular, intra-orbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, sub-articular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions. In some embodiments, the immune complex formulation of the present invention is administered to a subject extra-gastrointestinally. In certain embodiments, the immune complex formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

[0061] I. The present invention's immune complex preparation In this specification, the αPD-1 / IL2m immune complex formulation according to the present invention is also abbreviated as "the immune complex formulation of the present invention" or "the formulation of the present invention," and refers to a formulation comprising the αPD-1 / IL2m immune complex according to the present invention as an active ingredient and pharmaceutically usable excipients. The formulation of the present invention not only enables the formulation of the αPD-1 / IL2m immune complex as an active ingredient in a manner suitable for administration to a subject, but also enables the maintenance of its stability during storage and subsequent use.

[0062] Accordingly, in a first aspect, the present invention provides a liquid formulation containing an αPD-1 / IL2m immune complex, wherein (i) αPD-1 / IL2m immune complex, (ii) Buffering agent, (iii) Stabilizers, and (iv) Contains surfactants. The formulations of the present invention can be prepared as aqueous liquid formulations, such as ready-to-use pre-filled syringes, or as lyophilized formulations that are reconstituted (i.e., redissolved) by dissolving and / or suspending them in a physiologically acceptable solution immediately before use. In some embodiments, the formulations of the present invention are in liquid formulation form.

[0063] Properties of the Liquid Formulation of the Present Invention The liquid formulations of the present invention can be stored stably for a long period of time, for example, for at least 24 months or longer. In certain embodiments, the liquid formulations of the present invention can be stored stably for at least 5 days, at least 10 days, at least 20 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 36 months or longer, under conditions of about -80°C to about 45°C, for example, -80°C, about -30°C, about -20°C, about 0°C, about 0°C, about 25°C, about 35°C, about 38°C, about 40°C, about 42°C, or about 45°C.

[0064] In some embodiments, the liquid formulations of the present invention can be stored stably at approximately 2°C–8°C for at least 3 months, at least 12 months, or at least 24 months. In some embodiments, the liquid formulations of the present invention can be stored stably at, for example, room temperature or approximately 25°C for at least 1 month, at least 2 months, or at least 3 months. In some embodiments, the liquid formulations of the present invention can be stored stably at approximately 40°C for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month.

[0065] In certain embodiments, the stability of a stored formulation can be demonstrated by measuring changes in the appearance of the formulation, visible foreign matter, protein content, pH, purity, charge variants, and / or polysorbate surfactant content. In certain embodiments, the stability of the liquid formulation of the present invention can be measured by high-temperature stress forced experiments (e.g., after storage at 40°C ± 2°C for at least one week, two weeks, or one month), accelerated experiments (e.g., after storage at 25°C ± 2°C for at least one month, two months, or three months), or long-term experiments (e.g., after storage at 5°C ± 3°C for at least two months or three months).

[0066] In certain embodiments, the stability of the liquid formulation of the present invention is visually inspected after storage. Here, the liquid formulation of the present invention is a clear to slightly milky liquid in appearance, colorless to pale yellow, and free of foreign matter. In certain embodiments, a visual inspection is performed using a turbidimeter, and no visible foreign matter is present in the formulation. In certain embodiments, the stability of the liquid formulation of the present invention is measured by measuring the change in protein content after storage. Here, for example, by ultraviolet spectrophotometric (UV) spectroscopy, the rate of change in protein content relative to the initial value on day 0 of storage is 20% or less, preferably 10% or less, for example 7-8%, more preferably 5% or less. In certain embodiments, the stability of the liquid formulation of the present invention is measured by measuring the change in pH of the liquid formulation of the present invention after storage. Here, the change value relative to the initial value on day 0 of storage is 0.3 or less, preferably 0.2 or less, more preferably 0.1 or less. In certain embodiments, the stability of the liquid formulation of the present invention is measured by measuring the change in purity of the liquid formulation of the present invention after storage. Here, by size exclusion high-performance liquid chromatography (SEC-HPLC), the change in immunocomplex monomer purity relative to the initial value on day 0 of storage is 10% or less, for example, 5%, 4%, 3%, or 1-2%, preferably 1% or less. In a specific embodiment, the stability of the liquid formulation of the present invention is measured by measuring the change in purity of the liquid formulation of the present invention after storage. Here, by non-reduced sodium lauryl sulfate capillary electrophoresis (nrCE-SDS), the decrease in the change in immunocomplex monomer purity is 10% or less, for example, 5%, 4%, 3%, preferably 2% or less. In a specific embodiment, the stability of the liquid formulation of the present invention is measured by imaging capillary isoelectric focusing (iCIEF) after storage. Here, relative to the initial value on day 0 of storage, the total change in the charge variants of the immunocomplex (main component, acidic component, and basic component) is 50% or less, for example, 40%, 30%, 20%, 10%, 5%. In certain embodiments, the stability of the liquid formulation of the present invention is measured after storage by high-performance liquid chromatography-fluorescence detection (HPLC-FLD).Here, the change in the polysorbate surfactant content from the initial value on day 0 of storage is 0.3 mg / ml or less, for example, 0.2 mg / ml or less, or 0.1 mg / ml or less. Preferably, after storage of the formulation, the polysorbate 80 surfactant content is 0.1 mg / ml to 0.5 mg / ml, for example, 0.2 mg / ml to 0.4 mg / ml, and preferably about 0.3 mg / ml.

[0067] In certain embodiments, the formulation is stable after storage, for example, after being stored at 2-8°C for at least 24 months, or at room temperature for at least 3 months, or at 40°C ± 2°C for 1 month, and preferably has one or more of the following characteristics. (i) When measured by SEC-HPLC, the formulation shall have a purity of more than 90%, preferably more than 95%, 96%, or 97%. (ii) When measured by the non-reducing CE-SDS method, the formulation shall have a purity of more than 90%, preferably more than 92%, 94%, 96%, or 98%. (iii) When measured by the iCIEF method, the total change in the charge heterogeneity of the immune complex in the formulation (main component, acidic component, and basic component) relative to the initial value on day 0 of storage shall be 50% or less, for example, 40%, 30%, 20%, 10%, or 5% or less. (iv) When measured by HPLC-FLD method, the variation in the polysorbate 80 content in the formulation relative to the initial value on day 0 of storage shall be 0.3 mg / ml or less, for example, 0.2 mg / ml, 0.10 mg / ml, or 0.05 mg / ml or less, and (v) When measured by ELISA, the decrease in the relative binding activity of the immune complex in the formulation to the IL-2 receptor and PD-1 compared to the initial value on day 0 of storage is 30% or less, for example, 80%-110%, for example, about 70%, about 80%, about 90%, about 100%, or about 110%. The SEC-HPLC method, non-reducing CE-SDS method, iCIEF method, HPLC-FLD method, and ELISA method for measuring the stability of the formulation may be carried out by methods known to those skilled in the art or by the methods described in the examples.

[0068] The liquid formulations of the present invention can maintain stability under shaking and freeze-thaw conditions. In certain embodiments, the stability of the formulation under shaking and freeze-thaw conditions can be demonstrated by measuring changes in the appearance of the formulation, visible foreign matter, protein content, pH, purity, charge variants, and / or polysorbate surfactant content. In certain embodiments, the stability of the liquid formulation of the present invention can be measured in shaking experiments, for example, by shaking at a rotation speed of 650 rpm for 1, 2, or 3 days in a light-shielded room at room temperature. In certain embodiments, the stability of the liquid formulation of the present invention can be measured in freeze-thaw experiments, for example, after one, two, three, four, five, or six freeze-thaw cycles consisting of one, two, three, four, five, or six freeze-storage cycles at -20°C and thaw-cycles at 5°C ± 3°C.

[0069] The room-temperature accelerated experiment, high-temperature stress experiment, and long-term experiment for measuring the stability of the formulation of the present invention may be carried out by methods known to those skilled in the art, or by the methods described in the examples.

[0070] Liquid formulation components of the present invention (i) αPD-1 / IL2m immune complex The αPD-1 / IL2m immune complex contained in the formulation of the present invention comprises (i) an anti-PD-1 semiantibody and (ii) a mutant IL-2 fusion polypeptide. The anti-PD-1 semiantibody according to the present invention comprises a Fab fragment that fuses to the Fc region and specifically binds to PD-1, and the mutant IL-2 fusion polypeptide according to the present invention comprises a mutant IL-2 polypeptide that fuses to the Fc region. The mutant IL-2 polypeptide and the αPD-1 / IL2m immune complex applied to the present invention are published in PCT / CN2022 / 120265, and the full text thereof is incorporated into this application for reference.

[0071] The following describes in detail the components of the αPD-1 / IL2m immune complex of the present invention, the anti-PD-1 half-antibody and the mutant IL-2 fusion polypeptide, and the exemplary αPD-1 / IL2m immune complex. Those skilled in the art will understand that, unless the context explicitly indicates otherwise, any combination of the technical features described in these sections falls within the assumed scope of the present invention, and that the αPD-1 / IL2m immune complex of the present invention may include any combination of such features.

[0072] Mutant IL-2 polypeptide The mutant IL-2 fusion polypeptide of the present invention comprises a mutant IL-2 polypeptide fused to the Fc region. In some embodiments, the mutant IL-2 polypeptide according to the present invention has a mutation that weakens the binding affinity to the IL-2Rβγ receptor at the IL-2Rβγ binding site compared to wild-type IL-2, and has a shortened B'C' loop region that improves protein expression level and / or purity.

[0073] In this specification, the terms wild-type “interleukin-2” or “IL-2” refer to the parental IL-2 protein used as a template for introducing the mutation or combination of mutations of the present invention, and preferably refer to a natural IL-2 protein, such as a natural IL-2 protein from a human or non-human primate, including both an unprocessed (e.g., signal peptide not removed) and a processed (e.g., signal peptide removed) form. The full-length natural human IL-2 sequence including the signal peptide is shown in SEQ ID NO: 1, and the sequence of its mature protein is shown in SEQ ID NO: 2. This expression also includes variants of natural IL-2, for example, the natural human IL-2 protein with the C125S mutation introduced at position 125 (uniprot: P60568) belongs to the wild-type IL-2 of the present invention. An instance of the wild-type human IL-2 protein containing the C125S mutation is shown in SEQ ID NO: 3. Preferably, the wild-type IL-2 sequence has at least 85%, 95%, and moreover at least 96%, 97%, 98%, 99%, or higher amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, 2, or 3.

[0074] In this specification, when an amino acid position in an IL-2 polypeptide or IL-2 sequence segment is mentioned, it refers to wild-type human IL-2 protein (IL-2 WTThe amino acid positions are determined by referring to the amino acid sequence SEQ ID NO: 3 (also known as SEQ ID NO: 3). By aligning the amino acid sequence with SEQ ID NO: 3, the corresponding amino acid positions on other IL-2 proteins or polypeptides (full-length sequences or cleaved fragments) can be identified. Thus, in this invention, unless otherwise stated, the amino acid positions of IL-2 proteins or polypeptides refer to the amino acid positions by SEQ ID NO: 3 number. For example, when "F42" is mentioned, it refers to the 42nd phenylalanine residue F in SEQ ID NO: 3, or the amino acid residue at the corresponding position on other IL-2 polypeptide sequences after alignment. Also, for ease of understanding, if the mutant IL-2 of this invention involves cleavage or deletion of a specific region (e.g., the B'C' loop sequence, i.e., the 11 amino acid residues from 73 to 83 in SEQ ID NO: 3), the region can be visually inspected and a gap introduced as needed. This ensures that when aligning the mutant IL-2 with SEQ ID NO: 3, the deleted or cleaved region segment with added gaps and the corresponding segment on SEQ ID NO: 3 are in the corresponding positional region and have the corresponding amino acid residue numbers. For example, in the case of a mutant IL-2 polypeptide (SEQ ID NO: 4) with a B'C' loop sequence (AGDASIH) containing seven cleaved amino acid residues, visual inspection reveals that by introducing four gaps into the cleaved B'C' loop region, when aligning the mutant IL-2 polypeptide with SEQ ID NO: 3, the cleaved B'C' loop region with introduced gaps and the B'C' loop sequence shown on SEQ ID NO: 3 are in the corresponding positional segment and have amino acid residue numbers from 73 to 83. [ka]

[0075] Sequence alignment to determine amino acid positions can be performed using the Basic Local Alignment Search Tool, available from https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with default parameters.

[0076] In this specification, amino acid mutations in IL-2 mutants may include amino acid substitutions, deletions, insertions, and / or additions. Any combination of substitutions, deletions, insertions, and additions can yield a final mutant protein construct with desired properties (e.g., decreased IL-2Rα binding affinity, improved drug potential, and / or weakening of IL-2Rβγ). For example, the alanine residue at the first position of full-length human IL-2 can be deleted, or one or more amino acids can be deleted from the B'C' loop region to shorten the length of the loop region. Alternatively, a shortened B'C' loop sequence can be obtained by substituting, for example, all or part of the B'C' loop sequence of wild-type IL-2 with a different sequence (e.g., the B'C' loop of IL-15).

[0077] In this specification, when IL-2 mutant proteins are mentioned, single amino acid substitutions are denoted as follows: [original amino acid residue / position / substituted amino acid residue]. For example, if lysine at position 35 is substituted with glutamic acid, it can be denoted as K35E. If there are multiple selectable amino acid substitution methods (e.g., D, E) at a particular position (e.g., position K35), the amino acid substitution can be denoted as K35D / E. At the same time, a single amino acid substitution can be linked with a plus sign "+" or "-" to indicate a compound mutation at multiple specific positions. For example, a compound mutation at positions F42A, N88R, and S127E can be denoted as F42A+N88R+S127E, or F42A-N88R-S127E.

[0078] In this specification, the sequence differences between the IL-2 mutant polypeptide according to the present invention and the wild-type IL-2 polypeptide can be expressed as sequence identity or the number of different amino acids between them. In certain embodiments, the IL-2 mutant polypeptide according to the present invention and the wild-type IL-2 polypeptide have at least 85%, 86%, 87%, 88%, or 89% identity, preferably 90% or more identity, for example, at least 95%, 96%, or 97% identity. In some other embodiments, with the exception of the mutations specifically defined according to the present invention (IL-2Rα binding interface mutations, IL-2βγ binding interface, and / or shortened B'C' loop region), the IL-2 mutant polypeptide and the wild-type polypeptide may have 15 or fewer mutations, for example, 1-10 or 1-5 mutations, for example, 0, 1, 2, 3, or 4 mutations. In certain embodiments, the other mutations may be conservative substitutions.

[0079] In this specification, the terms “B'C'Loop,” “B'C'Loop Region,” or “B'C'Loop Sequence” may be used interchangeably and refer to the ligation sequence between the B helix and C helix of the IL-2 protein. The B'C' Loop Sequence of the IL-2 protein can be determined by crystal structure analysis of IL-2 (e.g., PDB:2ERJ). For the purposes of this invention, the B'C' Loop Sequence, as indicated by SEQ ID NO:3, refers to the sequence of residues 72 and 84 in the IL-2 polypeptide. In the wild-type IL-2 proteins shown in SEQ ID NO:1,2, and3, the ligation sequence contains a total of 11 amino acids from A73 to R83. Similarly, in this specification, the terms “shortened loop region” or “shortened B'C' loop region” refer to the mutant protein having a shortened B'C' loop sequence compared to the wild-type IL-2 protein, i.e., the length of the linked sequence between amino acid residues aa72 and aa84 is shortened, as indicated by SEQ ID NO: 3. The “shortened loop region” can be achieved by substitution or cleavage of the loop sequence. The substitution or cleavage can be performed in any region or portion of the B'C' loop sequence. For example, the substitution or cleavage may be a substitution of the loop region A73-R83 sequence (e.g., to substitute into the IL-15 B'C loop region) or a cleavage from one or more amino acid residues at the C-terminus of the sequence to obtain a B'C' loop sequence of less than 10, 9, 8, 7, 6, or 5 amino acids in length. Alternatively, for example, the substitution or cleavage may be a substitution of the loop region Q74-R83 sequence or a cleavage from one or more amino acid residues at the C-terminus of the sequence. After the aforementioned substitution or cleavage, further single amino acid substitutions may be introduced into the loop sequence as needed to further improve the performance of the mutant protein, such as drug potential, by removing glycosylated amino acid substitutions and / or using reverse mutations. Accordingly, in this specification, the shortened B'C' loop region after mutation can be described by a sequence linking the 72nd and 84th residues after mutation.

[0080] In this specification, the term “IL-2Rα binding interface” mutation refers to a mutation occurring at the amino acid site where IL-2 interacts with IL-2Rα (i.e., CD25). These interaction sites can be determined by analyzing the crystal structure of IL-2 and its receptor complex (e.g., PDB:1Z92). In some embodiments, the mutation refers specifically to a mutation in the amino acid residue region 35-72 of IL-2. Preferably, the IL-2 protein containing the mutation has reduced or removed IL-2Rα binding compared to the corresponding protein before the introduction of the mutation.

[0081] In this specification, the term “IL-2βγ binding interface” mutation refers to a mutation occurring at the amino acid sites where IL-2 interacts with IL-2Rβγ (i.e., CD122 and CD132). These interacting amino acid sites can be determined by analyzing the crystal structure of IL-2 and its receptor complex (e.g., PDB:2ERJ). In some embodiments, the mutation refers specifically to mutations in the amino acid residues 12-20, 84-95, and 126-130 regions of IL-2. Preferably, the IL-2 protein containing the mutation has weakened IL-2Rβγ binding compared to the corresponding protein before the mutation.

[0082] In some embodiments, the mutant IL-2 polypeptide according to the present invention contains the following mutations compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 containing the sequence shown in SEQ ID NO: 3). (i) Having a mutation that weakens binding to the IL-2Rβγ receptor at the IL-2-IL-2Rβγ binding interface, particularly at at least one position selected from positions 88, 127 and / or 130, and (ii) A shortened B'C' loop region (i.e., a sequence linked to amino acid residues aa72 and aa84), wherein the shortened loop region has a length of 10, 9, 8, 7, 6, or 5 amino acids, and preferably a length of 7 amino acids, and preferably the shortened B'C' loop region leads to an improvement in protein expression level and / or purity. Here, the amino acid position is determined by SEQ ID NO: 3.

[0083] Preferably, the IL-2Rβγ binding interface mutation includes one or more of the following mutations, or a combination thereof: N88D, N88R, S127E, S130R, N88R+S130R, N88R+S127E. Preferably, the shortened B'C' loop region has the sequence A(Q / G)SKN(F / I)H, or more preferably, a B'C' loop sequence selected from AQSKNFH, AGSKNFH, AQSANFH, and AQSANIH (i.e., a sequence linked to aa72~aa84).

[0084] Optionally, the mutant IL-2 polypeptides of the present invention may also include mutations that cause loss or reduction of binding affinity to the IL-2Rα receptor at the IL-2-IL-2Rα binding interface, particularly at position 35 and / or 42. Preferably, the IL-2Rα binding interface mutations include mutations K35E and / or F42A.

[0085] In some embodiments, the IL-2 mutant polypeptide according to the present invention is compared to wild-type IL-2. (i) N88R + S130R; N88D; N88R; F42A + N88R + S127E, or K35E+N88R + S127E, and (ii) B'C' loop sequence AGDASIH or AQSKNFH, and the optional (iii)T3A.

[0086] In certain preferred embodiments, the mutant IL-2 polypeptide comprises (i) T3A + N88R + S130R and (ii) B'C' loop sequence AGDASIH. Preferably, the mutant IL-2 comprises the amino acid sequence shown in SEQ ID NO: 4, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto.

[0087] Anti-PD-1 half antibody In certain embodiments, the anti-PD-1 semiantibody according to the present invention comprises three heavy chain CDR sequences (i.e., HCDR1, HCDR2, and HCDR3) contained in the heavy chain variable region sequence shown in SEQ ID NO: 8, and three light chain CDR sequences (i.e., LCDR1, LCDR2, and LCDR3) contained in the light chain variable region sequence shown in SEQ ID NO: 15. In some embodiments, the CDRs are defined by Chothia, AbM, Kabat, IMGT, or any combination thereof, for example, Kabat or Chothia or a combination thereof. In some embodiments, the CDRs are CDR sequences defined by the North scheme. In certain preferred embodiments, the light chain CDRs (LCDRs) are CDR sequences defined by the Kabat scheme, and the heavy chain CDRs (HCDRs) are CDR sequences defined by a combination of the Kabat and Chothia schemes.

[0088] In certain embodiments, the anti-PD-1 half-antibody according to the present invention is - Consists of HCDR1, HCDR2, and HCDR3 or combinations thereof, consisting of the amino acid sequences shown in SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11, respectively, and -Contains LCDR1, LCDR2, and LCDR3 or combinations thereof, consisting of the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, respectively.

[0089] In certain embodiments, the anti-PD-1 semi-antibody includes a heavy chain variable region sequence shown in SEQ ID NO: 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto, and a light chain variable region sequence shown in SEQ ID NO: 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto. Preferably, the anti-PD-1 semi-antibody includes the heavy chain variable region shown in SEQ ID NO: 8 and the light chain variable region shown in SEQ ID NO: 15.

[0090] Fc region of the αPD-1 / IL2m immune complex The αPD-1 / IL2m immune complex according to the present invention comprises (i) an anti-PD-1 semiantibody and (ii) a mutant IL-2 fusion polypeptide. Preferably, the anti-PD-1 semiantibody and the mutant IL-2 fusion polypeptide correctly pair with each other via their respective Fc regions and the hinge region located at the N-terminus of the Fc region to form the immune complex according to the present invention.

[0091] In certain embodiments, the Fc regions of the mutant IL-2 fusion polypeptide and the anti-PD-1 heavy chain may be IgG-class Fc regions, or IgG1, IgG2, IgG3, or IgG4 subclass Fc regions. In certain embodiments, the Fc regions include knob-in-hole mutations to facilitate accurate pairing of the anti-PD-1 semiantibody with the mutant IL-2 fusion polypeptide. In certain embodiments, the Fc region of the mutant IL-2 fusion polypeptide includes knob mutations, and the Fc region of the anti-PD-1 heavy chain includes hole mutations. In certain embodiments, the knob mutations include mutations T366W and S354C (EU numbering), and the hole mutations include mutations T366S, L368A, Y407V, and Y349C (EU numbering).

[0092] Exemplary αPD-1 / IL2m immune complex In some preferred embodiments, the αPD-1 / IL2m immune complex according to the present invention comprises or consists of (i) a mutant IL-2 fusion polypeptide, (ii) an anti-PD-1 heavy chain, and (iii) an anti-PD-1 light chain, where, -The mutant IL-2 fusion polypeptide contains the amino acid sequence shown in SEQ ID NO: 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity therewith. -Anti-PD-1 heavy chains include the amino acid sequence shown in SEQ ID NO: 14 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity therewith, and - The anti-PD-1 light chain contains the amino acid sequence shown in SEQ ID NO: 20 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto.

[0093] In some more preferred embodiments, the αPD-1 / IL2m immune complex according to the present invention comprises or consists of a mutant IL-2 fusion polypeptide having the amino acid sequence shown in SEQ ID NO: 7, an anti-PD-1 heavy chain having the amino acid sequence shown in SEQ ID NO: 14, and an anti-PD-1 light chain having the amino acid sequence shown in SEQ ID NO: 20.

[0094] In some embodiments, the αPD-1 / IL2m immune complex of the present invention used in the formulation of the present invention is produced by recombinant expression of HEK293 cells (including HEK293 cells and cells obtained by modifications based on HEK293 cells such as HEK293T, HEK293F, HEK293E cells) or CHO cells (including CHO cells and cells obtained by modifications based on CHO cells such as CHO-S, CHO-dhfr-, CHO / DG44, ExpiCHO cells) and obtained by purification. The αPD-1 / IL2m immune complex of the present invention can be recombinantly expressed using methods known in the art for recombinant expression of immune complex proteins or methods described in the examples. Techniques for purifying therapeutic proteins to pharmaceutical grade are well known in the art and include, but are not limited to, protein A capture, ion exchange chromatography, viral inactivation, viral filtration, ultrafiltration / dialysis filtration, and combinations thereof. Using such purification techniques, an immune complex active pharmaceutical ingredient with sufficient reproducibility and moderate purity for formulation can be obtained. As shown in the embodiments, the immune complex used in the liquid formulation of the present invention exhibits remarkable antitumor activity while reducing side effects.

[0095] The amount of αPD-1 / IL2m immune complex contained in the antibody preparation of the present invention may vary depending on the specific intended use of the preparation, the specific environment in which it is used, and the intended use of the preparation. In some embodiments, the formulation of the present invention is a liquid formulation and may contain the αPD-1 / IL2m immunocomplex of the present invention in an amount of about 0.5 to 150 mg / ml, preferably about 0.5 mg / ml to about 100 mg / ml, for example, about 0.5 mg / ml to about 50 mg / ml or about 1 mg / ml to about 20 mg / ml, for example, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml or about 20 mg / ml.

[0096] (ii) Buffer A buffering agent or buffering solution is generally a solution consisting of a buffer pair of a weak acid and its conjugate base, or a buffer pair of a weak base and its conjugate acid, that have a buffering effect. Examples include a mixed solution of a weak acid (e.g., acetic acid) and its salt (e.g., sodium acetate), or a mixed solution of a weak base (e.g., histidine) and its salt (e.g., histidine hydrochloride or histidine acetate).

[0097] In this specification, a buffer refers to a substance that can maintain the pH of a solution within an acceptable range. In some embodiments, the buffer is a weak acid or weak base compound that constitutes a buffer or buffer solution and can achieve the desired buffering effect by mixing it with an appropriate amount of the corresponding "conjugate acid / base" (the amount of which depends on a predetermined desired pH value) or by supplying the required amount of the corresponding "conjugate acid / base" natively. In some other embodiments, the buffer may be a buffer system consisting of a weak acid and its salt, or a weak base and its salt. For example, in the case of a histidine buffer, it may be prepared by titrating histidine (solid) with an acid (e.g., hydrochloric acid or acetic acid), or by using an appropriate amount of histidine and its salt (e.g., hydrochloride or acetate) to provide the desired pH. Therefore, when it is mentioned in this specification that a formulation of the present invention contains a particular weak acid or weak base buffer, it should be understood that this also covers cases where the formulation contains a buffer system consisting of the weak acid and its salt, or the weak base and its salt. For example, if a formulation is described as containing histidine or a histidine buffer, this should be understood to also include cases where the formulation contains a buffer system consisting of histidine and its salts (e.g., hydrochloride or acetate, but not limited to these).

[0098] In this specification, when a liquid formulation is referred to as containing a particular buffer at a particular concentration, this refers to the total amount of the weak acid or weak base present as a buffer in the liquid formulation and its corresponding conjugate acid / conjugate base. In the case of a histidine buffer, a person skilled in the art can easily calculate its concentration based on the amount of histidine and / or its salts used to prepare the formulation.

[0099] In some embodiments, the buffer used in the formulation of the present invention can control the pH of the formulation to a pH range of about 5.0 to about 6.5, for example, about 5.0 to about 5.5, preferably about 5.0. In some specific embodiments, the formulation of the present invention has pH values ​​of about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, and about 6.4. In some specific embodiments, the formulation of the present invention has a pH of 5.0 ± 0.2.

[0100] In certain embodiments, the buffer used in the formulations of the present invention may be selected from histidine buffers, glutamate buffers, phosphate buffers, acetate buffers, citrate buffers, trishydroxymethylaminomethane (Tris) buffers, and combinations thereof. In some embodiments, the (total) concentration of the buffer in the formulations of the present invention is about 0.5 mM to about 200 mM, about 5 mM to about 50 mM, or about 5 mM to about 20 mM.

[0101] In some embodiments, the buffer used in the formulation of the present invention is a histidine buffer, or a histidine buffer system containing histidine and its salts, particularly a histidine buffer system containing histidine and histidine hydrochloride. In some embodiments, the liquid formulation contains about 5 mM to about 50 mM of histidine. In certain embodiments, the liquid formulation contains about 10 mM to about 30 mM of histidine, for example, about 10 mM, about 15 mM, about 20 mM, about 25 mM, and about 30 mM. In certain embodiments, the liquid formulation of the present invention contains about 10 mM of histidine. In certain embodiments, the liquid formulation of the present invention contains a combination of about 0.75 mM of histidine and about 9.25 mM of histidine hydrochloride, or a combination of about 0.12 mg / ml of histidine and about 1.94 mg / ml of histidine hydrochloride.

[0102] (iii) Stabilizer Suitable stabilizers for use in the present invention may be selected from sugars, amino acids, and combinations thereof. Furthermore, the stabilizers of the present invention may also include chelating agents such as EDTA.

[0103] The saccharides used as stabilizers may be sugars, trisaccharides, and polysaccharides, and may be selected from, but are not limited to, sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, and glucan. In certain embodiments, the saccharides used as stabilizers are sucrose and / or trehalose.

[0104] In some embodiments, sugars as stabilizers are present in the liquid formulation of the present invention at concentrations of about 50 mM to about 500 mM, preferably about 100 mM to about 400 mM, for example, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, and about 400 mM. In some embodiments, the sugar as stabilizer is sucrose, and preferably the concentration of sucrose in the liquid formulation of the present invention is about 30 mg / ml to about 120 mg / ml, for example, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, or about 120 mg / ml. In some embodiments, the content of sugars (e.g., sucrose) can be further adjusted in conjunction with the osmotic pressure of the liquid formulation.

[0105] In some embodiments, in addition to sugars, the formulations of the present invention also include amino acids as stabilizers. The amino acid used as a stabilizer may be selected from, but is not limited to, homocysteine, cysteine, cystathionine, methionine, and arginine, and preferably the amino acid is methionine. In this case, the concentration of the sugars as stabilizers is about 50 mM to about 500 mM, preferably about 100 mM to about 400 mM, for example, about 100 mM, about 150 mM, about 200 mM, about 250 mM, about 300 mM, about 350 mM, and about 400 mM, and the concentration of the amino acid as a stabilizer is 10 mM to about 200 mM, preferably about 30 mM to about 100 mM, for example, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0106] In certain embodiments, the liquid formulation of the present invention contains sucrose as a stabilizer. The concentration of sucrose in the liquid formulation is about 50 mg / ml to about 120 mg / ml, for example, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, or about 120 mg / ml, and preferably about 80 mg / ml.

[0107] In certain embodiments, the liquid formulation of the present invention comprises sucrose and methionine as stabilizers. The concentration of sucrose in the liquid formulation is about 30 mg / ml to about 100 mg / ml, for example, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, or about 80 mg / ml. The concentration of methionine in the liquid formulation is about 20 mM to about 80 mM, for example, about 30 mM, about 40 mM, about 50 mM, or about 60 mM. Preferably, the concentration of sucrose is about 50 mg / ml to about 80 mg / ml, and the concentration of methionine is about 30 mM to about 60 mM. More preferably, the concentration of sucrose is about 70 mg / ml, and the concentration of methionine is about 50 mM. In certain preferred embodiments, the liquid formulation of the present invention comprises about 70 mg / ml of sucrose and about 7.49 mg / ml of methionine.

[0108] (iv) Surfactants In this specification, the term "surfactant" refers to an organic substance having an amphiphilic structure; that is, they are typically composed of groups with opposing solubility tendencies, such as an oil-soluble hydrocarbon chain and a water-soluble ionic group.

[0109] In certain embodiments, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, such as alkyl poly(ethylene oxide). Specific nonionic surfactants that may be included in the formulation of the present invention include polysorbate-based surfactants such as polysorbate-20, polysorbate-80, polysorbate-60, or polysorbate-40, and Pluronic. In certain preferred embodiments, the liquid formulation of the present invention contains polysorbate-80 as the surfactant.

[0110] The amount of surfactant contained in the antibody formulation of the present invention may vary depending on the specific intended use of the formulation, the specific environment, and the intended use of the formulation. In some preferred embodiments, the formulation may contain a polysorbate surfactant (e.g., polysorbate-80) in an amount of about 0.1 mg / l to about 1 mg / ml, preferably about 0.2 mg / ml to about 0.8 mg / ml, for example, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, about 0.5 mg / ml, about 0.6 mg / ml, about 0.7 mg / ml, or about 0.8 mg / ml. Preferably, the formulation contains about 0.1 mg / ml to about 0.5 mg / ml of polysorbate-80, more preferably about 0.3 mg / ml of polysorbate-80.

[0111] (v) Other excipients The liquid formulation of the present invention may or may not contain other excipients. For example, the liquid formulation of the present invention may contain a chelating agent such as EDTA, preferably the concentration of EDTA being about 0.01 mg / ml to about 0.1 mg / ml, more preferably about 0.01 mg / ml to about 0.05 mg / ml, for example, about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, or about 0.05 mg / ml. Alternatively, the liquid formulation of the present invention may also contain a tension modifier. The tension modifier may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride.

[0112] These and other known pharmaceutical excipients and / or additives applicable to the formulations of the present invention are well known in the art and are described, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., American Pharmaceuticals Association (2003) and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Lippincott Williams & Wilkins (2005)."

[0113] II. Uses of the Formulation Formulations of the present invention comprising the immune complex of the present invention may be used to treat or prevent cancer in a subject. In some embodiments, the cancer is a solid tumor or a hematological malignancy. In some embodiments, the cancer is a gastrointestinal tumor or melanoma, for example, colon cancer or colorectal cancer. In some embodiments, the tumor is a tumor or cancer resistant to known drugs, for example, a known anti-PD-1 antibody, for example, a refractory tumor or cancer. In some embodiments, the cancer is characterized by elevated protein levels and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, and / or PD-L2.

[0114] The present invention provides the use of a formulation of the present invention for delivering an immune complex according to the present invention to a mammal in the preparation of a drug, or for treating, preventing, or improving one or more of the above-mentioned cancers. Preferably, the mammal is a human.

[0115] The formulation of the present invention can be administered to a subject or patient by multiple routes. For example, it can be administered via infusion or syringe. In certain embodiments, the present invention provides a pharmaceutical container (e.g., a syringe container) and a delivery device (e.g., a syringe such as a pre-filled syringe) containing the formulation of the present invention. Through such administration, the patient receives an effective amount of the complex of the present invention, i.e., an amount sufficient to treat, improve, or prevent the disease or condition of interest.

[0116] Therapeutic effects may include the reduction of physiological symptoms. The optimal effective dose and concentration of the immune complex for a particular subject depends on a variety of factors, including the patient's age, weight, health status and / or sex, the nature and severity of the disease, the activity of the specific immune complex, its clearance in the body, and other therapies that may be administered in combination with this preparation. In specific circumstances, the effective dose administered may be determined at the discretion of the clinician.

[0117] The following embodiments are provided to aid in understanding the present invention. These embodiments are not intended to limit the scope of protection of the present invention, nor should they be construed in any way as such.

[0118] [Table 1] [Examples]

[0119] To ensure long-term stable storage and guarantee product quality within its shelf life (at least 24 months), we designed a formulation screening test to investigate the effects of different buffer pH values, buffer systems, and excipients on the stability of αPD-1 / IL2m immune complex injection formulations. The materials and methods used in the test are as follows.

[0120] Materials and methods 1.1. Immune complex samples used in formulation studies of the present invention αPD-1 / IL2mut immune complex protein 2149 was prepared and purified according to PCT application number PCT / CN2022 / 120265.

[0121] Specifically, the heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) of the anti-PD-1 antibody, as well as the IL-2m-Fc polypeptide chain (SEQ ID NO: 3, a configuration in which the IL-2m polypeptide is linked to the N-terminus of the IgG1 Fc knob chain via a linker), were constructed in pcDNA3.1 vectors. HEK293 cells were co-transfected using a mixture of the three constructed plasmid vectors and three times the mass of PEI (for example, 1 μg of heavy chain + 1 μg of light chain + 1 μg of IL-2m-Fc polypeptide chain + 9 μg of PEI were required to transfect 3 ml of HEK293). The supernatant containing the expressed immune complex proteins was collected from the culture of the transfected cells and purified. Table 2 below shows the information of the immune complex samples used in the following examples.

[0122] [Table 2]

[0123] 1.2. Reagents and materials used in formulation studies of the present invention [Table 3]

[0124] [Table 4]

[0125] 1.3. Equipment and devices used in the formulation study of the present invention [Table 5]

[0126] 1.4. Measurement items and methods for measuring formulation stability The following items were measured for the immunocomplex formulation: (1) appearance and presence or absence of visible foreign matter; (2) measurement of protein content in the formulation by ultraviolet (UV) method; (3) measurement of insoluble particle content in the formulation; (4) pH measurement of the formulation; (5) measurement of formulation purity by size exclusion high-performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of ​​immunocomplex monomers to the total peak area; (6) measurement of formulation purity by non-reduced sodium lauryl sulfate capillary electrophoresis (nrCE-SDS), expressed as the percentage of the area of ​​immunocomplex monomers to the total peak area; (7) measurement of charge variants in the formulation by imaging capillary isoelectric focusing (iCIEF method), expressed as the percentages of the main component, acidic component, and basic component; (8) measurement of polysorbate-80 content in the formulation by high-performance liquid chromatography-fluorescence detection (HPLC-FLD); (9) measurement of the relative binding activity of the immunocomplex in the formulation to PD-1 antigen and IL-2R by immunoassay methods such as direct ELISA.

[0127] Visible foreign matter measurement In accordance with the method described in the National Pharmacopoeia Committee, Pharmacopoeia of the People's Republic of China (2015 edition, General Rules 0904 "Method for Inspecting Visible Foreign Matter", Beijing: China Pharmaceutical and Technology Press, 2015), visible foreign matter in the sample was measured using a turbidimeter (manufactured by Tianjin Tianda Tianfa, model YB-2).

[0128] Insoluble particle measurement The insoluble particle content in the sample was measured using the photoresist method.

[0129] Protein content measurement The protein content in the sample was measured using a UV-Vis spectrophotometer (Unchained Labs, USA, model number Biglunatic).

[0130] pH measurement The pH of the sample was measured using a SevenCompact pH meter (manufactured by Swiss Mettler, model number S220).

[0131] Purity measurement (SEC-HPLC method) Measurements were performed using a size exclusion column. The column used was a TSKgel G3000Swxl (7.8 × 300 mm, 5 μm) analytical column, the guard column was a TSKgel guardcolumn Swxl (6.0 × 40 mm, 7 μm), the mobile phase was 20 mmol / L phosphate buffer + 400 mmol / L NaClO4, pH 6.8, the sample tray temperature was 10°C, the column temperature was 25°C, the injection volume was 50 μl, the flow rate was 0.5 ml / min, the sampling time was 30 minutes, and an FLD detector with a PMT value of 7, excitation wavelength of 285 nm, and fluorescence wavelength of 340 nm was used.

[0132] The target sample was diluted to 1.0 mg / ml in the mobile phase to prepare the test solution. The buffer solution of the formulation was taken and diluted using the same method as described above to prepare the blank solution. 50 μl each of the blank solution and the test solution were injected into the liquid chromatograph and measurements were started.

[0133] Purity measurement (non-reducing CE-SDS method) Measurements were performed using capillary gel electrophoresis. The capillaries used were uncoated capillaries with an inner diameter of 50 μm, a total length of 30.2 cm, and an effective length of 20.2 cm. Before electrophoresis, the capillary columns were flushed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi, respectively. The target sample was diluted to 1.0 mg / ml with an appropriate amount of ultrapure water. 50 μl of the diluted sample was placed in a 1.5 ml centrifuge tube, and 45 μl of pH 6.5 sample buffer (prepared by dissolving 0.32 g of citrate monohydrate and 2.45 g of sodium hydrogen phosphate dodecahydrate in 45 ml of ultrapure water, making up to 50 ml to obtain citrate-phosphate buffer; accurately weighing 200 μl of this buffer, adding 80 μl of 10% (w / v) sodium dodecyl sulfate solution, and adding water to make 1 ml, then mixing well) and 5 μl of 250 mmol / L NEM solution (prepared by weighing 62 mg of N-ethylmaleimide and dissolving it in 2 ml of ultrapure water) were added. After thorough mixing, the mixture was heated at 70±2°C for 10±2 minutes, cooled to room temperature, and then transferred to a sample vial to obtain the test solution. The same volume of formulation buffer as the test solution was taken and the same procedure was followed to obtain a blank solution. Sample injection conditions: ±5 kV for 20 seconds; separation voltage: ±10 kV for 35 minutes. The capillary column temperature was set to 25°C, and the detection wavelength was set to 220 nm.

[0134] Measurement of charge variants (iCIEF method) The results were measured by imaging capillary isoelectric focusing (iCIEF). The capillary column had an inner diameter of 100 μm and a total length of 5 cm. Before electrophoresis of the samples, the capillary column was flushed with 0.5% methylcellulose solution (hereinafter abbreviated as MC solution) and ultrapure water, respectively. A vacuum injection method was used, with a pre-focus voltage and time of 1.5 kV for 1 minute, a focus voltage and time of 3 kV for 8 minutes, an injection time of 55 seconds, a sample tray temperature of 10°C, and a detection wavelength of 280 nm. The cathode stabilizer was a 500 mmol / L arginine solution, and the 0.5% MC solution reduced adhesion between the protein and the capillary. After concentrating and replacing the test samples, they were diluted with water to 1.0 mg / ml. 20 μl of the diluted test solution was taken, and 77 μl of the premix solution (premix ratio: 3M urea-0.5% MC, amphoteric electrolyte, cathode stabilizer, pI Marker) was added and thoroughly mixed to obtain the target sample solution. The sample was injected and analyzed, and the content of the main component, acidic component, and basic component was calculated according to the area normalization method.

[0135] High-performance liquid chromatography-fluorescence detection (HPLC-FLD) The polysorbate 80 content of the sample was measured by HPLC-FLD. The chromatography column was a SUPELCO nitrile reaction coil (5 m × 0.50 mm ID), and the mobile phase was a mixed solution of 0.15 mol / L sodium chloride, 0.05 mol / L Tris, 5% acetonitrile, 5.0 μM NPN, and 15 ppm Brij23, with a pH of 8.0. Detection conditions: column temperature 30°C, injection volume 10 μl, collection time 3 minutes, flow rate 1.5 ml / min, excitation wavelength 350 nm, fluorescence wavelength 420 nm. After the analysis, the polysorbate 80 content in the final product was calculated using a calibration curve method.

[0136] Biological activity measurement (direct ELISA method) The following antigens were used. IL-2 receptor: Recombinant Human CD25-His Avi purchased from KACTUS, catalog number: CD5-HM425. PD-1: Recombinant human PD-1 purchased from Sinobiological, Inc., Catalog No.: 10377-H08H The relative binding activity of the IL-2 terminus and anti-PD-1 terminus of the immune complex protein was measured using the ELISA measurement program described below.

[0137] The antigen was diluted to 0.5 μg / ml with PBS and plated at 100 μl / well into a 96-well ELISA plate, and incubated overnight at 4°C. After washing, blocking solution (2% BSA-PBST, 300 μl / well) was added and the mixture was blocked at 37°C for 2 hours. The immune complex proteins were diluted to 300 μg / ml (IL2 receptor) or 10 μg / ml (PD-1) with 2% BSA-PBST and diluted to the 11th concentration using a 3-fold gradient. The gradient-diluted samples were added to the ELISA plate (blocking solution discarded) at 100 μl / well. As a negative control, only 100 μl of diluent (2% BSA-PBST) was added to each well, and the mixture was incubated at 37°C in a constant temperature incubator for 60 minutes. After washing the plates, HRP-complexed goat anti-human IgG-Fc fragment (BETHYL, USA, catalog number A80-104P) diluted with 2% BSA-PBST was added, and the reaction was carried out at 37°C for 30 minutes with a secondary antibody (120,000-fold (IL2 receptor) or 100,000-fold (PD-1) dilution, 100 μl / well). After washing the plates, 100 μl of TMB colorimetric solution was added to each well and allowed to develop for 10 minutes, then 100 μl of 1 mol / L H2SO4 was added to each well to stop the reaction. The OD value at 450 nm was measured with a reference wavelength of 620 nm. The concentration value of each concentration gradient sample was plotted on the x-axis, and the OD 450 nm-OD 620 nm value of each gradient sample was plotted on the y-axis, and the EC reflecting the binding activity of the immune complex protein to the antigen was measured using Prism 4 parameter fitting. 50 The result was calculated.

[0138] Example 1: Formulation study 1.1 Experimental Method Buffers containing 1.55 mg / ml histidine and 80.00 mg / ml sucrose were prepared, and their pH was adjusted to 5.0, 5.5, 6.0, and 6.5 with dilute hydrochloric acid, respectively, to form F1 to F4. 1.55 mg / ml histidine, 50.00 mg / ml sucrose, and 7.49 mg / ml methionine were prepared, and their pH was adjusted to 5.0 with dilute hydrochloric acid to form F5. 1.55 mg / ml histidine, 30.00 mg / ml sucrose, and 17.42 mg / ml arginine were prepared, and their pH was adjusted to 5.0 with dilute hydrochloric acid to form F6. 1.55 mg / ml histidine, 50.00 mg / ml sucrose, and 7.49 mg / ml methionine were prepared, and their pH was adjusted to 5.0 with dilute hydrochloric acid to form F7. Protein 2149 was ultrafiltered and transferred to each buffer solution, adjusting the protein content to approximately 1.0 mg / ml, and polysorbate-80 was added to a final concentration of 0.3 mg / ml. Disodium edetate was added to F7 to a final concentration of 0.03 mg / ml, and detailed formulation information is shown in Table 6. The solution was filtered, dispensed into vials, sealed, and cap-rolled. The samples were subjected to stability testing at 40°C ± 2°C, and the specific test plan is shown in Table 7.

[0139] [Table 6] [Table 7]

[0140] 1.2 Criteria Based on our understanding of root products and the precision of our equipment and methods, we established a criterion that the quality of a sample should not change compared to its initial value. See Table 8 for details.

[0141] [Table 8]

[0142] 1.3 Experimental Results See Table 9 for details of the formulation evaluation results and Figure 1 for the trend of change. The results showed that after being left at 40°C ± 2°C for 4 weeks, the appearance and visible foreign matter of the samples for each formulation were acceptable, and no significant changes were observed in protein content and pH. As the pH of the formulation increased, the content of SEC aggregates increased, and the decrease in main peak content increased. As the pH of the formulation decreased, the content of non-reducing CE fragments increased, and the decrease in main peak content increased slightly. Charge variants showed a significant increase in the acidic component, a significant decrease in the main component, and a significant increase in the basic component. Of F1 to F4, F1 was the best. Comparing F1 and F5 to F7, the polysorbate-80 content of F1 and F6 decreased significantly. There were significant changes in the purity and charge variants of F6.

[0143] Based on the results regarding purity and the stability of charge variants, F5 was selected for formulation confirmation experiments. [Table 9-1] [Table 9-2]

[0144] Example 2: Prescription Confirmation Test 2.1 Experimental Method Based on the F5 formulation selected in formulation validation experiments, this experiment primarily investigated the effects of additives (sucrose, histidine, polysorbate-80, and methionine) on the stability of the 2149 protein. See Table 10 for detailed formulation information. Here, the sucrose content was appropriately increased to adjust the osmotic pressure of the formulation.

[0145] The formulation buffer was prepared according to Table 10, and the 2149 protein was ultrafiltered and transferred to the formulation solution, adjusting the protein content to 1.0 mg / ml. Polysorbate-80 was added to bring the final concentration to 0.3 mg / ml. The solution was filtered, dispensed into vials, sealed, and cap-rolled. The samples were then subjected to stability testing under the conditions shown in Table 11.

[0146] [Table 10]

[0147] [Table 11]

[0148] 2.2 Judgment criteria For details on the criteria for evaluation, please refer to Table 8.

[0149] 2.3 Experimental Results Detailed results of the formulation confirmation stability test are shown in Tables 12 and 13, and the trend of change is shown in Figure 2. According to the results, the appearance and visible foreign matter of the samples were acceptable at all time points, the protein content and pH value did not change significantly, the SEC main peak decreased by 1.0% under high temperature conditions, the charged variant acidic component increased under accelerated and high temperature conditions, the charged variant main component decreased under accelerated and high temperature conditions, and the charged variant basic component slightly increased under high temperature conditions. The polysorbate-80 content remained unchanged. IL2 terminal activity significantly decreased under high temperature conditions for 4 weeks, while PD-1 terminal activity did not change significantly. Under shaking and freeze-thaw conditions, the protein quality did not change significantly.

[0150] [Table 12]

[0151] As shown in Table 12, after 4 weeks at 40°C ± 2°C, the main peak of SEC decreased by 1.0%, the main peak of unreduced CE-SDS decreased by 2.8%, the acidic component of the charge variant increased by 13.6%, the main component decreased by 17.7%, and the basic component increased slightly. After 3 months at 25°C ± 2°C, unreduced CE-SDS decreased by 2.2%, the acidic component of the charge variant increased by 7.3%, and the main component decreased by 9.4%. No significant changes were observed in any of the measured parameters after long-term storage for 3 months at 5°C ± 3°C.

[0152] [Table 13]

[0153] conclusion Based on the results of formulation review and formulation confirmation experiments, the confirmed formulation was selected as the final formulation for 2149. To avoid pH adjustment with hydrochloric acid during production, the buffer system was adjusted to histidine and histidine hydrochloride. Specifically, the formulation for 2149 consisted of 1.0 mg / ml recombinant αPD-1 / IL2m immune complex protein molecule, 0.12 mg / ml histidine, 1.94 mg / ml histidine hydrochloride, 70.00 mg / ml sucrose, 7.49 mg / ml methionine, and 0.3 mg / ml polysorbate-80, with a pH of 5.0.

[0154] Example 3: In vivo efficacy experiment of αPD-1 / IL2m immune complex The αPD-1 / IL2m immune complex of the present invention specifically binds to human PD-1 at its αPD-1 terminus, inhibiting the binding of PD-1 to PD-L1 and thereby releasing the immune braking mechanism. Simultaneously, it specifically binds to IL-2 receptors on T cells or NK cells at its IL-2m terminus, activating and amplifying T cells or NK cells, thereby enhancing the effects of PD-1 antibody immunotherapy.

[0155] To confirm the in vivo efficacy of the αPD-1 / IL2m immune complex of the present invention, MC38 cells (mouse colon cancer cell line, manufactured by Shanghai Heyuan Bio-Co., Ltd.) were inoculated into hPD-1 knock-in mice, and the antitumor effect of the immune complex (2149) of the present invention was measured. For the experiment, SPF-grade female hPD-1 knock-in mice (purchased from Shanghai Southern Model Bio-Co., Ltd.) were used, with a conformity certificate number of NO. 20170010006762.

[0156] For subsequent in vivo experiments, MC38 cells were subcultured using standard methods. The cells were harvested by centrifugation, and the MC38 cells were resuspended in PBS (1×) until the cell concentration reached 5×10⁶. 6A cell suspension was prepared at a concentration of cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right flank of hPD-1 knock-in mice to establish an MC38 tumor-bearing mouse model.

[0157] Eight days after tumor cell inoculation, the tumor volume of each mouse was detected and they were divided into groups (8 mice / group). The dosage and administration method are shown in Table 14.

[0158] [Table 14]

[0159] The concentrations used for h-IgG,2149,10 mg / kg, 20 mg / kg, and 40 mg / kg were 4 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml, respectively, administered once a week for a total of three times (QWx3). As shown in Figures 3A-B, the administration was performed on days 8, 15, and 22 after MC38 cell inoculation, and mouse tumor volume and body weight were monitored twice a week. When the tumor volume reached 2000 mm³... 3 Mice were euthanized once the tumor volume exceeded a certain threshold, and some mice were observed until the end of the experiment at 61 days. Some groups of mice had tumor volumes of 2000 mm³. 3 Since euthanasia was performed once the threshold was exceeded, the relative tumor growth inhibition rate (TGI%) was calculated based on 36 days after vaccination. The calculation formula is as follows: TGI% = 100% * (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-administration tumor volume). Measurement of tumor volume: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L * W 2 / 2. Weight was measured using an electronic balance.

[0160] The tumor growth curves and survival curves are shown in Figures 3A and 3B. The antitumor effect of 2149 molecules in different dose groups was dose-dependent, with complete tumor regression in mice in the 20 mg / kg and 40 mg / kg groups. This advantage is also reflected in the survival curve in Figure 3B. In the two mouse groups, tumors regressed 100%, but in the 10 mg / kg group, only 2 out of 8 mice experienced complete tumor regression. The results of tumor growth inhibition rates are shown in Table 15. At 36 days post-inoculation, the tumor growth inhibition rates for 2149, 10 mg / kg, 2149, 20 mg / kg, and 2149, 40 mg / kg were 84%, 103%, and 103%, respectively, compared to the h-IgG group. Simultaneously, results of the mouse body weight test (Figure 3C) showed no significant difference in mouse body weight at 36 days post-inoculation.

[0161] [Table 15]

[0162] To confirm the in vivo efficacy of immune complex 2149, hPD-1 knock-in mice were inoculated with PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475), and the antitumor effect of the dual-function PD-1 antibody and IL-2 mutant molecule fusion protein (2149) of the present invention was measured. For the experiment, SPF-grade female hPD-1 knock-in mice (purchased from Shanghai Southern Model Biotechnology Co., Ltd.) with certification number NO. 20170010007909 were used.

[0163] For subsequent in vivo experiments, B16F10 cells were subcultured using standard methods. The cells were harvested by centrifugation, and the B16F10 cells were resuspended in PBS (1×) to a cell concentration of 2.5×10⁶. 6 A cell suspension was prepared at a concentration of cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right flank of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0164] Six days after tumor cell inoculation, the tumor volume of each mouse was detected and they were divided into groups (8 mice / group). The dosage and administration method are shown in Table 16.

[0165]

Table 16

[0166] The use concentrations of h-IgG, IBI308 at 20 mg / kg, IBI308 at 40 mg / kg, 2149 at 20 mg / kg and 2149 at 40 mg / kg were 4 mg / ml, 2 mg / ml, 4 mg / ml, 2 mg / ml and 4 mg / ml respectively, and they were administered once a week for a total of 3 times (QWx3). As shown in Figures 4A - B, they were administered on the 8th, 15th and 22nd days after inoculation with B16F10 cells respectively, and the mouse tumor volume and body weight were monitored twice a week until 22 days later. Since B16F10 cells are prone to metastasis and induce mouse death, the relative tumor growth inhibition rate (TGI%) was calculated based on the 15th day after inoculation. The calculation formula is as follows: TGI% = 100% * (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group tumor volume before administration). Measurement of tumor volume: The maximum major axis (L) and maximum minor axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated by the following formula: V = L * W 2 / 2. The body weight was measured using an electronic balance. When the tumor volume exceeded 2000 mm 3 , the mice were euthanized.

[0167] The tumor growth curves of the mice are shown in Figures 4A and 4B. In the PD1-resistant model, IBI308 showed almost no effect, but the 20 mg / kg and 40 mg / kg groups of 2149 showed a certain degree of anti-tumor effect, and the anti-tumor effect of the high-dose group was better than that of the low-dose group. This effect was also reflected in the survival curve of the mice. At the end of the experiment, the tumors had completely regressed in two mice in the high-dose group (Figures 4C and Table 17).

[0168] The results for tumor growth inhibition rates are shown in Table 17: On day 15 after inoculation, the tumor growth inhibition rates for IBI308, 20 mg / kg, IBI308, 40 mg / kg, 2149, 20 mg / kg, and 2149, 40 mg / kg were 29%, 27%, 82%, and 86%, respectively, compared to the h-IgG, 40 mg / kg group. Simultaneously, no significant difference in mouse body weight was observed on day 22 after inoculation, as shown in the mouse body weight test (Figure 4D).

[0169] [Table 17]

[0170] To confirm that the in vivo efficacy of αPD-1 / IL2m immune complex 2149 is superior to that of the control drug PD-1-IL2v (molecular number 2061, sequence derived US20180326010A1, see also sequence listing), hPD-1 knock-in mice were inoculated with PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475), and the antitumor effect of the dual-function PD-1 antibody and IL-2 mutant molecule fusion protein (2149) of the present invention was measured. For the experiment, SPF-grade female hPD-1 knock-in mice (purchased from Shanghai Southern Model Biological Co., Ltd.) with certificate number NO. 20170010008942 were used.

[0171] For subsequent in vivo experiments, B16F10 cells were subcultured using standard methods. The cells were harvested by centrifugation, and MC38 cells were resuspended in PBS (1×) to a cell concentration of 2.5×10⁶. 6 A cell suspension was prepared at a concentration of cells / ml. On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right flank of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0172] Eight days after tumor cell inoculation, the tumor volume of each mouse was detected and they were divided into groups (7 mice / group). The dosage and administration method are shown in Table 18.

[0173] [Table 18]

[0174] h-IgG, IBI308, 40 mg / kg, 2061, 10 mg / kg, 2061, 20 mg / kg, 2061, 40 mg / kg, 2149, 10 mg / kg, 2149, 20 mg / kg, and 2149-40 mg / kg were administered at concentrations of 4 mg / ml, 2 mg / ml, 4 mg / ml, 2 mg / ml, and 4 mg / ml, respectively, once a week for a total of three doses (QWx3). As shown in Figure 5A, the doses were administered on days 8, 15, and 22 after B16F10 cell inoculation, and mouse tumor volume and body weight were monitored twice a week until 23 days later. Since B16F10 cells are highly metastatic and induce mouse death, the relative tumor growth inhibition rate (TGI%) was calculated based on day 19 after inoculation. The calculation formula is as follows: TGI% = 100% * (control group tumor volume - treatment group tumor volume) / (control group tumor volume - control group pre-treatment tumor volume). Tumor volume measurement: The maximum long axis (L) and maximum width axis (W) of the tumor were measured using calipers, and the tumor volume was calculated using the following formula: V = L * W² / 2. Body weight was measured using an electronic balance. Tumor volume was 2000 mm 3 Mice were euthanized when the threshold was exceeded. If more than half of the mice in a group died, the tumor growth curve for the entire group at that point was not shown.

[0175] The results for tumor growth inhibition rates are shown in Table 19: On day 19 after inoculation, the tumor growth inhibition rates for IBI308, 40 mg / kg, 2061, 10 mg / kg, 2149, 10 mg / kg, 2149, 20 mg / kg, and 2149-40 mg / kg were 21%, 96%, 79%, 87%, and 97%, respectively, compared to the h-IgG, 40 mg / kg group. In the 2061 20 mg / kg and 40 mg / kg groups, significant weight loss occurred after the first dose, and mice died, so TGI was not calculated for these groups.

[0176] Furthermore, the survival rate of the mice was statistically analyzed (Figure 5B). Comparing the maximum dose / maximum tolerated dose of 2061 and 2149 in this experiment, more mice survived at a dose of 40 mg / kg with 2149 and at 10 mg / kg with 2061. In the 2149 group, the tumors completely regressed in 3 out of 7 mice, while in the 2061 group, the tumors completely regressed in only 1 mouse. Simultaneously, in the measurement of mouse body weight (Figure 5C), the body weight of mice in all dose groups of 2149 did not decrease 29 days after inoculation, but the body weight of mice in the 2061 group did decrease. At low doses (10 mg / kg), the average body weight of mice decreased by more than 5%, and at medium doses (20 mg / kg) and high doses (40 mg / kg), mice died. A total of 6 out of 7 mice died in each group. See Table 12 for details. On the other hand, 2149 was relatively safe, and no significant weight loss was observed at any of the low, medium, or high doses. Only one mouse death occurred at the low and medium doses, but there were no mouse deaths at the high dose. 2149 showed a higher complete tumor remission rate than 2061 (Table 19). Therefore, 2149 is more effective and safer than 2061 and has a broader therapeutic range.

[0177] [Table 19]

[0178] [Table 20-1] [Table 20-2] [Table 20-3]

Claims

1. (i) immune complexes; (ii) Buffering agent, (iii) Stabilizers, and (iv) An immunocomplex liquid formulation containing a surfactant, Here, the immune complex comprises (a) an anti-PD-1 semiantibody and (b) a mutant IL2 fusion polypeptide. Here, the anti-PD-1 semiantibody includes a Fab fragment fused to the Fc region, and also includes three heavy chain CDR sequences contained in the heavy chain variable region sequence shown in SEQ ID NO: 8, and three light chain CDR sequences contained in the light chain variable region sequence shown in SEQ ID NO: 15, Here, the mutant IL2 fusion polypeptide comprises a mutant IL-2 polypeptide fused to the Fc region, and the mutant IL-2 polypeptide comprises (i) N88R + S130R, (ii) B'C' loop sequence AGDASIH or AQSKNFH, and optionally (iii) T3A. Herein, the pH of the liquid formulation is about 5.0 to about 6.5, for example, the pH is about 5.0, about 5.5, about 6.0 or about 6.5, preferably the pH is about 5.0, an immunocomplex liquid formulation.

2. The liquid formulation according to claim 1, characterized in that the concentration of the immune complex is about 0.5 mg / ml to about 150 mg / ml, preferably about 0.5 to about 10 mg / ml, for example, about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 3 mg / ml, about 4 mg / ml, about 5 mg / ml, about 6 mg / ml, about 7 mg / ml, about 8 mg / ml, about 9 mg / ml, or about 10 mg / ml.

3. The liquid formulation according to claim 1 or claim 2, wherein the buffer comprises a histidine buffer, preferably comprising histidine in a concentration of about 5 mM to about 50 mM, preferably histidine in a concentration of about 10 mM to about 30 mM, for example, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM histidine.

4. The aforementioned stabilizer is - Sugars, especially sucrose, preferably with a concentration of sucrose of about 50 mg / ml to about 120 mg / ml, for example, about 80 mg / ml, or - A combination of sugars and amino acids, particularly a combination of sucrose and methionine, preferably with a sucrose concentration of about 30 mg / ml to about 100 mg / ml, for example, about 50 mg / ml, about 60 mg / ml, about 70 mg / ml, or about 80 mg / ml, and a methionine concentration of about 20 mM to about 80 mM, for example, about 30 mM, about 40 mM, about 50 mM, or about 60 mM. A liquid formulation according to any one of claims 1 to 3, characterized by containing the following:

5. The liquid formulation according to any one of claims 1 to 4, characterized in that the surfactant in the liquid formulation is selected from polysorbate-based surfactants, and preferably polysorbate-80.

6. The liquid formulation according to any one of claims 1 to 5, characterized in that the concentration of the surfactant is about 0.1 mg / ml to about 1 mg / ml, preferably about 0.1 mg / ml to about 0.5 mg / ml, for example, about 0.1 mg / ml, about 0.2 mg / ml, about 0.3 mg / ml, about 0.4 mg / ml, or about 0.5 mg / ml.

7. The liquid formulation according to any one of claims 1 to 6, wherein the liquid formulation contains EDTA, preferably the concentration of EDTA is about 0.01 mg / ml to about 0.1 mg / ml, more preferably about 0.01 mg / ml to about 0.05 mg / ml, for example, about 0.01 mg / ml, about 0.02 mg / ml, about 0.03 mg / ml, about 0.04 mg / ml, or about 0.05 mg / ml.

8. The aforementioned immune complex contains a knob-in-hole mutation, Preferably, the liquid formulation according to any one of claims 1 to 7, wherein the Fc region of the fusion polypeptide contains a knob mutation, and the Fc region of the semi-antibody contains a hole mutation.

9. The anti-PD-1 semi-antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 15 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. Preferably, the anti-PD-1 semiantibody comprises a heavy chain and a light chain, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 14 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, and the light chain comprises the amino acid sequence shown in SEQ ID NO: 20 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, the liquid formulation according to any one of claims 1 to 8.

10. The mutant IL2 protein comprises the amino acid sequence shown in SEQ ID NO: 4 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto. Preferably, the mutant IL-2 fusion polypeptide comprises the amino acid sequence shown in SEQ ID NO: 7 or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity thereto, according to any one of claims 1 to 9.

11. The liquid formulation according to any one of claims 1 to 10, characterized in that the immune complex is recombinantly expressed in HEK293 cells or CHO cells.

12. The liquid preparation is an injectable preparation, preferably an injectable preparation used for subcutaneous injection or intravenous injection, or an injectable preparation used for intravenous infusion, as described in any one of claims 1 to 11.

13. (i) Immune complexes of approximately 1–50 mg / ml, (ii) Histidine of approximately 5–50 mM, (iii) Sucrose at approximately 50–120 mg / ml, or a combination of sucrose at approximately 30–100 mg / ml and methionine at approximately 20–80 mM, (iv) A liquid formulation containing approximately 0.1 to 0.5 mg / ml of polysorbate 80, Here, the pH of the liquid formulation is approximately 5.0-5.5, preferably approximately pH 5.

0. For example, the liquid formulation is (i) Immune complexes of approximately 1–10 mg / ml, (ii) Histidine at approximately 10–30 mM, (iii) Sucrose at approximately 70–90 mg / ml, or a combination of sucrose at approximately 50–80 mg / ml and methionine at approximately 40–60 mM, (iv) Contains approximately 0.2–0.4 mg / ml of polysorbate 80, Here, the pH of the liquid formulation is approximately 5.0-5.5, preferably approximately pH 5.

0. Alternatively, the liquid formulation is (i) Immune complexes of approximately 1.0 mg / ml, (ii) Approximately 10 mM histidine, (iii) Sucrose at approximately 80 mg / ml, or a combination of sucrose at approximately 70 mg / ml and methionine at approximately 50 mM, (iv) Contains approximately 0.3 mg / ml of polysorbate 80, Here, the pH of the liquid formulation is approximately 5.0-5.5, preferably approximately pH 5.

0. Alternatively, the liquid formulation is (i) Immune complexes of approximately 1.0 mg / ml, (ii) Approximately 0.12 mg / ml of histidine and approximately 1.94 mg / ml of histidine hydrochloride, (iii) Approximately 70 mg / ml of sucrose and approximately 7.49 mg / ml of methionine, (iv) Contains approximately 0.3 mg / ml of polysorbate 80, The liquid formulation according to any one of claims 1 to 12, characterized in that the pH of the liquid formulation is about 5.0-5.5, preferably about 5.

0.

14. The formulation is stable after storage, for example, after being stored at 2-8°C for at least 3 months, or at room temperature for at least 3 months, or at 40°C ± 2°C for 1 month, preferably, (i) When measured by SEC-HPLC, the formulation must have a purity of 90%, 95%, 96%, or 97% or higher. (ii) When measured by the non-reducing CE-SDS method, the formulation must have a purity of 90%, 92%, 94%, 96%, or 98% or higher. (iii) When measured by the iCIEF method, the total change in charge heterogeneity (main component, acidic component, and basic component) in the formulation relative to the initial value on day 0 of storage shall be 50% or less, for example, 40%, 30%, 20%, 10%, or 5% or less, and (iv) The liquid formulation according to any one of claims 1 to 13, characterized in that, when measured by HPLC-FLD, the variation in the polysorbate 80 content in the formulation relative to the initial value on day 0 of storage is 0.3 mg / ml or less, for example, 0.2 mg / ml, 0.10 mg / ml, or 0.05 mg / ml or less.

15. A solid preparation obtained by solidifying a liquid preparation according to any one of claims 1 to 14, wherein the solid preparation is, for example, in the form of a lyophilized powder injection.

16. A pharmaceutical injection container comprising a liquid preparation according to any one of claims 1 to 14 or a solid preparation according to claim 15.

17. A pre-filled syringe for intravenous or intramuscular injection comprising a liquid formulation according to any one of claims 1 to 14 or a solid formulation according to claim 15.

18. Use of a liquid formulation according to any one of claims 1 to 14 or a solid formulation according to claim 15 in the prevention or treatment of cancer, or in the preparation of a drug for the prevention or treatment of cancer, wherein the cancer is a solid tumor or hematological malignancy, for example, a gastrointestinal tumor or melanoma, for example, colorectal cancer or colon cancer, and for example, the cancer is PD-1 antibody-resistant cancer.

Images