Preparation containing Anti-PD-1 / mutant il-2 immunoconjugate

Abstract

The present invention relates to a preparation containing an immunoconjugate comprising a recombinant anti-programmed cell death protein 1 (PD-1) antibody and mutant interleukin 2 (IL2), and in particular to a stable liquid preparation, a method for preparing the preparation, and a use of the preparation in the treatment and / or prevention of diseases.

Description

Anti-PD-1 and mutant IL-2 immunoconjugate formulations Technical Field

[0001] The present invention relates to the field of immunoconjugate preparations. More specifically, the present invention relates to preparations comprising an immunoconjugate of a recombinant anti-programmed death receptor 1 (PD-1) antibody and a mutant interleukin 2 (IL2) (hereinafter also referred to as "αPD-1 / IL2m"), in particular a stable liquid preparation, as well as methods for preparing the preparations, and the use of the preparations for treating and / or preventing diseases. Background Art

[0002] Interleukin-2 (IL-2), also known as T cell growth factor (TCGF), is a multifunctional cytokine produced primarily by activated T cells, particularly CD4+ T helper cells. IL-2 mediates multiple actions in the immune response by binding to IL-2 receptors on different cells. On the one hand, IL-2 has an immune system-stimulating effect, stimulating the proliferation and differentiation of T cells and natural killer (NK) cells. Therefore, IL-2 has been approved as an immunotherapy agent for the treatment of cancer and chronic viral infections. On the other hand, IL-2 can also promote 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)), causing immunosuppression in patients caused by activated Treg cells.

[0003] Programmed death protein-1 (PD-1) is an important immune checkpoint protein, a 55kDa type I transmembrane protein that is primarily expressed inductively on the surface of activated T cells, and is also expressed on cells such as B cells, NK cells, monocytes, and DCs. Two cell surface glycoprotein ligands for PD-1 have been identified: programmed death protein ligand 1 (PD-L1) and programmed death protein ligand 2 (PD-L2). PD-1 ligands are highly expressed on many cancer cells. Binding of PD-1 to PD-1 ligands can lead to T cell apoptosis, immune anergy, T cell "exhaustion," and IL-10 secretion. Therefore, blocking the PD1 pathway can restore T cell function in cancer patients (Sheridan, Nature Biotechnology 30 (2012), 729-730). Monoclonal antibodies targeting PD-1 have been described, for example, Nivolumab from Bristol-Myers Squibb (BMS) and Pembrolizumab from Merck. ) is a fully humanized IgG4 antibody molecule, pembrolizumab (trade name ) is a humanized IgG4 antibody molecule. The anti-PD-1 monoclonal antibody, upon binding to PD-1 on T lymphocytes, can inhibit the binding of PD-1 to its ligands PD-L1 and PD-L2, thereby promoting T lymphocyte activation, proliferation, and production of immune-activating cytokines, and relieving PD-1's inhibition of T lymphocyte immune surveillance with anti-tumor activity.

[0004] Given the importance of IL-2 and PD-1 in regulating immune responses, the present inventors, through intensive research, have developed an anti-PD-1 / IL-2 mutein immunoconjugate that targets both PD-1 and the IL-2 receptor (see patent application number PCT / CN2022 / 120265, which is hereby incorporated by reference in its entirety). This immunoconjugate exhibits enhanced anti-tumor effects while also exhibiting reduced side effects, making it a promising candidate for clinical tumor treatment.

[0005] For clinical medications, drug stability is a key indicator for ensuring both efficacy and safety. Obtaining a formulation that confers excellent drug stability is crucial for maintaining drug safety and efficacy throughout its shelf life. Therefore, to meet the demand for drug stability, further targeted research and development of pharmaceutical formulations suitable for anti-PD-1 / IL-2 mutein immunoconjugates is needed in this field.

[0006] SUMMARY OF THE INVENTION

[0007] To address these issues, the inventors designed a formulation screening experiment for recombinantly expressed anti-PD-1 / IL-2 mutein immunoconjugates. High-temperature experiments were used to screen buffer pH, buffer systems, and excipients. The formulation was then evaluated and confirmed based on testing for appearance, visible foreign matter, protein content, pH, purity, charge variants, polysorbate 80 content, and biological activity. This ultimately resulted in the development of the liquid formulation of the present invention. This formulation not only ensures controlled product quality throughout its shelf life but can also be prepared using a stable and easily implemented aseptic formulation process.

[0008] Therefore, in a first aspect, the present invention provides a liquid formulation comprising an αPD-1 / IL2m immunoconjugate. In some embodiments, the liquid formulation of the present invention is an injection, preferably an injection for intravenous infusion.

[0009] In some embodiments, the liquid formulation of the present invention comprises:

[0010] (i) αPD-1 / IL2m immunoconjugate;

[0011] (ii) a buffer;

[0012] (iii) stabilizers; and

[0013] (iv) a surfactant,

[0014] The pH of the liquid formulation is about 5.0 to about 6.5, such as about 5.0 to about 5.5, preferably about 5.0.

[0015] In some embodiments, the αPD-1 / IL2m immunoconjugate according to the present invention comprises: (a) an anti-PD-1 half antibody and (b) a mutant IL2 fusion polypeptide,

[0016] The anti-PD-1 half antibody comprises a Fab fragment fused to an Fc region, and comprises the three heavy chain CDR sequences contained in the heavy chain variable region sequence of SEQ ID NO: 8; and the three light chain CDR sequences contained in the light chain variable region sequence of SEQ ID NO: 15; and

[0017] The mutant IL2 fusion polypeptide comprises a mutant IL-2 polypeptide fused to an Fc region, and the mutant IL-2 polypeptide comprises (i) N88R+S130R; and (ii) the B'C' loop sequence AGDASIH or AQSKNFH, and optionally (iii) T3A.

[0018] In some embodiments, the liquid formulation comprises about 0.5 mg / ml to about 150 mg / ml of the immunoconjugate, preferably, the concentration of the immunoconjugate is about 0.5 mg / ml to about 5 mg / ml.

[0019] In some embodiments, the buffer is selected from histidine buffer, glutamate buffer, phosphate buffer, acetate buffer, citrate buffer and tris (hydroxymethylaminomethane) (Tris) buffer and their combination. Preferably, the liquid preparation according to the present invention comprises histidine buffer. In some embodiments, in the liquid preparation according to the present invention, the (total) concentration of buffer is about 0.5mM to about 200mM, about 5mM to about 50mM or about 5mM to about 20mM.

[0020] In some embodiments, the stabilizer is selected from a carbohydrate (e.g., sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin, dextran), an amino acid (e.g., methionine, arginine, and salts thereof), or a combination thereof. Preferably, the stabilizer comprises sucrose, methionine, or a combination thereof. In some embodiments, the (total) concentration of the stabilizer is from about 1 mM to about 1000 mM, or from about 50 mM to about 500 mM.

[0021] In some embodiments, the surfactant is selected from polysorbate surfactants, such as polysorbate 80 and polysorbate 20, 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.

[0022] In some embodiments, the liquid formulation of the present invention comprises:

[0023] (i) αPD-1 / IL2m immunoconjugate;

[0024] (ii) histidine buffer;

[0025] (iii) sucrose and methionine; and

[0026] (iv) polysorbate 80,

[0027] and optionally (v) EDTA,

[0028] The pH of the liquid formulation is about 5.0 to about 6.5, such as about 5.0 to about 5.5, preferably about 5.0.

[0029] In some embodiments, the liquid formulation of the present invention comprises:

[0030] (i) αPD-1 / IL2m immunoconjugate;

[0031] (ii) histidine and histidine hydrochloride;

[0032] (iii) sucrose and methionine; and

[0033] (iv) polysorbate 80,

[0034] and optionally (v) EDTA,

[0035] The pH of the liquid formulation is about 5.0 to about 6.5, such as about 5.0 to about 5.5, preferably about 5.0.

[0036] Preferably, the liquid preparation of the present invention comprises:

[0037] (i) about 1 mg / ml to about 10 mg / ml of an αPD-1 / IL2m immunoconjugate;

[0038] (ii) about 10 mM to about 30 mM histidine buffer;

[0039] (iii) about 70 mg / ml to about 90 mg / ml of sucrose, or about 50 mg / ml to about 80 mg / ml of sucrose in combination with about 40 mM to about 60 mM methionine; and

[0040] (iv) about 0.2 mg / ml to about 0.4 mg / ml of polysorbate 80;

[0041] wherein the pH of the liquid formulation is about 5.0-5.5, preferably about pH 5.0;

[0042] Alternatively, the liquid preparation comprises

[0043] (i) about 1.0 mg / ml of αPD-1 / IL2m immunoconjugate;

[0044] (ii) about 10 mM histidine buffer;

[0045] (iii) about 80 mg / ml sucrose, or a combination of about 70 mg / ml sucrose and about 50 mM methionine, and

[0046] (iv) about 0.3 mg / ml polysorbate 80;

[0047] The pH of the liquid preparation 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 comprising histidine and its salts, especially a histidine buffer system comprising histidine and histidine hydrochloride.

[0048] In some more preferred embodiments, the liquid formulation of the present invention comprises:

[0049] (i) about 1.0 mg / ml of αPD-1 / IL2m immunoconjugate;

[0050] (ii) about 0.12 mg / ml histidine and about 1.94 mg / ml histidine hydrochloride;

[0051] (iii) about 70 mg / ml sucrose and about 7.49 mg / ml methionine, and

[0052] (iv) about 0.3 mg / ml polysorbate 80;

[0053] The pH of the liquid formulation is about 5.0-5.5, preferably about pH 5.0.

[0054] In a second aspect, the present invention provides a solid preparation obtained by subjecting the liquid preparation of the present invention to a solidification treatment. The solidification treatment is carried out, for example, by crystallization, spray drying, or freeze drying. In a preferred embodiment, the solid preparation is, for example, in the form of a lyophilized powder injection. The solid preparation can be reconstituted in an appropriate solvent prior to use to form a reconstituted preparation of the present invention. The reconstituted preparation is also a liquid preparation of the present invention. In one embodiment, the appropriate solvent is selected from water for injection, an organic solvent for injection, including but not limited to oil for injection, ethanol, propylene glycol, etc., or a combination thereof.

[0055] In a third aspect, the present invention provides a pharmaceutical container comprising the liquid formulation of the present invention. In some embodiments, the container is an injection container.

[0056] In a fourth aspect, the present invention provides a delivery device comprising a liquid or solid formulation of the present invention. In one embodiment, the delivery device of the present invention is provided in the form of a prefilled syringe comprising a liquid or solid formulation of the present invention, e.g., for intravenous, subcutaneous, intradermal or intramuscular injection, or intravenous infusion.

[0057] In a fifth aspect, the present invention provides a method for delivering an αPD-1 / IL2m immunoconjugate to a subject, such as a mammal, comprising administering to the subject a liquid formulation or solid formulation of the present invention, such as by using a delivery device such as a pre-filled syringe.

[0058] In a sixth aspect, the present invention provides a use of a liquid or solid formulation of the present invention for treating or preventing cancer in a subject; or for preparing a medicament or delivery device (e.g., a prefilled syringe) for treating or preventing cancer in a subject. Preferably, the cancer is a solid tumor or a hematological tumor, such as a gastrointestinal tumor or melanoma, such as colorectal cancer or colon cancer; for example, the cancer is a cancer resistant to PD-1 antibody treatment.

[0059] Other embodiments of the present invention will become apparent by reference to the detailed description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The preferred embodiments of the present invention described in detail below will be better understood when read in conjunction with the following drawings. For the purpose of illustrating the present invention, the drawings show presently preferred embodiments. However, it should be understood that the present invention is not limited to the precise arrangements and means of the embodiments shown in the drawings.

[0061] Figure 1 shows the trend graphs of the results of the formulation study. (A) Protein content (UV method); (B) Purity (SEC-HPLC method); (C) Purity (nrCE-SDS method); (D) Charge variant-acidic component (iCIEF method); (E) Charge variant-principal component (iCIEF method); and (F) Charge variant-basic component (iCIEF method).

[0062] Figure 2 shows the trend graphs of the results of the formulation confirmation study. (A)-(E) show the results of the 40°C stability study, and (F)-(J) show the results of the 25°C stability study. (A) and (F) show the trend graphs of purity (SEC-HPLC method); (B) and (G) show the trend graphs of purity (nrCE-SDS method); (C) and (H) show the trend graphs of charge variant-acidic component (iCIEF method); (D) and (I) show the trend graphs of charge variant-principal component (iCIEF method); and (E) and (J) show the trend graphs of charge variant-basic component (iCIEF method).

[0063] Figure 3 shows the in vivo efficacy of the immunoconjugate 2149 protein. (A) shows a tumor volume change curve reflecting the anti-tumor effect of 2149 on MC38 tumors in mice; (B) shows a survival curve reflecting the anti-tumor effect of 2149 on MC38 tumors in mice; (C) shows the effect of 2149 on mouse body weight.

[0064] Figure 4 shows the in vivo efficacy of the immunoconjugate 2149 protein. (A) shows a tumor volume change curve reflecting the anti-tumor effect of 2149 on mouse B16F10 tumors; (B) shows a tumor volume change curve reflecting the anti-tumor effect of 2149 on mouse B16F10 tumors; (C) shows a survival curve reflecting the anti-tumor effect of 2149 on mouse B16F10 tumors; (D) shows the effect of 2149 on mouse body weight.

[0065] Figure 5 shows a comparison of the in vivo efficacy of the control molecule 2061 and the immunoconjugate protein 2149. (A) shows a curve of changes in individual mouse tumor volume reflecting the anti-tumor effects of 2061 and 2149 against B16F10 tumors in mice; (B) shows a survival curve reflecting the anti-tumor effects of 2061 and 2149 against B16F10 tumors in mice; (C) shows the effects of 2061 and 2149 on mouse body weight.

[0066] FIG6 shows a schematic structural diagram of the αPD-1 / IL2m immunoconjugate of the present invention. [0066.1] [Corrected 06.01.2025 according to Rule 91] Figure 7 shows a schematic diagram of the sequence alignment.

[0067] Detailed Description of the Invention

[0068] 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 in this specification, because the methods and conditions can be varied. In addition, the terminology used herein is only for describing specific embodiments and is not intended to be limiting.

[0069] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present invention, the following terms are defined below.

[0070] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0071] The term "and / or" when used to link two or more alternatives should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0072] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0073] As used herein, the term "antibody" refers to a polypeptide comprising at least light and heavy chain immunoglobulin variable regions that specifically recognize and bind to an antigen. The term encompasses various antibody structures, including but not limited to monoclonal antibodies, chimeric or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0074] As used herein, a "whole antibody" (used interchangeably with "full-length antibody," "complete antibody," and "intact antibody") refers to a protein molecule comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains: CH1, CH2, and CH3. Therefore, an antibody heavy chain typically comprises, from N-terminus to C-terminus: a heavy chain variable region, heavy chain constant domains CH1, CH2, and CH3. Herein, the C-terminal region of the heavy chain located at the C-terminus of the CH1 domain and comprising the CH2 and CH3 domains is also referred to as the Fc region or Fc fragment. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. Therefore, an antibody light chain typically comprises, from N-terminus to C-terminus: a light chain variable region and a light chain constant domain, CL. The variable regions of the heavy and light chains of antibodies pair to form an antigen-binding domain that is responsible for binding to the antigen. The constant regions of the heavy and light chains are not directly involved in binding the antibody to the antigen, but exhibit various effector functions.

[0075] As used herein, the term "anti-PD-1 half-antibody" refers to a monovalent antigen-binding polypeptide comprising one heavy chain and one light chain of a full-length anti-PD-1 antibody. In such a half-antibody, typically, the VH-CH1 region of the heavy chain pairs with the VL-CL region of the light chain to form a Fab fragment that specifically binds to the PD-1 antigen. The heavy chain Fc region is covalently linked to the C-terminus of the CH1 domain via an immunoglobulin hinge region. Therefore, an anti-PD-1 half-antibody is a polypeptide comprising a Fab fragment that specifically binds to PD-1 fused to an Fc region.

[0076] As used herein, 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 the C-terminus. Typically, the fusion polypeptide comprises, from the N-terminus to the C-terminus, a mutant IL-2 polypeptide, an optional linker sequence, a hinge region, and an Fc region.

[0077] As used herein, an αPD-1 / IL2m immunoconjugate refers to an immunoconjugate comprising, consisting essentially of, or consisting of (i) an anti-PD-1 half antibody and (ii) a mutant IL-2 fusion polypeptide. An exemplary structure of an αPD-1 / IL2m immunoconjugate is shown in Figure 6. As shown in Figure 6, in the αPD-1 / IL2m immunoconjugate, 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 and dimerizes with the Fc region of the anti-PD-1 heavy chain. It should be understood that the antibody constant region contained in the immunoconjugate of the present invention can belong to any class or subclass based on its sequence and can be a native sequence constant region or a variant sequence constant region.

[0078] In this article, the class and subclass of antibody constant region (such as Fc region) refer to the classification determined by constant region sequence. Generally, antibody light chain constant region can be divided into two types (called kappa (κ) and lambda (λ)) based on its amino acid sequence. Antibody heavy chain constant region can be divided into main 5 kinds of different types based on its amino acid sequence: IgA, IgD, IgE, IgG and IgM, and several of these types can be further divided into subclasses, such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA2. Therefore, when mentioning that immunoconjugate has the Fc region of IgG class, it is meant that the immunoconjugate has the Fc region belonging to the IgG type based on its amino acid sequence. Similarly, when mentioning that immunoconjugate has kappa light chain constant region, it is meant that the immunoconjugate has the light chain constant region belonging to the kappa type based on its amino acid sequence.

[0079] In this article, the term "native sequence Fc region" encompasses naturally occurring various immunoglobulin Fc region sequences, such as various Ig subtypes and the Fc region sequences of their allotypes (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, human IgG heavy chain Fc region has an amino acid sequence extending to the heavy chain carboxyl terminus from Cys226 or from Pro230. However, the C-terminal terminal lysine (Lys447) in the Fc region may be present or absent. In further embodiments, human IgG heavy chain Fc region carries a hinge sequence or a partial hinge sequence of a natural immunoglobulin at the N-terminus, such as, according to EU numbering, a sequence from E216 to T225 or a sequence from D221 to T225.

[0080] As used herein, the term "variant sequence Fc region" refers to an Fc region polypeptide comprising modifications relative to a native sequence Fc region polypeptide. The modifications may be additions, deletions, or substitutions of amino acid residues. Substitutions may include naturally occurring amino acids and non-naturally occurring amino acids. The purpose of the modifications may be to alter the binding of the Fc region to its receptor and the effector functions thereby elicited.

[0081] As used herein, the terms "complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" are used interchangeably and refer to regions in the antibody variable domain that are highly variable in sequence and form structurally defined loops ("hypervariable loops") and / or contain antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. Herein, the CDRs of the antibody heavy and light chains are numbered sequentially from the N-terminus and are commonly referred to as CDR1, CDR2, and CDR3. The CDRs located within the antibody heavy chain variable domain are also referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the CDR sequences can be determined using a variety of methods well known in the art. A variety of schemes are known in the art that can be used to determine the CDR sequence within a given VH or VL amino acid sequence, including, for example, 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 ed., 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 a large number of crystal structures. Alternatively, a CDR may be identified based on having the same Kabat numbering position as a reference CDR sequence.Unless otherwise indicated, in the present invention, when referring to residue positions in the antibody heavy chain / light chain variable region, the numbering position is according to 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)).

[0082] In this article, " sequence identity " refers to the identical degree of sequence based on Nucleotide or amino acid by Nucleotide in a comparison window. " sequence identity percentage ratio " can be calculated in the following manner: the sequence of two optimal comparisons is compared in a comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G, I) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) position in two sequences to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce sequence identity percentage ratio. For determining the optimal comparison carried out of sequence identity percentage ratio, can realize by several ways known in the art, for example, use 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 algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of interest.

[0083] In this document, with respect to antibody sequences, the percentage of amino acid sequence identity is determined by optimally aligning the candidate antibody sequence with the given antibody sequence, preferably according to the Kabat numbering convention. In this document, without specifying a comparison window (i.e., the region of the target antibody to be compared), the comparison is performed over the entire length of the given antibody sequence.

[0084] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the biological function of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Typical conservative amino acid substitutions refer to substitutions of one amino acid with another amino acid having similar chemical properties (e.g., charge or hydrophobicity). Conservative substitution tables of functionally similar amino acids are well known in the art.

[0085] As used herein, the term "immunoconjugate formulation" refers to a preparation that is in a form that allows the biological activity of the immunoconjugate as the active ingredient to be effectively exerted and does not contain other components that are unacceptably toxic to the subject to whom the preparation is to be administered. Such preparations are generally sterile. Typically, a pharmaceutically acceptable excipient is included in the preparation. A "pharmaceutically acceptable" excipient is an agent that can be reasonably administered to a subject mammal so that an effective dose of the active ingredient used in the preparation can be delivered to the subject. The concentration of the excipient is adapted to the mode of administration, for example, it may be acceptable for injection.

[0086] As used herein, a "stable" immunoconjugate formulation refers to an immunoconjugate in the formulation that maintains an acceptable degree of physical stability and / or chemical stability after storage under specific conditions. Although the immunoconjugate contained in the immunoconjugate formulation may not maintain 100% of its chemical structure after storage for a specific period of time, the immunoconjugate formulation is generally considered "stable" if about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of the immunoconjugate structure or function is maintained after storage for a specific period of time. In some embodiments, the immunoconjugates of the present invention substantially retain their physical and chemical stability after storage.

[0087] A variety of analytical techniques are known in the art that can be used to determine the stability of proteins, 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 time can be selected based on the expected shelf life of the formulation. Alternatively, accelerated stability tests or high temperature stress forced experiments can be used. In some embodiments, stability testing is performed by subjecting the formulation to various stress tests. These tests can represent extreme conditions that the formulated formulation may encounter during manufacture, storage, or transportation, or conditions that may accelerate the instability of the immunoconjugate in the formulation during non-manufacturing, storage, or transportation. For example, the formulated immunoconjugate formulation can be filled into glass vials to test the stability of the immunoconjugate under high temperature stress.

[0088] After a period of storage, if the preparation shows no aggregation, precipitation, turbidity and / or denaturation; or shows very little aggregation, precipitation, turbidity and / or denaturation, the immunoconjugate can be considered to have "maintained its physical stability" in the preparation. Aggregation of immunoconjugates in the preparation can potentially lead to an increased immune response in the patient, thereby causing safety issues. Therefore, it is necessary to minimize or prevent aggregation of the immunoconjugates in the preparation. Light scattering can be used to measure visible aggregates in the preparation. SEC can be used to measure soluble aggregates in the preparation. In addition, the stability of the preparation can be indicated by visual inspection of the appearance, color and / or clarity of the preparation, or by detecting the turbidity of the preparation by the OD350nm method, or by measuring the purity of the preparation by the non-reduced CE-SDS method. In one embodiment, the stability of the preparation is measured by measuring the percentage of immunoconjugate protein monomers in the preparation after storage at a specific temperature for a specific time, wherein the higher the percentage of immunoconjugate protein monomers in the preparation, the higher the stability of the preparation.

[0089] As used herein, the expression "immunoconjugate protein monomer" (or "immunoconjugate monomer" or "monomer") in relation to formulation stability refers to a single intact functional immunoconjugate protein molecule. An immunoconjugate protein monomer is distinguished from a protein aggregate composed of a plurality of said protein monomers, and is also distinguished from an immunoconjugate protein fragment, such as a half-antibody portion or an IL-2 mutant fusion protein portion that is part of an immunoconjugate protein. Monomeric forms of immunoconjugate proteins can be distinguished from aggregate forms of immunoconjugate proteins by SEC-HPLC analysis. Monomeric forms of immunoconjugate proteins can also be distinguished from fragment forms of immunoconjugate proteins by non-reducing CE-SDS analysis.

[0090] An "acceptable degree" of physical stability can mean that at least about 92% of the immunoconjugate protein monomers are detected in the formulation after storage at a specific temperature for a specific time. In some embodiments, an acceptable degree of physical stability means that at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the immunoconjugate protein monomers remain in the formulation 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 more. When evaluating physical stability, the specific temperature at which the pharmaceutical formulation is stored can be any temperature between about -80°C and about 45°C, for example, storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C, about 40°C, about 42°C, or about 45°C. For example, if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the immunoconjugate protein monomers are detected after storage at about 40°C ± 2°C for 2 weeks or 4 weeks, the pharmaceutical formulation is considered stable. The pharmaceutical formulation is considered stable if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the immunoconjugate protein monomer is detected after storage at about 25°C ± 2°C for 4 weeks, 2 months, or 3 months. The pharmaceutical formulation is considered stable if at least about 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the immunoconjugate protein monomer is detected after storage at about 5°C ± 3°C for 3 months.

[0091] After a period of storage, if the immunoconjugate protein in the formulation does not show significant chemical changes, the immunoconjugate protein can be considered to "maintain its chemical stability" in the formulation. Most chemical instabilities arise from the formation of covalently modified forms of the immunoconjugate protein (e.g., charge variants of the immunoconjugate protein). For example, basic variants can be formed by aspartic acid isomerization, N- and C-terminal modifications; acidic variants can be produced by deamidation, sialylation, and saccharification. Chemical stability can be assessed by detecting and / or quantifying chemically altered forms of the immunoconjugate protein. For example, the charge variants of the immunoconjugate protein in the formulation can be detected by imaging capillary isoelectric focusing (iCIEF). In one embodiment, the stability of the formulation is measured by determining the charge variant percentage change value of the immunoconjugate protein in the formulation after storage at a specific temperature for a specific time, wherein the smaller the change value, the higher the stability of the formulation.

[0092] An "acceptable level" of chemical stability can be indicated by a percentage change of no more than 50% in the charge variants (e.g., the principal component or acidic component or basic component) of the immunoconjugate protein in the formulation after storage at a specific temperature for a specific period of time, e.g., no more than 30%, no more than 20%. In some embodiments, an acceptable level of chemical stability can be indicated by a percentage change of no more than about 50%, 40%, 30%, 20%, or 15% in the charge variants of the principal component 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 more. When evaluating chemical stability, the pharmaceutical formulation can be stored at any temperature between about -80°C and about 45°C, for example, at 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 pharmaceutical formulation can be considered stable if the percent change in the principal component charge variant after storage at 5°C is less than about 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%. The pharmaceutical formulation can also be considered stable if the percent change in the principal component charge variant is less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% after storage at 25°C for 2 months. The pharmaceutical formulation can also be considered stable if the percent change in the principal component charge variant is less than about 50%, 40%, 30%, 20%, 10%, 5%, or 4% after storage at 40°C for 1 month.

[0093] The term "lyophilized preparation" refers to a composition obtained or obtainable by freeze-drying a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0094] The term "reconstituted formulation" refers to a liquid formulation obtained by dissolving and / or suspending a solid formulation (eg, a lyophilized formulation) in a physiologically acceptable solution.

[0095] The term "room temperature" as used herein refers to a temperature of 15°C to 30°C, preferably 20°C to 27°C, more preferably 25°C.

[0096] "Stress conditions" refers to an environment that is chemically and / or physically unfavorable for the immunoconjugate, which may lead to unacceptable destabilization of the immunoconjugate protein. "High temperature stress" refers to storing the immunoconjugate formulation at room temperature or even higher temperatures (e.g., 40°C ± 2°C) for a period of time. The stability of the immunoconjugate formulation can be examined by room temperature accelerated tests or high temperature stress forced tests.

[0097] As used herein, the term "parenteral administration" refers to administration other than enteral and topical administration, typically by injection or infusion, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion. In some embodiments, the immunoconjugate formulation of the present invention is administered to a subject parenterally. In one embodiment, the immunoconjugate formulation of the present invention is administered to a subject by subcutaneous, intradermal, intramuscular, or intravenous injection.

[0098] I. Immunoconjugate Formulations of the Invention

[0099] Herein, the formulation of the αPD-1 / IL2m immunoconjugate according to the present invention, also referred to as the "immunoconjugate formulation of the present invention" or "formulation of the present invention," refers to a preparation comprising the αPD-1 / IL2m immunoconjugate according to the present invention as an active ingredient and a pharmaceutically acceptable excipient. The formulation of the present invention not only enables the αPD-1 / IL2m immunoconjugate, as the active ingredient, to be formulated in a manner suitable for administration to a subject, but also maintains its stability during storage and subsequent use.

[0100] Therefore, in a first aspect, the present invention provides a liquid formulation comprising an αPD-1 / IL2m immunoconjugate, comprising:

[0101] (i) αPD-1 / IL2m immunoconjugate;

[0102] (ii) a buffer;

[0103] (iii) stabilizers; and

[0104] (iv) Surfactants.

[0105] The formulations of the present invention can be prepared as liquid preparations in aqueous form, for example, for use in pre-filled syringes, or can be prepared as lyophilized preparations that are reconstituted (i.e., reconstituted) by dissolving and / or suspending in a physiologically acceptable solution immediately before use. In some embodiments, the formulations of the present invention are in the form of liquid preparations.

[0106] Properties of the liquid preparation of the present invention

[0107] Liquid preparation of the present invention can be stored stably for a long time, for example, at least 24 months or longer.In one embodiment, liquid preparation of the present invention can be at about-80 ℃ to about 45 ℃, for example, under the conditions of -80 ℃, about-30 ℃, about-20 ℃, about 0 ℃, about 5 ℃, about 25 ℃, about 35 ℃, about 38 ℃, about 40 ℃, about 42 ℃ or about 45 ℃, store 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, and be stable.

[0108] In some embodiments, the liquid formulations of the present invention are stable at about 2° C. to 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 are stable at room temperature, or for example, about 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 are stable at about 40° C. for at least 1 week, at least 2 weeks, at least 3 weeks, or at least 1 month.

[0109] In one embodiment, the stability of the formulation after storage can be indicated by detecting changes in the appearance, visible foreign matter, protein content, pH value, purity, charge variants and / or polysorbate surfactant content of the formulation. In one embodiment, the stability of the liquid formulation of the present invention can be detected in a forced experiment of high temperature stress, for example, after storage at 40°C ± 2°C for at least 1 week, 2 weeks or 1 month, or in an accelerated experiment, for example, after storage at 25°C ± 2°C for at least 1 month, 2 months or 3 months, or in a long-term experiment, for example, after storage at 5°C ± 3°C for at least 2 months or 3 months.

[0110] In one embodiment, after storage, the stability of the liquid preparation of the present invention is checked by visual inspection, wherein the liquid preparation of the present invention remains clear to slightly opalescent in appearance, is a colorless to light yellow liquid, and is free of foreign matter. In one embodiment, visual inspection under a clarity detector shows that no visible foreign matter is present in the preparation. In one embodiment, after storage, the stability of the liquid preparation of the present invention is checked by measuring the change in protein content, wherein, for example, by ultraviolet spectrophotometry (UV), the protein content rate of change is no more than 20%, preferably no more than 10%, such as 7-8%, more preferably no more than 5%, relative to the initial value of storage day 0. In one embodiment, after storage, the stability of the liquid preparation of the present invention is checked by measuring the change in pH value of the liquid preparation of the present invention, wherein, relative to the initial value of storage day 0, the change is no more than 0.3, preferably no more than 0.2, and more preferably no more than 0.1. In one embodiment, after storage, the stability of the liquid formulation of the present invention is tested by measuring the change in purity of the liquid formulation of the present invention, wherein the change in the purity of the immunoconjugate monomer by size exclusion high performance liquid chromatography (SEC-HPLC) relative to the initial value on storage day 0 is no more than 10%, such as no more than 5%, 4%, 3%, such as a change of no more than 1-2%, preferably no more than 1%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is tested by measuring the change in purity of the liquid formulation of the present invention, wherein the change in the purity of the immunoconjugate monomer by non-reduced sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS) is no more than 10%, such as no more than 5%, 4%, 3%, preferably no more than 2%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is tested by imaging capillary isoelectric focusing (iCIEF), wherein the sum of the change in the charge variants (main component, acidic component, and basic component) of the immunoconjugate relative to the initial value on storage day 0 is no more than 50%, such as no more than 40%, 30%, 20%, 10%, or 5%. In one embodiment, after storage, the stability of the liquid formulation of the present invention is detected by high performance liquid chromatography fluorescence detection method (HPLC-FLD method), wherein the polysorbate surfactant content changes by no more than 0.3 mg / ml, for example, no more than 0.2 mg / ml, or no more than 0.1 mg / ml relative to the initial value on storage day 0. Preferably, after storage of the formulation, the polysorbate 80 surfactant content is between 0.1 mg / ml and 0.5 mg / ml, for example, between 0.2 mg / ml and 0.4 mg / ml, preferably about 0.3 mg / ml.

[0111] In one embodiment, the formulation is stable after storage, e.g., after storage at 2-8°C for at least 24 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ± 2°C for 1 month, preferably having one or more of the following characteristics:

[0112] (i) the preparation has a purity greater than 90%, preferably greater than 95%, 96%, or 97% as measured by SEC-HPLC;

[0113] (ii) the preparation has a purity greater than 90%, preferably greater than 92%, 94%, 96%, or 98% as measured by non-reduced CE-SDS;

[0114] (iii) the sum of the changes in the charge variant components (main component, acidic component, and basic component) of the immunoconjugate in the formulation relative to the initial value on storage day 0, as measured by the iCIEF method, does not exceed 50%, for example, does not exceed 40%, 30%, 20%, 10%, or 5%;

[0115] (iv) the change in the polysorbate 80 content of the formulation relative to the initial value on storage day 0 as measured by HPLC-FLD is no more than 0.3 mg / ml, e.g., no more than 0.2 mg / ml, 0.10 mg / ml, or 0.05 mg / ml; and

[0116] (v) The relative binding activity of the immunoconjugate to the IL-2 receptor and PD-1 in the formulation, as measured by ELISA, decreases by no more than 30%, for example, 80%-110%, for example, about 70%, about 80%, about 90%, about 100%, or about 110%, relative to the initial value on storage day 0. The SEC-HPLC method, non-reduced CE-SDS method, iCIEF method, HPLC-FLD method, and ELISA method used to determine the stability of the formulation can be performed as known in the art or as described in the Examples.

[0117] The liquid preparation of the present invention can remain stable under vibration and freeze-thaw conditions. In one embodiment, the stability of the preparation under vibration and freeze-thaw conditions can be indicated by detecting changes in the appearance, visible foreign matter, protein content, pH value, purity, charge variants and / or polysorbate surfactant content of the preparation. In one embodiment, the stability of the liquid preparation of the present invention can be detected in an vibration experiment, for example, after 1 day, 2 days or 3 days of vibration at a speed of 650 rpm at room temperature in the dark. In one embodiment, the stability of the liquid preparation of the present invention can be detected in a freeze-thaw experiment, for example, after 1, 2, 3, 4, 5 or 6 freeze-thaw cycles consisting of -20°C freezing and 5°C ± 3°C thawing.

[0118] The room temperature accelerated test, high temperature stress test and long-term test for examining the stability properties of the preparation of the present invention can be carried out in a manner known in the art or in a manner described in the examples.

[0119] Components of the liquid preparation of the present invention

[0120] (i) αPD-1 / IL2m immunoconjugate

[0121] The αPD-1 / IL2m immunoconjugate contained in the formulation of the present invention comprises (i) an anti-PD-1 half-antibody and (ii) a mutant IL-2 fusion polypeptide. The anti-PD-1 half-antibody according to the present invention comprises a Fab fragment that specifically binds to PD-1 fused to an Fc region, and the mutant IL-2 fusion polypeptide according to the present invention comprises a mutant IL-2 polypeptide fused to an Fc region. Mutant IL-2 polypeptides and αPD-1 / IL2m immunoconjugates suitable for use in the present invention are disclosed in PCT / CN2022 / 120265, which is hereby incorporated by reference in its entirety.

[0122] The components of the αPD-1 / IL2m immunoconjugate of the present invention, the anti-PD-1 half-antibody and the mutant IL-2 fusion polypeptide, and an exemplary αPD-1 / IL2m immunoconjugate are described in detail below. Those skilled in the art will understand that, unless the context clearly indicates otherwise, any combination of any technical features described in these sections is within the scope of the present invention, and the αPD-1 / IL2m immunoconjugate of the present invention may include any such combination of features.

[0123] Mutant IL-2 polypeptide

[0124] The mutant IL-2 fusion polypeptides according to the present invention comprise a mutant IL-2 polypeptide fused to an Fc region. In some embodiments, the mutant IL-2 polypeptides according to the present invention have mutations that weaken binding affinity for the IL-2Rβγ receptor at the IL-2-IL-2Rβγ binding interface compared to wild-type IL-2; and have a shortened B'C' loop region that improves protein expression and / or purity.

[0125] Herein, wild-type "interleukin-2" or "IL-2" refers to a parent IL-2 protein that serves as a template for introducing a mutation or combination of mutations of the present invention, preferably a naturally occurring IL-2 protein, such as a natural IL-2 protein derived from humans or non-human primates, including unprocessed (e.g., signal peptide not removed) and processed (e.g., signal peptide removed) forms. A full-length natural human IL-2 sequence including a 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, a natural human IL-2 protein (uniprot: P60568) with the mutation C125S introduced at position 125 belongs to the wild-type IL-2 of the present invention. An example of a wild-type human IL-2 protein comprising a C125S mutation is shown in SEQ ID NO: 3. Preferably, the wild-type IL-2 sequence has at least 85%, 95%, or even at least 96%, 97%, 98%, or 99% or higher amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1 or 2 or 3.

[0126] [Corrected 06.01.2025 according to Rule 91] In this document, when referring to amino acid positions in IL-2 polypeptides or IL-2 sequence segments, reference is made to the wild-type human IL-2 protein (also known as IL-2 WT) is identified by the amino acid sequence of SEQ ID NO:3. The corresponding amino acid positions in other IL-2 proteins or polypeptides (including full-length sequences or truncated fragments) can be identified by amino acid sequence alignment with SEQ ID NO:3. Therefore, in the present invention, unless otherwise indicated, amino acid positions of IL-2 proteins or polypeptides are those numbered according to SEQ ID NO:3. For example, reference to "F42" refers to the phenylalanine residue F at position 42 of SEQ ID NO:3, or to the amino acid residue at the corresponding position in another IL-2 polypeptide sequence. At the same time, for ease of understanding, when the mutant IL-2 of the present invention involves truncation or deletion of certain specific segments (for example, the sequence of the B'C' loop region, i.e., 11 amino acid residues at positions 73-83 of SEQ ID NO: 3), if necessary, the region can be visually inspected and gaps can be introduced to ensure that when the mutant IL-2 is aligned with SEQ ID NO: 3, the deleted or truncated segment with the gap added is in the corresponding position region and has the corresponding amino acid residue numbering as the corresponding segment in SEQ ID NO: 3. For example, for a mutant IL-2 polypeptide (SEQ ID NO: 4) comprising a B'C' loop sequence truncated to 7 amino acid residues (AGDASIH), four gaps can be introduced into the truncated B'C' loop region by visual inspection, such that when the mutant IL-2 polypeptide is aligned with SEQ ID NO: 3, the truncated B'C' loop region with the gaps introduced is in a corresponding positional segment with the B'C' loop sequence of SEQ ID NO: 3, and has amino acid residue numbering of positions 73-83, as shown in Figure 7 below.

[0127] Sequence alignment for amino acid position determination can be performed using the Basic Local Alignment Search Tool available at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi using default parameters.

[0128] In this article, the amino acid mutation in the IL-2 mutant can be an amino acid substitution, deletion, insertion, and / or addition. Any combination of substitution, deletion, insertion, and addition can be performed to obtain a final mutant protein construct with desired properties (e.g., reduced IL-2Rα binding affinity and / or improved drugability and / or weakened IL-2Rβγ). For example, an alanine residue can be deleted at position 1 of the full-length human IL-2, or one or more amino acids can be deleted in the B'C' loop region to shorten the length of the loop region. For another example, the B'C' loop region sequence of wild-type IL-2 can be replaced in whole or in part with a different sequence (e.g., the B'C' loop of IL-15) to obtain a B'C' loop region sequence with a shortened length.

[0129] When referring to IL-2 muteins herein, single amino acid substitutions are described as follows: [original amino acid residue / position / substituted amino acid residue]. For example, a substitution of lysine at position 35 with glutamic acid can be represented as K35E. When multiple alternative amino acid substitutions (e.g., D, E) are possible at a given position (e.g., K35), the amino acid substitution can be represented as: K35D / E. Accordingly, single amino acid substitutions can be linked by a plus sign (+) or a minus sign (-) to represent combined mutations at multiple given positions. For example, a combined mutation at positions F42A, N88R, and S127E can be represented as: F42A+N88R+S127E, or F42A-N88R-S127E.

[0130] Herein, the sequence difference 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 as the number of different amino acids between the two. In one embodiment, the IL-2 mutant polypeptide according to the present invention has at least 85%, 86%, 87%, 88%, 89% identity with the wild-type IL-2 polypeptide, preferably more than 90% identity, for example at least 95%, 96% or 97% identity. In other embodiments, in addition to the specifically specified mutations 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 may also have no more than 15 mutations, for example 1-10, or 1-5 mutations, for example, 0, 1, 2, 3, 4 mutations, between the wild-type polypeptide. In one embodiment, the remaining mutations may be conservative substitutions.

[0131] As used herein, the terms "B'C' Loop" or "B'C' loop region" or "B'C' loop sequence" are used interchangeably and refer to the connecting sequence between the B helix and the C helix of the IL-2 protein. The B'C' loop sequence of an IL-2 protein can be determined by crystal structure analysis of IL-2 (e.g., PDB: 2ERJ). For the purposes of the present invention, the B'C' loop sequence refers to the sequence connecting the residue at position 72 and the residue at position 84 in the IL-2 polypeptide according to the numbering of SEQ ID NO: 3. In the wild-type IL-2 proteins of SEQ ID NOs: 1, 2, and 3, the connecting sequence includes A73-R83, a total of 11 amino acids. Accordingly, as used herein, the term "shortened loop region" or "shortened B'C' loop region" refers to a mutant protein having a B'C' loop sequence of reduced length relative to the wild-type IL-2 protein, i.e., the length of the connecting sequence between amino acid residues aa72 and aa84 is shortened according to the numbering of SEQ ID NO: 3. A "shortened loop region" can be achieved by replacing or truncating the loop sequence. The replacement or truncation can occur in any region or portion of the B'C' loop sequence. For example, the replacement or truncation can be a replacement of the loop region A73-R83 sequence (e.g., replacement with the IL-15 B'C loop region) or a truncation of one or more amino acid residues from the C-terminus of the sequence to obtain a B'C' loop region sequence of less than 10, 9, 8, 7, 6, or 5 amino acids in length. For another example, the replacement or truncation can be a replacement of the loop region Q74-R83 sequence or a truncation of one or more amino acid residues from the C-terminus of the sequence. After performing the replacement or truncation, if necessary, single amino acid substitutions can be further introduced into the loop region sequence, such as amino acid substitutions for eliminating glycosylation, and / or back mutations, to further improve the performance of the mutant protein, such as druggability. Therefore, in this article, the shortened B'C' loop region after mutation can be described by the sequence between the residue at position 72 and the residue at position 84 after the introduction of the mutation.

[0132] In this article, "IL-2Rα binding interface" mutations refer to mutations that occur at amino acid sites 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 mutations particularly refer to mutations in the amino acid residues 35-72 region of IL-2. Preferably, the IL-2 protein comprising the mutation has reduced or eliminated IL-2Rα binding compared to the corresponding protein before the introduction of the mutation.

[0133] In this article, "IL-2βγ binding interface" mutations refer to mutations that occur at amino acid sites where IL-2 interacts with IL-2Rβγ (ie, CD122 and CD132). These interacting amino acid sites can be determined by crystal structure analysis of IL-2 and its receptor complex (e.g., PDB:2ERJ). In some embodiments, the mutations refer in particular to mutations in the amino acid residues 12-20, amino acid residues 84-95, and amino acid residues 126-130 regions of IL-2. Preferably, the IL-2 protein comprising the mutation has a weakened IL-2Rβγ binding compared to the corresponding protein before the introduction of the mutation.

[0134] In some embodiments, the mutant IL-2 polypeptide according to the present invention comprises the following mutations compared to wild-type IL-2 (preferably human IL-2, more preferably IL-2 comprising the sequence of SEQ ID NO: 3):

[0135] (i) having a mutation that weakens the binding to the IL-2Rβγ receptor at the IL-2 binding interface, particularly at at least one position selected from positions 88, 127 and / or 130;

[0136] as well as

[0137] (ii) a shortened B'C' loop region (i.e., the sequence connecting amino acid residues aa72 and aa84), preferably, the shortened loop region has a length of less than 10, 9, 8, 7, 6, or 5 amino acids, and preferably is 7 amino acids in length; preferably, the shortened B'C' loop region results in improved protein expression and / or purity,

[0138] The amino acid positions are numbered according to SEQ ID NO: 3.

[0139] Preferably, the IL-2Rβγ binding interface mutation comprises one or more or a combination of the following mutations:

[0140] N88D, N88R, S127E, S130R, N88R+S130R, N88R+S127E.

[0141] Preferably, the shortened B'C' loop region has the sequence A(Q / G)SKN(F / I)H, or more preferably, has a B'C' loop sequence (ie, the sequence connecting aa72 to aa84) selected from the group consisting of: AQSKNFH; AGSKNFH; AQSANFH; and AQSANIH.

[0142] Optionally, the mutant IL-2 polypeptide of the present invention may further comprise a mutation at the IL-2 and IL-2Rα binding interface, particularly at positions 35 and / or 42, that eliminates or reduces binding affinity to the IL-2Rα receptor. Preferably, the IL-2Rα binding interface mutation comprises mutations K35E and / or F42A.

[0143] In some embodiments, the IL-2 mutant polypeptide according to the present invention, relative to wild-type IL-2, comprises:

[0144] (i) N88R+S130R;

[0145] N88D;

[0146] N88R;

[0147] F42A+N88R+S127E; or

[0148] K35E+N88R+S127E; and

[0149] (ii) B'C' loop sequence AGDASIH or AQSKNFH;

[0150] and optionally (iii) T3A.

[0151] In a preferred embodiment, the mutant IL-2 polypeptide comprises: (i) T3A+N88R+S130R; and (ii) the B'C' loop sequence AGDASIH. Preferably, the mutant IL-2 comprises the amino acid sequence of SEQ ID NO:4, or has at least 85%, 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto.

[0152] Anti-PD-1 half-antibody

[0153] In one embodiment, the anti-PD-1 half antibody 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 of SEQ ID NO: 8; and three light chain CDR sequences (i.e., LCDR1, LCDR2, and LCDR3) contained in the light chain variable region sequence of SEQ ID NO: 15. In some embodiments, the CDRs are defined according to Chothia, AbM, Kabat, IMGT, or any combination thereof, for example, according to Kabat or Chothia, or a combination thereof. In some embodiments, the CDRs are defined according to the CDR sequences defined by the North scheme. In a preferred embodiment, the light chain CDRs (LCDRs) are CDR sequences defined according to the Kabat scheme; and the heavy chain CDRs (HCDRs) are CDR sequences defined according to a combination of the Kabat and Chothia schemes.

[0154] In one embodiment, the anti-PD-1 half-antibody according to the present invention comprises:

[0155] - HCDR1, HCDR2 and HCDR3 comprising or consisting of the amino acid sequences of SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively; and

[0156] - LCDR1, LCDR2 and LCDR3 comprising or consisting of the amino acid sequences shown in SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

[0157] In one embodiment, the anti-PD-1 half antibody comprises: a heavy chain variable region sequence of 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 of 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 half antibody comprises: a heavy chain variable region of SEQ ID NO: 8 and a light chain variable region of SEQ ID NO: 15.

[0158] Fc region of αPD-1 / IL2m immunoconjugate

[0159] The αPD-1 / IL2m immunoconjugate according to the present invention comprises (i) an anti-PD-1 half-antibody and (ii) a mutant IL-2 fusion polypeptide. Preferably, the anti-PD-1 half-antibody and the mutant IL-2 fusion polypeptide are properly paired via their respective Fc regions and the hinge region located N-terminally to the Fc region to form the immunoconjugate according to the present invention.

[0160] In one embodiment, the Fc region of the mutant IL-2 fusion polypeptide and the anti-PD-1 heavy chain can be an Fc region of the IgG class, or an Fc region of the IgG1, IgG2, IgG3 or IgG4 subclass. In one embodiment, the Fc region comprises a knob-in-hole mutation to promote the correct pairing of the anti-PD-1 half antibody with the mutant IL-2 fusion polypeptide. In one embodiment, the Fc region of the mutant IL-2 fusion polypeptide comprises a knob mutation, and the Fc region of the anti-PD-1 heavy chain comprises a hole mutation. In one embodiment, the knob mutation comprises mutations T366W and S354C (EU numbering), and the hole mutation comprises mutations T366S, L368A, Y407V and Y349C (EU numbering).

[0161] Exemplary αPD-1 / IL2m immunoconjugates

[0162] In some preferred embodiments, the αPD-1 / IL2m immunoconjugate 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, wherein:

[0163] - the fusion polypeptide of the mutant IL-2 comprises the amino acid sequence of SEQ ID NO: 7, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto;

[0164] - the anti-PD-1 heavy chain comprises the amino acid sequence of SEQ ID NO: 14, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto; and

[0165] - The anti-PD-1 light chain comprises the amino acid sequence of SEQ ID NO: 20, or a sequence having at least 90%, 91%, 92%, 93%, 94%, 96%, or 96% amino acid sequence identity thereto.

[0166] In some more preferred embodiments, the αPD-1 / IL2m immunoconjugate according to the present invention comprises or consists of a mutant IL-2 fusion polypeptide having the amino acid sequence of SEQ ID NO: 7, an anti-PD-1 heavy chain having the amino acid sequence of SEQ ID NO: 14, and an anti-PD-1 light chain having the amino acid sequence of SEQ ID NO: 20.

[0167] In some embodiments, the αPD-1 / IL2m immunoconjugate of the present invention used in the formulation of the present invention is produced by recombinant expression and purified from HEK293 cells (including HEK293 cells and cells modified based on HEK293 cells, such as HEK293T, HEK293F, and HEK293E cells); or from CHO cells (including CHO cells and cells modified based on CHO cells, such as CHO-S, CHO-dhfr-, CHO / DG44, and ExpiCHO cells). The αPD-1 / IL2m immunoconjugate of the present invention can be recombinantly expressed using methods well known in the art for recombinant expression of immunoconjugate proteins, or using the 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 / diafiltration, and combinations thereof. Such purification techniques can be used to provide immunoconjugate drug substances with sufficient reproducibility and moderate purity for formulation. As shown in the Examples, the immunoconjugates used in the liquid formulations of the present invention exhibit significant anti-tumor activity with reduced side effects.

[0168] The amount of αPD-1 / IL2m immunoconjugate included in the antibody formulations of the present invention may vary depending on the specific intended properties of the formulation, the specific environment, and the specific purpose for which the formulation is to be used. In some embodiments, the formulation of the invention is a liquid formulation, which may contain about 0.5-150 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 of the αPD-1 / IL2m immunoconjugate of the invention.

[0169] (ii) Buffer

[0170] A buffer solution or buffer solution is typically a solution composed of a "weak acid and its conjugate base" buffer pair or a "weak base and its conjugate acid" buffer pair having a buffering effect, for example, 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).

[0171] In this article, buffer refers to a substance that can maintain the pH of a solution within an acceptable range. In some cases, a buffer can be a weak acid or weak base compound constituting a buffer or a buffered solution, which can achieve the desired buffering effect when mixed with a suitable amount of its corresponding "conjugate acid / base" (the amount depends on the predetermined desired pH value), or when adding a strong acid or a strong base to provide the required amount of the corresponding "conjugate acid / base" in situ. In other cases, a buffer can be a buffer system comprising a weak acid and its salt or a weak base and its salt. For example, for a histidine buffer, histidine (solid) can be titrated with an acid (e.g., hydrochloric acid or acetic acid) to prepare it; or a suitable amount of histidine and its salt (e.g., hydrochloride or acetate) can be prepared to provide the desired pH value. Therefore, in this article, when mentioning that the preparation of the present invention comprises a specific weak acid or weak base buffer, it should be understood that this also covers the case where the preparation comprises a buffer system consisting of the weak acid and its salt or the weak base and its salt. For example, when referring to a formulation comprising histidine or a histidine buffer, it is understood that this also encompasses the case where the formulation comprises a buffer system consisting of histidine and a salt thereof (such as, but not limited to, hydrochloride or acetate).

[0172] As used herein, when a liquid formulation is referred to as containing a certain concentration of a buffer, this refers to the combined amount of the weak acid or weak base and its corresponding conjugate acid / conjugate base present as the buffer in the liquid formulation. For a histidine buffer, one skilled in the art can readily calculate this concentration based on the amount of histidine and / or its salt used to prepare the formulation.

[0173] In some embodiments, the buffer used in the formulations of the present invention can control the pH of the formulations of the present invention in a pH range of about 5.0 to about 6.5, for example, a pH of about 5.0 to about 5.5, preferably a pH of about 5.0. In some specific embodiments, the formulations of the present invention have a pH of about 5.0, about 5.2, about 5.4, about 5.6, about 5.8, about 6.0, about 6.2, about 6.4. In some specific embodiments, the formulations of the present invention have a pH of 5.0 ± 0.2.

[0174] In one embodiment, the buffer used in the formulation of the present invention can be selected from the group consisting of histidine buffer, glutamate buffer, phosphate buffer, acetate buffer, citrate buffer and tris (Tris) buffer and combinations thereof. In some embodiments, the (total) concentration of the buffer in the formulation of the present invention is from about 0.5 mM to about 200 mM, from about 5 mM to about 50 mM or from about 5 mM to about 20 mM.

[0175] In some embodiments, the buffer for the formulation of the present invention is a histidine buffer, or a histidine buffer system comprising histidine and its salts, especially a histidine buffer system comprising histidine and histidine hydrochloride. In some embodiments, the liquid formulation comprises about 5mM to about 50mM histidine. In one embodiment, the liquid formulation comprises about 10mM to about 30mM, for example, about 10mM, about 15mM, about 20mM, about 25mM, about 30mM histidine. In one embodiment, the liquid formulation of the present invention comprises about 10mM histidine. In another embodiment, the liquid formulation of the present invention comprises a combination of about 0.75mM histidine and about 9.25mM histidine hydrochloride, or a combination comprising about 0.12mg / ml histidine and about 1.94mg / ml histidine hydrochloride.

[0176] (iii) Stabilizer

[0177] Suitable stabilizers for use in the present invention may be selected from saccharides, amino acids, and combinations thereof. Furthermore, the stabilizer of the present invention may further comprise a chelating agent, such as EDTA.

[0178] The saccharide as the stabilizer can be a disaccharide, a trisaccharide and a polysaccharide, and the saccharide can be selected from, but not limited to: sucrose, dextrose, lactose, maltose, trehalose, cyclodextrin, maltodextrin and dextran. In one embodiment, the saccharide as the stabilizer is sucrose and / or trehalose.

[0179] In some embodiments, the saccharide as a stabilizer is present in the liquid preparation of the present invention at a concentration of about 50mM to about 500mM, preferably about 100mM to about 400mM, for example, about 100mM, about 150mM, about 200mM, about 250mM, about 300mM, about 350mM, or about 400mM. In some embodiments, the saccharide as a stabilizer is sucrose, and preferably, its concentration in the liquid preparation of the present invention is about 30mg / ml to about 120mg / ml, for example, about 50mg / ml, about 60mg / ml, about 70mg / ml, about 80mg / ml, about 90mg / ml, about 100mg / ml, or about 120mg / ml. In some embodiments, the content of saccharide (e.g., sucrose) can be further adjusted in conjunction with the osmotic pressure of the liquid preparation.

[0180] In some embodiments, in addition to carbohydrates, the formulations of the present invention further comprise amino acids as stabilizers. The amino acids used as stabilizers can be selected from, but are not limited to, homocysteine, cysteine, cystathionine, methionine, and arginine. Preferably, the amino acid is methionine. In this case, the concentration of the carbohydrate as a stabilizer is from about 50 mM to about 500 mM, preferably from 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, or about 400 mM, and the concentration of the amino acid as a stabilizer is from about 10 mM to about 200 mM, preferably from 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.

[0181] In one embodiment, the liquid formulation of the present invention comprises 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, preferably about 80 mg / ml.

[0182] In one embodiment, 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 a preferred embodiment, the liquid formulation of the present invention comprises about 70 mg / ml sucrose and about 7.49 mg / ml methionine.

[0183] (iv) Surfactants

[0184] As used herein, the term "surfactant" refers to organic substances with an amphiphilic structure; that is, they are composed of groups with opposite solubility tendencies, usually a hydrocarbon chain for oil solubility and an ionic group for water solubility.

[0185] In one embodiment, the surfactant in the liquid formulation of the present invention is a nonionic surfactant, for example, an alkyl poly(ethylene oxide). Specific nonionic surfactants that can be included in the formulation of the present invention include, for example, polysorbate surfactants such as polysorbate 20, polysorbate 80, polysorbate 60, or polysorbate 40; Pluronic, etc. In a preferred embodiment, the liquid formulation of the present invention includes polysorbate 80 as a surfactant.

[0186] The amount of surfactant contained in the antibody formulation of the present invention can change with the specific purpose characteristics of the formulation, the specific environment and the specific purpose of using the formulation. In preferred embodiments, the formulation can contain 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, about 0.8 mg / ml of polysorbate surfactant (e.g., polysorbate 80). Preferably, the formulation includes about 0.1 mg / ml to about 0.5 mg / ml of polysorbate 80, more preferably about 0.3 mg / ml of polysorbate 80.

[0187] (v) Other excipients

[0188] The liquid preparation of the present invention may or may not include other excipients. For example, the liquid preparation of the present invention may also include a chelating agent, such as EDTA, preferably, the concentration of EDTA is from about 0.01 mg / ml to about 0.1 mg / ml, preferably from about 0.01 mg / ml to about 0.05 mg / ml, such as 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 preparation of the present invention may also include a tension regulator. The tension regulator may be selected from the group consisting of sodium acetate, sodium lactate, sodium chloride, potassium chloride and calcium chloride.

[0189] These and further known pharmaceutical excipients and / or additives suitable for use in the formulations of the present invention are well known in the art and are listed, for example, in "The Handbook of Pharmaceutical Excipients, 4th edition, Rowe et al., ed., American Pharmaceuticals Association (2003); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, ed., Lippincott Williams & Wilkins (2005)".

[0190] II. Use of the Preparation

[0191] The preparations of the present invention comprising the immunoconjugates of the present invention can be used to treat or prevent cancer in a subject. In some embodiments, the cancer may be a solid tumor or a hematological tumor. In some embodiments, the cancer is a gastrointestinal tumor or melanoma, such as colon cancer or colorectal cancer. In some embodiments, the tumor is a tumor or cancer that is resistant to a known drug, such as a known anti-PD-1 antibody, such as a refractory tumor or cancer. In some embodiments, the cancer is a cancer characterized by elevated protein levels and / or nucleic acid levels (e.g., elevated expression) of PD-1, PD-L1, and / or PD-L2.

[0192] The present invention also provides the use of the formulation of the present invention in the preparation of a medicament, wherein the medicament is used to deliver the immunoconjugate according to the present invention to a mammal, or to treat, prevent or ameliorate one or more of the above-mentioned cancers. Preferably, the mammal is a human.

[0193] The formulations of the present invention can be administered to a subject or patient in a variety of ways. For example, administration can be by infusion or by syringe. Thus, in one aspect, the present invention provides a pharmaceutical container (e.g., an injection container) and a delivery device (e.g., a syringe, such as a prefilled syringe) comprising the formulation of the present invention. By such administration, the patient will receive an effective amount of the conjugate of the present invention, i.e., an amount sufficient to treat, ameliorate, or prevent the disease or condition of interest.

[0194] The therapeutic effect may include a reduction in physiological symptoms. The optimal effective amount and concentration of the immunoconjugate for any particular subject will depend on a variety of factors, including the patient's age, weight, health status and / or sex, the nature and extent of the disease, the activity of the specific immunoconjugate, its clearance rate by the body, and also any other possible treatments administered in combination with the formulation. The effective amount delivered for a specific situation can be determined within the clinician's discretion.

[0195] The following examples are described to assist understanding of the present invention. The examples are not intended to, and should not be interpreted in any way as, limiting the scope of protection of the present invention.

[0196] Table 1. Abbreviations and full names Example

[0197] To provide an αPD-1 / IL2m immunoconjugate injection formulation with long-term stability and ensure quality control within its shelf life (at least 24 months), a formulation screening study was designed to investigate the effects of different buffer pH values, buffer systems, and excipients on the stability of the αPD-1 / IL2m immunoconjugate formulation. The materials and methods used in the study are as follows:

[0198] Materials and methods

[0199] 1.1 Immunoconjugate Samples Used in the Formulation Studies of the Present Invention

[0200] The αPD-1 / IL2mut immunoconjugate protein 2149 was prepared and purified as described in PCT application number PCT / CN2022 / 120265.

[0201] Specifically, the heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) of the anti-PD-1 antibody and the IL-2m-Fc polypeptide chain (SEQ ID NO: 3, consisting of the IL-2m polypeptide linked to the N-terminus of the IgG1 Fc Knob chain via a linker) were separately constructed into the pcDNA3.1 vector. A mixture of the three plasmid vectors generated by the construction and 3 times the mass of PEI was used to co-transfect HEK293 cells (e.g., to transfect 3 ml of HEK293, 1 ug of heavy chain + 1 ug of light chain + 1 ug of IL-2m-Fc polypeptide chain + 9 ug of PEI was required). From the culture of the transfected cells, the supernatant containing the expressed immunoconjugate protein was harvested and purified. Table 2 below lists information on the immunoconjugate samples used in the following examples.

[0202] Table 2. Sample information

[0203] 1.2. Reagents and Materials Used in the Preparation Studies of the Present Invention

[0204] Table 3. Reagent information

[0205] Note: N / A means not applicable.

[0206] Table 4. Material information

[0207] 1.3. Instruments and Equipment Used in the Preparation Studies of the Present Invention

[0208] Table 5. Instrument and equipment information

[0209] 1.4. Test items and methods for preparation stability

[0210] The immunoconjugate preparations were tested for the following items: (1) appearance and presence of visible foreign matter; (2) protein content of the preparations by ultraviolet light (UV); (3) insoluble particulate matter content of the preparations; (4) pH value of the preparations; (5) purity of the preparations by size exclusion high performance liquid chromatography (SEC-HPLC), expressed as the percentage of the area of ​​the immunoconjugate monomer to the sum of all peak areas; (6) purity of the preparations by non-reduced sodium dodecyl sulfate capillary electrophoresis (nrCE-SDS). (7) Determination of the purity of the preparation, expressed as the percentage of the area of ​​the immunoconjugate monomer to the sum of all peak areas; (8) Determination of the polysorbate-80 content in the preparation by high performance liquid chromatography with fluorescence detection (HPLC-FLD); (9) Determination of the relative binding activity of the immunoconjugate in the preparation to PD-1 antigen and IL-2R by immunoassay, for example, direct ELISA.

[0211] Visible foreign matter detection

[0212] According to the method described in the Pharmacopoeia of the People's Republic of China (2015 edition, General Chapter 0904 of Part Four "Inspection Method for Visible Foreign Matter", Beijing: China Medical Science and Technology Press, 2015), a clarity tester (produced by Tianjin Tianda Tianfa, model YB-2) was used to inspect visible foreign matter in the samples.

[0213] Insoluble particulate matter determination

[0214] The light obscuration method was used to determine the content of insoluble particles in the sample.

[0215] Protein content determination

[0216] The protein content in the samples was determined using a UV / visible spectrophotometer (Biglunatic, Unchained Labs, USA).

[0217] pH determination

[0218] The pH value of the samples was measured using a SevenCompact pH meter (Mettler, Switzerland, model S220).

[0219] Purity determination (SEC-HPLC method)

[0220] Size exclusion chromatography was used for detection, where the chromatographic column was a TSKgel G3000Swxl (7.8×300mm, 5μm) analytical column, the guard column was a TSKgel guardcolumn Swxl (6.0×40mm, 7μm), the mobile phase was 20mmol / L phosphate buffer + 400mmol / LNaClO4, pH 6.8, the injection plate temperature was 10℃, the column temperature was 25℃, the injection volume was 50μl, the flow rate was 0.5ml / min, the acquisition time was 30 minutes, and an FLD fluorescence detector was used with a PMT value of 7, an excitation wavelength of 285nm, and an emission wavelength of 340nm.

[0221] Dilute the sample to be tested to 1.0 mg / ml with mobile phase to serve as the test solution. Dilute the formulation buffer using the same method as above to serve as the blank solution. Inject 50 μl of each of the blank solution and the test solution into the liquid chromatograph and begin testing.

[0222] Purity determination (non-reduced CE-SDS method)

[0223] Capillary gel electrophoresis was used for detection. The capillary was uncoated, 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 column was flushed with 0.1 mol / L sodium hydroxide, 0.1 mol / L hydrochloric acid, ultrapure water, and electrophoresis gel at 70 psi. Dilute the sample to be tested to 1.0 mg / ml with an appropriate amount of ultrapure water. Place 50 μl of this diluted sample in a 1.5 ml centrifuge tube and add 45 μl of pH 6.5 sample buffer (weigh 0.32 g of citric acid monohydrate and 2.45 g of disodium hydrogen phosphate dodecahydrate in 45 ml of ultrapure water, dilute to 50 ml to prepare citric acid-phosphate buffer. Accurately measure 200 μl of this buffer, add 80 μl of 10% (w / v) sodium lauryl sulfate solution, add water to 1 ml, and mix thoroughly) and 5 μl of 250 mmol / L NEM solution (weigh 62 mg of N-ethylmaleimide and dissolve in 2 ml of ultrapure water). Mix thoroughly, heat at 70 ± 2°C for 10 ± 2 minutes, cool to room temperature, and transfer to a sample vial as the test sample solution. Prepare a blank solution by taking the same volume of formulation buffer as the test sample and following the same procedure as above. Sample injection conditions: -5 kV for 20 seconds; separation voltage: -10 kV for 35 minutes. The capillary column temperature was controlled at 25°C, and the detection wavelength was 220 nm.

[0224] Charge variant determination (iCIEF method)

[0225] Imaging capillary isoelectric focusing (iCIEF) was used for detection. The inner diameter of the capillary was 100 μm and the total length was 5 cm. Before sample electrophoresis, the capillary column was rinsed with 0.5% methyl cellulose solution (hereinafter referred to as MC solution) and ultrapure water, respectively. A vacuum injection method was used, with a pre-focusing voltage and time of 1.5 kV for 1 minute, a focusing voltage and time of 3 kV for 8 minutes, an injection time of 55 seconds, a sample plate temperature of 10°C, and a detection wavelength of 280 nm. The cathodic stabilizer was a 500 mmol / L arginine solution, and a 0.5% MC solution was used to reduce the adhesion between the protein and the capillary. After concentrating the test sample, dilute it with water to 1.0 mg / ml. Add 77 μl of a premix (3 M urea-0.5% MC, ampholyte, cathode stabilizer, pI marker) to 20 μl of the diluted sample solution and mix thoroughly to prepare the test sample solution. Inject the sample for analysis and calculate the contents of the main component, acidic components, and basic components using the area normalization method.

[0226] High performance liquid chromatography with fluorescence detection (HPLC-FLD)

[0227] The polysorbate 80 content of the sample was determined using HPLC-FLD. The chromatographic column was a SUPELCO Knitted Reaction Coil (5m×0.50mm ID), and the mobile phase was a mixture of 0.15mol / L sodium chloride, 0.05mol / L Tris, 5% acetonitrile, 5.0μM NPN, and 15ppm Brij23, pH 8.0. Detection conditions: column temperature 30°C, injection volume 10μl, acquisition time 3 minutes, flow rate 1.5ml / min, excitation wavelength 350nm, emission wavelength 420nm. After the run, the polysorbate 80 content in the finished product was calculated using the standard curve method.

[0228] Biological activity detection (direct ELISA method)

[0229] The following antigens were used:

[0230] IL-2 receptor: Recombinant Human CD25-His Avi purchased from KACTUS, product number: CD5-HM425,

[0231] PD-1: Recombinant human PD-1 purchased from Sinobiological, Catalog No.: 10377-H08H,

[0232] The relative binding activities of the IL-2 end and the anti-PD-1 end of the immunoconjugate protein were detected according to the ELISA assay procedure described below.

[0233] Dilute the antigen to 0.5 μg / ml with PBS, 100 μl / well, and coat on a 96-well ELISA plate overnight at 4°C. After washing, add blocking solution (2% BSA-PBST, 300 μl / well) and block at 37°C for 2 hours. Dilute the immunoconjugate protein to 300 μg / ml (IL2 receptor) or 10 μg / ml (PD-1) with 2% BSA-PBST, and dilute 3-fold to the 11th concentration. Add the serially diluted test sample at 100 μl / well to the ELISA plate from which the blocking solution has been discarded. Set up a negative control by adding only 100 μl of diluent (2% BSA-PBST) to each well and incubate in a 37°C constant temperature incubator for 60 minutes. After washing the plate, add HRP-conjugated goat anti-human IgG-Fc fragment (BETHYL, USA, product number A80-104P) diluted in 2% BSA-PBST as the secondary antibody (120,000 times (IL2 receptor) or 100,000 times (PD-1) dilution, 100 μl / well), and react at 37°C for 30 minutes. After washing the plate, add 100 μl of TMB color development solution to each well. After 10 minutes of color development, add 100 μl of 1 mol / L H2SO4 to each well to terminate the reaction. Use 620 nm as the reference wavelength and measure the OD value at 450 nm. Use the concentration value of each concentration gradient sample as the horizontal axis and the OD450 nm-OD620 nm value of each gradient sample as the vertical axis. Use Prism four-parameter fitting to calculate EC 50 Reflects the binding activity of immunoconjugate protein to antigen.

[0234] Example 1. Prescription study

[0235] 1.1 Experimental plan

[0236] Prepare buffers containing 1.55 mg / ml histidine and 80.00 mg / ml sucrose, and adjust the pH to 5.0, 5.5, 6.0, and 6.5 with dilute hydrochloric acid, respectively. These are F1-F4. Prepare 1.55 mg / ml histidine, 50.00 mg / ml sucrose, and 7.49 mg / ml methionine, and adjust the pH to 5.0 with dilute hydrochloric acid to obtain F5. Prepare 1.55 mg / ml histidine, 30.00 mg / ml sucrose, and 17.42 mg / ml arginine, and adjust the pH to 5.0 with dilute hydrochloric acid to obtain F6. Prepare 1.55 mg / ml histidine, 50.00 mg / ml sucrose, and 7.49 mg / ml methionine, and adjust the pH to 5.0 with dilute hydrochloric acid to obtain F7. Ultrafiltration of the 2149 protein was performed into various buffers, and the protein content was adjusted to approximately 1.0 mg / ml. Polysorbate 80 was added to a final concentration of 0.3 mg / ml. Edetate disodium was added to F7 to a final concentration of 0.03 mg / ml. Detailed prescription information is provided in Table 6. Filter and dispense into vials, stopper, and cap. Stability testing of these samples was performed at 40°C ± 2°C. The specific protocol is provided in Table 7.

[0237] Table 6. Alternative prescription information table

[0238] Note: Use hydrochloric acid to adjust pH.

[0239] Table 7. Experimental plan

[0240] Note: (1)√ indicates sampling at that time. (2) After sampling at the above time points, all samples were first frozen at -40℃ and thawed as needed for testing.

[0241] 1.2 Judgment Criteria

[0242] Based on the understanding of the product and the precision of the instruments and methods, the judgment criteria for whether the sample test value has not changed compared with the initial value are set, as shown in Table 8.

[0243] Table 8. Criteria for judging if quality has not changed

[0244] Note: “*” indicates that this standard is only applicable to prescription determination experiments.

[0245] 1.3 Experimental Results

[0246] The results of the formulation study are detailed in Table 9, and the trend of change is shown in Figure 1. The results show that after being placed at 40℃±2℃ for 4 weeks, the appearance and visible foreign matter of the samples of each formulation were qualified; the protein content and pH did not change significantly. As the formulation pH increased, the content of SEC aggregates increased, and the decrease in the main peak content increased. As the formulation pH decreased, the content of non-reduced CE fragments increased, and the decrease in the main peak content increased slightly. The charge variant-acidic component of each sample increased significantly, the main component decreased significantly, and the alkaline component increased significantly. Among F1-F4, F1 was better. Compared with F1, F5-F7, the polysorbate-80 content of F1 and F6 decreased significantly. The purity and charge variant of F6 changed significantly.

[0247] Based on the results of purity and charge variant stability, F5 was selected for formulation confirmation experiments.

[0248] Table 9. Prescription study results

[0249] Example 2. Prescription Confirmation Study

[0250] 2.1 Experimental plan

[0251] Based on the F5 formulation selected in the formulation research experiment, this experiment mainly investigated the effects of excipients (sucrose, histidine, polysorbate 80, and methionine) on the stability of the 2149 protein. Detailed formulation information is shown in Table 10. In order to adjust the osmotic pressure of the preparation, the sucrose content was appropriately increased.

[0252] Prepare the prescribed buffer according to Table 10. Ultrafilter the 2149 protein into the prescribed solution and adjust the protein content to 1.0 mg / ml. Add polysorbate 80 to a final concentration of 0.3 mg / ml. Filter and dispense into vials, stopper, and cap. Stability testing of these samples was performed under the conditions in Table 11.

[0253] Table 10. Alternative Prescription Information

[0254] Note: Use hydrochloric acid to adjust pH.

[0255] Table 11. Stability study plan

[0256] Note: After sampling at the above time points, the samples were first placed in a -40℃ refrigerator for testing and thawed and sent for testing as needed.

[0257] 2.2 Judgment criteria

[0258] The specific judgment criteria are shown in Table 8.

[0259] 2.3 Experimental Results

[0260] The results of the formulation confirmation stability study are detailed in Tables 12 and 13, with the trends shown in Figure 2. The results showed that the sample appearance and visible foreign matter were acceptable at all time points. Protein content and pH remained unchanged. The main SEC peak decreased by 1.0% under high temperature conditions. The charge variant acidic component increased under accelerated and high temperature conditions, the charge variant main component decreased under accelerated and high temperature conditions, and the charge variant basic component increased slightly under high temperature conditions. Polysorbate 80 content remained unchanged. IL-2 activity decreased significantly after four weeks at high temperature, while PD-1 activity remained unchanged. Protein quality remained unchanged under oscillation and freeze-thaw conditions.

[0261] Table 12. Long-term, accelerated, and high-temperature stability study results

[0262] As shown in Table 12, after four weeks at 40°C ± 2°C, the main peak in SEC decreased by 1.0%, the main peak in non-reduced 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 three months at 25°C ± 2°C, the non-reduced 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 test items after three months at 5°C ± 3°C.

[0263] Table 13. Results of shaking and freeze-thaw studies

[0264] in conclusion

[0265] Combining the results of the formulation research and formulation confirmation experiments, the confirmed formulation was selected as the final formulation for 2149. To avoid the use of hydrochloric acid to adjust the pH during production, the buffer system was adjusted to histidine and histidine hydrochloride. The formulation for 2149 is: 1.0 mg / ml recombinant αPD-1 / IL2m immunoconjugate 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, pH 5.0.

[0266] Example 3. In vivo efficacy study of αPD-1 / IL2m immunoconjugate

[0267] The αPD-1 / IL2m immunoconjugate of the present invention can specifically bind to human PD-1 at its αPD-1 end to block the binding of PD-1 to PD-L1, thereby releasing the immune brake mechanism. At the same time, its IL-2m end can specifically bind to the IL-2 receptor on T cells or NK cells, thereby activating and amplifying T or NK cells, thereby enhancing the effect of PD-1 antibody immunotherapy.

[0268] To demonstrate the in vivo efficacy of the αPD-1 / IL2m immunoconjugate of the present invention, the anti-tumor efficacy of the immunoconjugate (2149) of the present invention was determined by inoculating hPD-1 knock-in mice with MC38 cells (a mouse colon cancer cell line, obtained from Shanghai Heyuan Biotechnology Co., Ltd.). SPF female hPD-1 knock-in mice (purchased from Shanghai Southern Model Organisms) with a certificate number of NO.20170010006762 were used in the experiment.

[0269] MC38 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in PBS (1×) to a cell concentration of 5×10 6On day 0, 0.2 ml of cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish an MC38 tumor-bearing mouse model.

[0270] Eight days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (8 mice per group). The dosage and method of administration are shown in Table 14.

[0271] Table 14: Grouping, dosage and method of administration for in vivo experiments

[0272] *: h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.

[0273] h-IgG, 2149, 10 mg / kg, 2149, 20 mg / kg, and 2149, 40 mg / kg were administered at concentrations of 4 mg / ml, 1 mg / ml, 2 mg / ml, and 4 mg / ml, respectively, and were administered once a week for a total of 3 times (QWx3). Dosing was performed on days 8, 15, and 22 after MC38 cell inoculation, and the tumor volume and body weight of the mice were monitored twice a week, as shown in Figures 3A-B. Tumors exceeding 2000 mm 3 The mice were euthanized and the whole experiment was monitored until the end of the 61st day. Because the tumor volume of some groups of mice exceeded 2000mm 3 The animals were euthanized, so the relative tumor inhibition rate (TGI%) was calculated on day 36 after inoculation using the following formula: TGI% = 100% * (tumor volume of the control group – tumor volume of the treatment group) / (tumor volume of the control group – tumor volume of the control group before administration). Tumor volume measurement: The maximum long axis (L) and maximum wide axis (W) of the tumor were measured using a vernier caliper, and the tumor volume was calculated using the following formula: V = L * W 2 / 2. Body weight was measured using an electronic balance.

[0274] Tumor growth and survival curves are shown in Figures 3A and 3B. The antitumor effects of 2149 at different doses were dose-dependent, with complete tumor regression in mice in the 20 mg / kg and 40 mg / kg groups. This superiority is also reflected in the survival curves in Figure 3B, where 100% of mice in these two groups experienced tumor regression, while only 2 of 8 mice in the 10 mg / kg group experienced complete tumor regression. Tumor inhibition rates are shown in Table 15. On day 36 after inoculation, compared to the h-IgG group, the tumor inhibition rates for 2149 at 10 mg / kg, 2149 at 20 mg / kg, and 2149 at 40 mg / kg were 84%, 103%, and 103%, respectively. Mouse body weights were also measured (Figure 3C), showing no significant differences on day 36 after inoculation.

[0275] Table 15. Antitumor efficacy statistics

[0276] *Complete response rate: The tumor completely disappears and the tumor volume is 0.

[0277] To demonstrate the in vivo efficacy of immunoconjugate 2149, hPD-1 knock-in mice were inoculated with PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) to determine the anti-tumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant fusion protein (2149). SPF female hPD-1 knock-in mice (purchased from Shanghai Southern Model Organisms) were used. The study was conducted with a quality control (SPF) standard of 20170010007909.

[0278] B16F10 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in PBS (1×) to a cell concentration of 2.5×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0279] Six days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (8 mice per group). The dosage and method of administration are shown in Table 16.

[0280] Table 16: Grouping, dosage and administration method of in vivo experiments

[0281] *: h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656;

[0282] & : IBI308 is the parental anti-PD-1 monoclonal antibody (Sintilimab).

[0283] h-IgG, IBI308 (20 mg / kg), IBI308 (40 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, and were administered once weekly for a total of three doses (QW x 3). Dosing was performed on days 8, 15, and 22 after B16F10 cell inoculation. Tumor volume and body weight were monitored twice weekly, as shown in Figures 4A-B, until day 22. Because B16F10 cells readily metastasize and induce mouse mortality, the relative tumor inhibition rate (TGI%) was calculated on day 15 post-inoculation using the following formula: TGI% = 100% * (control group tumor volume – treatment group tumor volume) / (control group tumor volume – control group pre-dose tumor volume). Tumor volume was measured using a vernier caliper to measure the longest axis (L) and widest axis (W). Tumor volume was calculated using the following formula: V = L * W 2 / 2. Weight is measured using an electronic balance. Tumors larger than 2000 mm 3 The mouse will be euthanized.

[0284] The mouse tumor growth curves are shown in Figures 4A and 4B. In the PD1-resistant model, IBI308 showed little efficacy. However, the 20 mg / kg and 40 mg / kg groups of 2149 exhibited some anti-tumor effect, with the high-dose group exhibiting superior anti-tumor effects compared to the low-dose group. This effect was also reflected in the mouse survival curves, with two mice in the high-dose group experiencing complete tumor regression at the end of the experiment (Figure 4C and Table 17).

[0285] The tumor inhibition rates are shown in Table 17. On day 15 post-inoculation, compared to the h-IgG 40 mg / kg group, the tumor inhibition rates of IBI308 20 mg / kg, IBI308 40 mg / kg, 2149 20 mg / kg, and 2149 40 mg / kg were 29%, 27%, 82%, and 86%, respectively. Simultaneous measurement of mouse body weight (Figure 4D) showed no significant differences in mouse body weight on day 22 post-inoculation.

[0286] Table 17. Antitumor efficacy statistics

[0287] To demonstrate that the in vivo efficacy of the αPD-1 / IL2m immunoconjugate 2149 is superior to that of the control drug PD-1-IL2v (molecule number 2061, sequence source US20180326010A1, also see the sequence listing), hPD-1 knock-in mice were inoculated with PD-1 antibody-resistant B16F10 cells (mouse melanoma cell line, ATCC CRL-6475) to determine the anti-tumor efficacy of the bifunctional PD-1 antibody and IL-2 mutant molecule fusion protein (2149). The experiment used SPF-grade female hPD-1 knock-in mice (purchased from Shanghai Southern Model Organisms), with a certificate number of NO.20170010008942.

[0288] B16F10 cells were routinely subcultured for subsequent in vivo experiments. Cells were collected by centrifugation and resuspended in PBS (1×) to a cell concentration of 2.5×10 6 On day 0, 0.2 ml of the cell suspension was subcutaneously inoculated into the right abdominal region of hPD-1 knock-in mice to establish a B16F10 tumor-bearing mouse model.

[0289] Eight days after tumor cell inoculation, the tumor volume of each mouse was measured and the mice were divided into groups (7 mice per group). The dosage and method of administration are shown in Table 18.

[0290] Table 18. Grouping, dosage and administration method of in vivo experiments

[0291] *: h-IgG is an isotype control antibody purchased from Equitech-Bio, lot number 161206-0656.

[0292] 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. Dosing was performed once weekly for three doses (QW x 3). Dosing was performed on days 8, 15, and 22 after B16F10 cell inoculation. Tumor volume and body weight were monitored twice weekly until day 33, as shown in Figure 5A. Because B16F10 cells are prone to metastasis and can cause mouse mortality, the relative tumor inhibition rate (TGI%) was calculated on day 19 after inoculation using the following formula: TGI% = 100% * (control group tumor volume – treatment group tumor volume) / (control group tumor volume – control group pre-dose tumor volume). Tumor volume measurement: Vernier calipers were used to measure the maximum longitudinal axis (L) and maximum width axis (W) of the tumor. Tumor volume was calculated using the following formula: V = L*W2 / 2. Body weight was measured using an electronic balance. Tumors exceeding 2000 mm 3 If more than half of the mice in a group have died, the tumor growth curve for the entire group at that time point will not be displayed.

[0293] Tumor inhibition results are shown in Table 19. On day 19 post-inoculation, compared to the h-IgG 40 mg / kg group, tumor 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. TGIs for 2061 20 mg / kg and 40 mg / kg were not calculated because mice died after the first dose, resulting in significant weight loss.

[0294] We also performed a statistical analysis of mouse survival (Figure 5B). Comparing the maximum administered / tolerated doses of 2061 and 2149 in this experiment, we can see that more mice survived at the 40 mg / kg dose of 2149 and the 10 mg / kg dose of 2061. For 2149, three of seven mice experienced complete tumor regression, while only one mouse achieved complete tumor regression with 2061. Simultaneous mouse body weight measurements (Figure 5C) showed that on day 29 after inoculation, no weight loss was observed in the 2149 group at any dose, while mice in the 2061 group experienced weight loss. At the low dose (10 mg / kg), the average weight loss exceeded 5%. Deaths occurred at the medium dose (20 mg / kg) and high dose (40 mg / kg), with six deaths in each group out of seven mice. Details are shown in Table 12. 2149 was relatively safe, with no significant weight loss at low, medium, or high doses. Only one mouse died at the low and medium doses, while no mice died at the high dose. Furthermore, 2149 achieved a higher complete tumor remission rate than 2061 (Table 19). Therefore, 2149 is more effective and safer than 2061, with a wider therapeutic window.

[0295] Table 19. Antitumor efficacy statistics

[0296] Sequence Listing

Claims

1. An immunoconjugate liquid preparation, comprising: (i) immunoconjugates, (ii) a buffer, (iii) a stabilizer, and (iv) a surfactant, The immunoconjugate comprises: (a) an anti-PD-1 half antibody and (b) a mutant IL2 fusion polypeptide, in, The anti-PD-1 half antibody comprises a Fab fragment fused to an Fc region, and comprises three heavy chain CDR sequences contained in the heavy chain variable region sequence of SEQ ID NO: 8; and three light chain CDR sequences contained in the light chain variable region sequence of SEQ ID NO: 15; and wherein the mutant IL2 fusion polypeptide comprises a mutant IL-2 polypeptide fused to an Fc region, and the mutant IL-2 polypeptide comprises (i) N88R+S130R; and (ii) a B'C' loop sequence AGDASIH or AQSKNFH, and optionally (iii) T3A; The pH of the liquid preparation is about 5.0 to about 6.5, for example, about 5.0, about 5.5, about 6.0, or about 6.5, preferably about 5.

0.

2. The liquid formulation of claim 1, characterized in that the concentration of the immunoconjugate is about 0.5 mg / ml to about 150 mg / ml, preferably about 0.5 to about 10 mg / mL, such as 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 2, characterized in that the buffer comprises a histidine buffer, preferably, the buffer comprises about 5 mM to about 50 mM histidine, preferably about 10 mM to about 30 mM histidine, for example, about 10 mM, about 15 mM, about 20 mM, about 25 mM, or about 30 mM histidine.

4. The liquid preparation according to any one of claims 1 to 3, characterized in that the stabilizer comprises - a sugar, in particular sucrose, preferably in a concentration of about 50 mg / ml to about 120 mg / ml, such as about 80 mg / ml; or - a combination of sugars and amino acids, in particular a combination of sucrose and methionine, preferably, the concentration of sucrose 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, and the concentration of methionine is about 20 mM to about 80 mM, for example, about 30 mM, about 40 mM, about 50 mM, or about 60 mM.

5. The liquid preparation according to any one of claims 1 to 4, characterized in that the surfactant in the liquid preparation is selected from polysorbate surfactants, 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, such as 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 preparation according to any one of claims 1 to 6, characterized in that the liquid preparation comprises EDTA, preferably, the concentration of EDTA is about 0.01 mg / ml to about 0.1 mg / ml, 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 liquid formulation according to any one of claims 1 to 7, wherein the immunoconjugate comprises a knob-in-hole mutation, Preferably, the Fc region of the fusion polypeptide comprises a knob mutation, and the Fc region of the half antibody comprises a hole mutation.

9. The liquid preparation according to any one of claims 1 to 8, wherein the anti-PD-1 half antibody comprises a heavy chain variable region and a light chain variable region, wherein: The heavy chain variable region comprises the amino acid sequence of 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 of 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 half antibody 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.

10. The liquid formulation according to any one of claims 1 to 9, wherein 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.

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

12. The liquid preparation according to any one of claims 1 to 11, characterized in that the liquid preparation is an injection, preferably for subcutaneous injection or intravenous injection, or an infusion, such as for intravenous infusion.

13. The liquid preparation according to any one of claims 1 to 12, comprising: (i) about 1-50 mg / ml of the immunoconjugate; (ii) about 5-50 mM histidine; (iii) about 50-120 mg / ml sucrose, or about 30-100 mg / ml sucrose in combination with about 20-80 mM methionine; and (iv) about 0.1-0.5 mg / ml polysorbate 80; wherein the pH of the liquid formulation is about 5.0-5.5, preferably about pH 5.0; For example, the liquid formulation comprises (i) about 1-10 mg / ml of the immunoconjugate; (ii) about 10-30 mM histidine; (iii) about 70-90 mg / ml sucrose, or about 50-80 mg / ml sucrose in combination with about 40-60 mM methionine; and (iv) about 0.2-0.4 mg / ml of polysorbate 80; wherein the pH of the liquid formulation is about 5.0-5.5, preferably about pH 5.0; Alternatively, the liquid preparation comprises (i) about 1.0 mg / ml of the immunoconjugate; (ii) about 10 mM histidine; (iii) about 80 mg / ml sucrose, or a combination of about 70 mg / ml sucrose and about 50 mM methionine, and (iv) about 0.3 mg / ml polysorbate 80; wherein the pH of the liquid formulation is about 5.0-5.5, preferably about pH 5.0; Alternatively, the liquid preparation comprises (i) about 1.0 mg / ml of the immunoconjugate; (ii) about 0.12 mg / ml histidine and about 1.94 mg / ml histidine hydrochloride; (iii) about 70 mg / ml sucrose and about 7.49 mg / ml methionine, and (iv) about 0.3 mg / ml polysorbate 80; The pH of the liquid formulation is about 5.0-5.5, preferably about pH 5.

0.

14. The liquid preparation according to any one of claims 1 to 13, characterized in that The formulation is stable after storage, for example after storage at 2-8°C for at least 3 months, or after storage at room temperature for at least 3 months, or after storage at 40°C ± 2°C for 1 month, and preferably has one or more of the following characteristics: (i) the preparation has a purity of greater than 90%, 95%, 96%, or 97% as measured by SEC-HPLC; (ii) the preparation has a purity of greater than 90%, 92%, 94%, 96%, or 98% as measured by non-reduced CE-SDS; (iii) the sum of the changes in the charge variant components (main component, acidic component, and basic component) in the formulation relative to the initial value on storage day 0 as measured by the iCIEF method does not exceed 50%, such as 40%, 30%, 20%, 10%, or 5%; and (iv) the change in the polysorbate 80 content in the formulation relative to the initial value on storage day 0 as measured by HPLC-FLD method is no more than 0.3 mg / ml, such as no more than 0.2 mg / ml, 0.10 mg / ml, or 0.05 mg / ml.

15. A solid preparation obtained by solidifying the 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 the liquid preparation according to any one of claims 1 to 14 or the solid preparation according to claim 15.

17. A prefilled syringe comprising the liquid preparation according to any one of claims 1 to 14 or the solid preparation according to claim 15, for intravenous injection or intramuscular injection.

18. Use of the liquid preparation according to any one of claims 1 to 14 or the solid preparation according to claim 15 for preventing or treating cancer or for preparing a medicament for preventing or treating cancer, preferably, the cancer is a solid tumor or a blood tumor, such as a gastrointestinal tumor or a melanoma, such as colorectal cancer or colon cancer; for example, the cancer is a cancer resistant to PD-1 antibody treatment.

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