Stable pharmaceutical composition of mixed antibodies

Abstract

Provided is a pharmaceutical composition containing anti-CTLA-4 and anti-PD-1 mixed antibodies, which has an excellent effect in terms of the stability, the safety, and the therapeutic efficacy, thereby effectively solving the problems of poor stability and high toxicity of a pharmaceutical composition containing anti-CTLA-4 and anti-PD-1 mixed antibodies.

Description

Stable mixed antibody pharmaceutical composition

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on December 1, 2023, with application number CN2023116423306 and invention name “Stable mixed antibody pharmaceutical composition”, and the Chinese patent application filed with the China Patent Office on November 21, 2024, with application number CN2024116757918 and invention name “Stable mixed antibody pharmaceutical composition”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present disclosure relates to a stable pharmaceutical composition containing mixed antibodies of anti-PD-1 and anti-CTLA-4 and uses thereof, belonging to the field of biotechnology. Background Art

[0003] Programmed death receptor-1 (PD1 or PD-1) is a 288-amino acid protein receptor expressed on activated T and B cells, natural killer cells, and monocytes. Binding to the PD-1 ligands PD-L1 or PD-L2 activates the PD-1 inhibitory signal, thereby suppressing T cell-mediated immune responses against tumor cells. To counteract this PD-1 pathway-mediated suppression of anti-tumor immune responses, several companies have developed monoclonal antibodies that bind to human PD-1 and block the interaction between PD-1 and its ligands. Immune checkpoint therapies targeting the programmed death receptor 1 (PD-1) axis have led to breakthrough improvements in clinical responses in a variety of human cancers.

[0004] Cytotoxic T lymphocyte-associated protein 4 (CTLA4 or CTLA-4) is a member of the immunoglobulin superfamily and consists of an extracellular V region, a transmembrane region, and a cytoplasmic region. CTLA-4 shares homology with the co-stimulatory molecule receptor CD28 on the surface of T cells, and the two compete for binding to their ligands B7-1 (CD80) and B7-2 (CD86). These ligands are mainly expressed on the surface of antigen-presenting cells. CTLA-4 is usually expressed on the surface of regulatory T cells (Treg) and conventional T cells in an activated state. After binding to B7 molecules, it inhibits the activation of T cells, participates in the negative regulation of immune responses, acts as an immune checkpoint, and downregulates immune responses. Therefore, CTLA-4 plays a very important role in immune regulation.

[0005] ZPML265 is a hybrid antibody drug formulation composed of a recombinant humanized IgG1 monoclonal antibody targeting human CTLA-4 and a recombinant humanized IgG4 monoclonal antibody targeting human PD-1. These two different antibodies are produced by a single host cell. This hybrid antibody can simultaneously and specifically bind to CTLA-4 and PD-1, thereby blocking the immune checkpoint signaling pathways between CTLA-4 and B7-1 / B7-2, and between PD-1 and PD-L1. This relieves the inhibitory effects of these two pathways on T lymphocytes, restoring their functional activity and anti-tumor immune responses, thereby enabling the body to fight and kill tumors.

[0006] Compared to traditional monoclonal antibodies, hybrid antibodies offer improved safety and tolerability, are easy to administer, offer significant efficacy, and exhibit significantly superior efficacy compared to single antibodies. Furthermore, compared to bispecific antibodies, hybrid antibody combinations require less time to develop. Furthermore, the development of higher-concentration antibody formulations can shorten injection times and reduce injection volumes, increasing patient compliance. This can also reduce the frequency of antibody administration and improve production and storage efficiency. However, due to their large molecular weight and complex structure, antibody drugs are susceptible to degradation, aggregation, and undesirable chemical modifications, making them unstable. Hybrid antibodies, in particular, exist as a mixture of two antibodies at low concentrations. Developing high-concentration formulations presents the challenge of maintaining appropriate activity and delivery parameters at higher antibody or protein concentrations. Currently, there is an urgent need to develop stable pharmaceutical compositions at both low and high concentrations that are suitable for the production and administration of hybrid antibodies, maintain stability during storage and subsequent use, and achieve enhanced therapeutic efficacy.

[0007] SUMMARY OF THE INVENTION

[0008] The present disclosure aims to provide a pharmaceutical composition of mixed antibodies that is suitable for transportation and storage and has high long-term stability.

[0009] The present disclosure provides an aqueous pharmaceutical composition containing a mixed anti-CTLA-4 and anti-PD-1 antibody, wherein the pharmaceutical composition comprises the mixed antibody, a buffer, a stabilizer, and a surfactant.

[0010] In some embodiments, the heavy chain amino acid sequence of the anti-CTLA-4 antibody is shown in SEQ ID NO: 1, the light chain amino acid sequence of the anti-CTLA-4 antibody is shown in SEQ ID NO: 2, the heavy chain amino acid sequence of the anti-PD-1 antibody is shown in SEQ ID NO: 3, and the light chain amino acid sequence of the anti-PD-1 antibody is shown in SEQ ID NO: 4.

[0011] In some embodiments, the molar ratio of anti-CTLA-4 antibody to anti-PD-1 antibody in the antibody cocktail is about 1:2.

[0012] In some embodiments, the total concentration of the mixed antibodies in the pharmaceutical composition is about 20-200 mg / ml.

[0013] In some embodiments, the mixed antibody is at a low concentration in the pharmaceutical composition, for example, about 20-30 mg / ml, including but not limited to the following concentrations: about 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml or any value in between.

[0014] In some embodiments, the hybrid antibody is at a high concentration in the pharmaceutical composition, for example, about 100-200 mg / ml, including but not limited to the following concentrations: about 100 mg / ml, 105 mg / ml, 110 mg / ml, 115 mg / ml, 120 mg / ml, 125 mg / ml, 130 mg / ml, 135 mg / ml, 140 mg / ml, 145 mg / ml, 150 mg / ml, 155 mg / ml, 160 mg / ml, 165 mg / ml, 170 mg / ml, 175 mg / ml, 180 mg / ml, 185 mg / ml, 190 mg / ml, 195 mg / ml, 200 mg / ml or any value between any two numbers.

[0015] In some embodiments, the buffer is selected from acetic acid-sodium hydroxide, histidine-hydrochloric acid, and histidine-acetic acid, and its concentration is about 5-50 mM, preferably about 10-20 mM, and more preferably about 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM or any value in between any two numbers.

[0016] In some embodiments, the stabilizer is selected from sucrose or trehalose, and its concentration is about 50-100 mg / ml, preferably about 60-100 mg / ml, more preferably about 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml or any value in between.

[0017] In some embodiments, the surfactant is selected from polysorbate 20, polysorbate 80 and poloxamer 188, and its concentration is about 0.1-2 mg / ml, preferably about 0.1-1 mg / ml, more preferably about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml or any value in between.

[0018] In some embodiments, the pH of the pharmaceutical composition is in the range of about 4.0-6.0, preferably in the range of about 4.7-5.6, and more preferably in the range of about 5.0-5.4.

[0019] In some embodiments, the pharmaceutical composition further comprises an antioxidant and / or a diffusing agent.

[0020] In some embodiments, the antioxidant is selected from methionine and its concentration is about 0-50 mM, preferably about 5-50 mM, and more preferably about 10 mM.

[0021] In some embodiments, the spreading agent is selected from hyaluronidase, and its concentration is about 0-4000 IU / ml, preferably about 500-4000 IU / ml, and more preferably about 2000 IU / ml.

[0022] In some embodiments, the pharmaceutical composition may optionally further comprise arginine hydrochloride at a concentration of about 0-150 mM, preferably about 20-150 mM, and more preferably about 100 mM.

[0023] The low-concentration aqueous pharmaceutical composition disclosed herein comprises about 20-30 mg / ml of a mixed antibody; about 50-100 mg / ml of sucrose or about 50-100 mg / ml of trehalose; about 0.1-2 mg / ml of polysorbate 80 or about 0.1-2 mg / ml of polysorbate 20 or about 0.1-2 mg / ml of poloxamer 188; about 5-50 mM of acetic acid-sodium hydroxide or about 5-50 mM of histidine-hydrochloric acid or about 5-50 mM of histidine-acetic acid; wherein the pH of the aqueous pharmaceutical composition is about 4.0-6.0.

[0024] Further preferably, the low-concentration aqueous pharmaceutical composition described in the present disclosure comprises about 20-30 mg / ml of the mixed antibody; about 80-100 mg / ml of sucrose or about 80-100 mg / ml of trehalose; about 0.1-0.5 mg / ml of polysorbate 80 or about 0.1-0.5 mg / ml of polysorbate 20 or about 0.1-0.5 mg / ml of poloxamer 188; about 10-50 mM acetic acid-sodium hydroxide or about 10-50 mM histidine-hydrochloric acid or about 10-50 mM histidine-acetic acid; wherein the pH of the aqueous pharmaceutical composition is about 4.7-5.3.

[0025] The low-concentration aqueous pharmaceutical composition disclosed herein comprises a component selected from the following:

[0026] Aqueous pharmaceutical composition A: about 20 mg / ml of mixed antibody, about 50 mg / ml of sucrose, about 0.1 mg / ml of polysorbate 20, about 5 mM acetic acid-sodium hydroxide, pH about 4.0;

[0027] Aqueous pharmaceutical composition B: about 20 mg / ml of mixed antibody, about 100 mg / ml of sucrose, about 2 mg / ml of polysorbate 20, about 50 mM acetic acid-sodium hydroxide, pH about 5.3;

[0028] Aqueous pharmaceutical composition C: about 25 mg / ml of the mixed antibody, about 60 mg / ml of sucrose, about 0.3 mg / ml of polysorbate 20, about 10 mM acetic acid-sodium hydroxide, pH about 5.8;

[0029] Aqueous pharmaceutical composition D: about 25 mg / ml of the mixed antibody, about 70 mg / ml of sucrose, about 0.5 mg / ml of polysorbate 20, about 20 mM acetic acid-sodium hydroxide, pH about 5.2;

[0030] Aqueous pharmaceutical composition E: about 25 mg / ml of the mixed antibody, about 90 mg / ml of sucrose, about 0.2 mg / ml of polysorbate 20, about 10 mM acetic acid-sodium hydroxide, pH about 5.0;

[0031] Aqueous pharmaceutical composition F: about 25 mg / ml of the mixed antibody, about 100 mg / ml of sucrose, about 1 mg / ml of polysorbate 20, about 30 mM acetic acid-sodium hydroxide, pH about 6.0;

[0032] Aqueous pharmaceutical composition G: about 30 mg / ml of mixed antibody, about 80 mg / ml of sucrose, about 0.6 mg / ml of polysorbate 20, about 20 mM of acetic acid-sodium hydroxide, pH about 4.7;

[0033] Aqueous pharmaceutical composition H: about 30 mg / ml of mixed antibody, about 100 mg / ml of sucrose, about 0.4 mg / ml of polysorbate 20, about 40 mM of acetic acid-sodium hydroxide, pH about 5.6.

[0034] The high-concentration aqueous pharmaceutical composition disclosed herein comprises about 100-200 mg / ml of a mixed antibody; about 50-100 mg / ml of sucrose or about 50-100 mg / ml of trehalose; about 0.1-2 mg / ml of polysorbate 80 or about 0.1-2 mg / ml of polysorbate 20 or about 0.1-2 mg / ml of poloxamer 188; about 5-50 mM of acetic acid-sodium hydroxide or about 5-50 mM of histidine-hydrochloric acid or about 5-50 mM of histidine-acetic acid; wherein the pH of the aqueous pharmaceutical composition is about 4.0-6.0; and optionally, further comprises about 0-50 mM of methionine, and / or, about 0-4000 IU / ml of hyaluronidase, and / or, about 0-150 mM of arginine hydrochloride.

[0035] Further preferably, the high-concentration aqueous pharmaceutical composition described in the present disclosure comprises about 100-200 mg / ml of the mixed antibody; about 50-100 mg / ml of sucrose or about 50-100 mg / ml of trehalose; about 0.1-2 mg / ml of polysorbate 80 or about 0.1-2 mg / ml of polysorbate 20 or about 0.1-2 mg / ml of poloxamer 188; about 5-50 mM of acetic acid-sodium hydroxide or about 5-50 mM of histidine-hydrochloric acid or about 5-50 mM of histidine-acetic acid; about 5-50 mM of methionine; about 500-4000 IU / ml of hyaluronidase; wherein the pH of the aqueous pharmaceutical composition is about 4.0-6.0; and optionally, further comprises about 20-150 mM of arginine hydrochloride.

[0036] Still further preferably, the high-concentration aqueous pharmaceutical composition disclosed herein comprises about 100-200 mg / ml of the mixed antibody; about 60-90 mg / ml of sucrose or about 60-90 mg / ml of trehalose; about 0.2-1 mg / ml of polysorbate 80 or about 0.2-1 mg / ml of polysorbate 20 or about 0.2-1 mg / ml of poloxamer 188; about 10-20 mM acetic acid-sodium hydroxide or about 10-20 mM histidine-hydrochloric acid or about 10-20 mM histidine-acetic acid; about 10 mM methionine; about 2000 IU / ml of hyaluronidase; wherein the pH of the aqueous pharmaceutical composition is about 5.0-5.8; and optionally, further comprises about 100 mM arginine hydrochloride.

[0037] The high-concentration aqueous pharmaceutical composition of the present disclosure comprises a component selected from the following:

[0038] Aqueous pharmaceutical composition I: about 100 mg / ml of mixed antibody, about 50 mg / ml of sucrose, about 0.2 mg / ml of polysorbate 20, about 10 mM histidine-hydrochloric acid, pH about 5.0;

[0039] Aqueous pharmaceutical composition J: about 100 mg / ml of mixed antibody, about 82 mg / ml of sucrose, about 0.8 mg / ml of polysorbate 20, about 15 mM histidine-hydrochloric acid, pH about 5.4;

[0040] Aqueous pharmaceutical composition K: about 100 mg / ml of mixed antibody, about 90 mg / ml of sucrose, about 1.5 mg / ml of polysorbate 20, about 40 mM histidine-hydrochloric acid, pH about 5.6;

[0041] Aqueous pharmaceutical composition L: about 120 mg / ml of mixed antibody, about 80 mg / ml of sucrose, about 0.4 mg / ml of polysorbate 20, about 13 mM histidine-hydrochloric acid, pH about 5.0;

[0042] Aqueous pharmaceutical composition M: about 120 mg / ml of the mixed antibody, about 70 mg / ml of sucrose, about 0.6 mg / ml of polysorbate 20, about 10 mM of histidine-HCl, about 10 mM of methionine, pH about 5.6;

[0043] Aqueous pharmaceutical composition N: about 120 mg / ml of mixed antibody, about 63 mg / ml of sucrose, about 0.5 mg / ml of polysorbate 20, about 18 mM histidine-HCl, about 10 mM methionine, pH about 5.4;

[0044] Aqueous pharmaceutical composition O: about 120 mg / ml of the mixed antibody, about 90 mg / ml of sucrose, about 1 mg / ml of polysorbate 20, about 20 mM histidine-HCl, about 10 mM methionine, pH about 5.0;

[0045] Aqueous pharmaceutical composition P: about 150 mg / ml of the mixed antibody, about 100 mg / ml of sucrose, about 2 mg / ml of polysorbate 20, about 25 mM histidine-HCl, about 10 mM methionine, pH about 5.8;

[0046] Aqueous pharmaceutical composition Q: about 150 mg / ml of the mixed antibody, about 60 mg / ml of sucrose, about 0.5 mg / ml of polysorbate 20, about 18 mM histidine-HCl, about 10 mM methionine, pH about 5.0;

[0047] Aqueous pharmaceutical composition R: about 150 mg / ml of the mixed antibody, about 76 mg / ml of sucrose, about 0.8 mg / ml of polysorbate 20, about 30 mM histidine-HCl, about 10 mM methionine, pH about 5.4;

[0048] Aqueous pharmaceutical composition S: about 200 mg / ml of mixed antibody, about 90 mg / ml of sucrose, about 0.7 mg / ml of polysorbate 20, about 50 mM histidine-HCl, about 10 mM methionine, pH about 5.2;

[0049] Aqueous pharmaceutical composition T: about 200 mg / ml of mixed antibody, about 70 mg / ml of sucrose, about 1 mg / ml of polysorbate 20, about 15 mM histidine-HCl, about 10 mM methionine, pH about 5.4;

[0050] Aqueous pharmaceutical composition U: about 200 mg / ml of mixed antibody, about 82 mg / ml of sucrose, about 0.5 mg / ml of polysorbate 20, about 10 mM of histidine-HCl, about 10 mM of methionine, about 2000 IU / ml of hyaluronidase, pH about 5.4;

[0051] Aqueous pharmaceutical composition V: about 150 mg / ml of the mixed antibody, about 76 mg / ml of sucrose, about 0.8 mg / ml of polysorbate 20, about 18 mM of histidine-HCl, about 10 mM of methionine, and about 2000 IU / ml of hyaluronidase, with a pH of about 5.4;

[0052] Aqueous pharmaceutical composition W: about 150 mg / ml of the mixed antibody, about 70 mg / ml of sucrose, about 0.6 mg / ml of polysorbate 20, about 15 mM of histidine-HCl, about 10 mM of methionine, and about 2000 IU / ml of hyaluronidase, with a pH of about 5.2;

[0053] Aqueous pharmaceutical composition X: about 120 mg / ml of a mixed antibody, about 63 mg / ml of sucrose, about 0.5 mg / ml of polysorbate 20, about 13 mM of histidine-HCl, about 10 mM of methionine, and about 2000 IU / ml of hyaluronidase, with a pH of about 5.4;

[0054] Aqueous pharmaceutical composition Y: about 120 mg / ml of the mixed antibody, about 70 mg / ml of sucrose, about 0.8 mg / ml of polysorbate 20, about 25 mM histidine-HCl, about 10 mM methionine, about 2000 IU / ml of hyaluronidase, pH about 5.2;

[0055] Aqueous pharmaceutical composition Z: about 100 mg / ml of mixed antibody, about 60 mg / ml of sucrose, about 0.6 mg / ml of polysorbate 20, about 20 mM of histidine-HCl, about 10 mM of methionine, about 2000 IU / ml of hyaluronidase, and a pH of about 5.2.

[0056] The present disclosure also provides a lyophilized preparation, which is obtained by lyophilizing the aqueous pharmaceutical composition of the present disclosure, or the aqueous pharmaceutical composition of the present disclosure is obtained by reconstitution of the lyophilized preparation.

[0057] The present disclosure also provides use of the aqueous pharmaceutical composition or lyophilized preparation in preparing a drug for treating tumors, wherein the tumor is selected from esophageal squamous cell carcinoma, melanoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, liver cancer, and kidney cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 shows the changes in tumor volume after treatment with ZPML265, anti-PD-1 antibody, anti-CTLA-4 antibody, and IgG.

[0059] Figure 2 shows the changes in tumor volume of mice in different groups.

[0060] FIG3 shows the tumor weights of mice in different groups.

[0061] FIG4 shows the body weight changes of mice in different groups. Specific implementation plan

[0062] the term

[0063] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0064] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present disclosure belongs.

[0065] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that numerical ranges or descriptions in terms of ranges are intended for simplicity and convenience and should not be considered as strict limitations on the scope of the present disclosure. Therefore, descriptions in terms of ranges should be considered to specifically disclose all possible subranges and all possible specific numerical points within the range, as these subranges and numerical points have been clearly stated herein. Regardless of the width of the numerical value, the above principles apply equally. When describing in terms of ranges, the range includes the endpoints of the range.

[0066] "About" includes and describes the value or parameter itself. For example, "about x" includes and describes "x" itself. As used herein, when used in conjunction with a measurement or to modify a value, unit, constant, or series of values, the term "about" includes, in addition to the value or parameter itself, a variation within ±20%, or in some cases within ±10%, or in some cases within ±5%, or in some cases within ±1%, or in some cases within ±0.1% of the specified value.

[0067] As used herein, the three-letter and one-letter codes for amino acids are as described in J. Biol. Chem, 243, p3558 (1968).

[0068] The term "antibody," as used herein, typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Natural IgG antibodies have this structure. Each light chain comprises a variable domain (VL) and a constant domain (CL). Each heavy chain comprises a variable domain (VH) and a constant domain (CH).

[0069] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM. The corresponding heavy chain constant domains are called α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two clearly distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0070] As used herein, the types of "antibodies" in a broad sense may include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including muteins and variants thereof), and the like.

[0071] The term "monoclonal antibody" (or "mAb") refers to a substantially homogeneous antibody produced by a single cell clone that is directed against a specific antigenic epitope. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or a combination of these techniques.

[0072] The term "hybrid antibody" refers to an antibody containing a limited number of primary antibody species, optionally no more than two, three, four, five, six, seven, eight, nine, or ten, produced from host cells (optionally cells from a single host cell line) that have been transfected with DNA encoding at least two different antibodies (optionally full-length primate IgG antibodies) with different binding specificities. In some embodiments, DNA encoding at least two different heavy chains (HCs) and at least two different light chains (LCs) can be introduced into the same host cell. For example, a host cell can be transfected with DNA encoding at least two but no more than four different antibodies with different binding specificities. In some embodiments, the sequences of all transfected DNA encoding HCs and LCs can be mutated to alter the amino acid sequence of the antibody, disfavoring non-homologous HC / LC pairing and strongly favoring homologous HC / LC pairing. When two different HCs are introduced into the host cell, one or both of the two different HCs can be optionally altered to disfavor heterodimer formation. In some embodiments, only one heavy chain is altered to prevent heterodimer formation. In some embodiments, when DNA encoding only two different antibodies is introduced into a host cell, only one of the antibodies encoded by the DNA comprises one or more partner-directed changes that favor cognate HC / LC pairing, while the other antibody does not comprise such changes. In some embodiments, the host cell produces only two major antibody species, wherein each HC predominantly pairs with its cognate LC, and most antibodies are tetramers containing two heavy chains having the same amino acid sequence and two light chains having the same amino acid sequence (see PCT / US2017 / 030676).

[0073] The terms "pharmaceutical preparation" or "preparation" or "preparation formulation" are used interchangeably herein to refer to a preparation that is in a form that permits the effective presence of the pharmaceutically active ingredient and that contains no other components that are toxic to the subject to which the preparation is to be administered.

[0074] The term "pharmaceutical composition" refers to a formulation or combination of formulations containing one, two, or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. When a "pharmaceutical composition" is present as a combination of separate formulations containing two or more different active ingredients, they can be administered simultaneously, sequentially, separately, or at intervals, with the goal of exerting the biological activities of the multiple active ingredients for combined treatment of a disease.

[0075] The term "ZPML265" refers to a mixed antibody comprising an anti-CTLA-4 antibody and an anti-PD-1 antibody, wherein the heavy chain of the anti-CTLA-4 antibody is set forth in SEQ ID NO:1, the light chain of the anti-CTLA-4 antibody is set forth in SEQ ID NO:2, the heavy chain of the anti-PD-1 antibody is set forth in SEQ ID NO:3, and the light chain of the anti-PD-1 antibody is set forth in SEQ ID NO:4. "ZPML265 injection" refers to a low-concentration formulation of ZPML265, intended for administration to a subject for the treatment of a disease. "ZPML265H injection" refers to a high-concentration formulation of ZPML265, intended for administration to a subject for the treatment of a disease.

[0076] The term "high concentration" formulation refers to a formulation comprising the active pharmaceutical ingredient (API) at a concentration of about 100 mg / ml to 200 mg / ml.

[0077] The term "effective amount" refers to a dose of a pharmaceutical formulation comprising an active ingredient of the present disclosure that, after administration to a patient in a single or multiple doses, produces the desired effect in the patient being treated. The effective amount can be readily determined by the attending physician, who is skilled in the art, by considering a variety of factors such as ethnic differences; weight, age, and health status; the specific disease involved; the severity of the disease; the response of the individual patient; the specific antibody being administered; the mode of administration; the bioavailability characteristics of the administered formulation; the selected dosing regimen; and the use of any concomitant therapy.

[0078] The term "pharmaceutically acceptable excipient" is a substance / agent that can be reasonably administered to a subject to provide an effective amount of an active pharmaceutical ingredient in a stable formulated form. Suitable pharmaceutically acceptable excipients are well known in the art and include, but are not limited to, buffers, stabilizers, surfactants, and the like.

[0079] The term "buffer" refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation. Suitable buffers are well known in the art and can be found in the literature.

[0080] The term "stabilizer" is a pharmaceutically acceptable excipient used to protect the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during production, storage, and use. The term "surfactant" refers to a pharmaceutically acceptable excipient used to protect protein formulations from mechanical stresses such as agitation and shear.

[0081] The term "antioxidant" refers to a compound that prevents oxidation of the active ingredient under storage conditions.

[0082] The term "diffusing agent" refers to a compound used to increase the permeability of active substances into tissues (such as skin) and help the active substances diffuse effectively, such as hyaluronidase, which can hydrolyze hyaluronic acid, thereby reducing the viscosity of hyaluronic acid in the extracellular matrix and making the drug preparation easier to disperse in the tissue.

[0083] The term "lyophilized preparation" refers to a composition obtained or obtainable by a freeze-drying process of a liquid preparation. Preferably, it is a solid composition having a water content of less than 5%, preferably less than 3%.

[0084] The term "treatment" refers to clinical intervention intended to alter the disease process in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or relieving the condition, and alleviating or improving the prognosis.

[0085] The term "individual" or "subject" or "patient" refers to any animal, such as a mammal or marsupial. Individuals of the present disclosure include, but are not limited to, humans, non-human primates (e.g., cynomolgus or rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and any type of poultry.

[0086] The terms "disease," "condition," or "disorder" refer to any change or disorder that damages or interferes with the normal function of a cell, tissue, or organ. For example, "disease" includes, but is not limited to, tumors, pathogen infection, autoimmune diseases, T-cell dysfunction, or immune tolerance deficiency (e.g., transplant rejection).

[0087] The term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion into other tissues or organs. Tumor growth is typically uncontrolled and progressive, without inducing or inhibiting normal cell proliferation. Tumor includes "cancer" and refers broadly to all malignant tumors.

[0088] The term "combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. The therapies can be physical, such as radiotherapy, or chemical, such as administering a drug to a subject, and the drug also includes a combination drug. "Combination drug" refers to a combination of two or more pharmaceutical preparations, each with an active ingredient, that need to be used in combination when administered to a subject. The active ingredients can be mixed together to form a single administration unit, or they can be independently administered as administration units and used separately: when administered, the different pharmaceutical preparations can be administered essentially synchronously, simultaneously or sequentially.

[0089] Relapse-free survival (RFS) was defined as the time from randomization to the first documented disease recurrence (local, regional, or distant) or death from any cause, whichever occurred first.

[0090] Overall survival (OS) is the time from the start of study treatment to death from any cause. Patients who were still in follow-up at the cut-off date were censored based on the cut-off date. Subjects lost to follow-up were censored based on the date of last contact.

[0091] Progression-free survival (PFS) refers to the time from the start of study treatment to the first imaging assessment of disease progression or death from any cause (whichever occurs first).

[0092] The objective response rate (ORR) was defined as the best confirmed ORR during the study period, including cases of complete response (CR) and partial response (PR). According to RECIST v1.1, when PR or CR first occurred, additional imaging examination of the lesion was required for confirmation at ≥4 weeks.

[0093] Disease control rate (DCR) refers to the percentage of cases with the best efficacy evaluation as "CR+PR+SD".

[0094] Complete remission (CR): All target lesions disappear and the short diameter of all pathological lymph nodes (including target nodules and non-target nodules) must be reduced to <10mm.

[0095] Partial response (PR): The sum of the target lesion diameters decreased by at least 30% compared with the baseline level.

[0096] Disease progression (PD): The minimum value of the sum of all target lesion diameters measured during the entire experimental study is used as a reference, and the sum of diameters increases by at least 20% (if the baseline measurement value is the minimum, the baseline value is used as a reference): In addition, the absolute value of the sum of diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered as disease progression).

[0097] Stable disease (SD): The target lesion has not decreased to the level of PR, nor increased to the level of PD, but is somewhere in between. The minimum sum of the diameters can be used as a reference for research.

[0098] Duration of response (DOR) was defined as the time from the first tumor assessment as CR or PR (whichever was recorded first) to the first assessment as PD or death.

[0099] Treatment-emergent adverse events (TEAEs) refer to adverse events that occur during treatment.

[0100] Treatment-related adverse events (TRAEs) are adverse events related to study drugs that occur during treatment.

[0101] A serious adverse event (SAE) refers to an adverse medical event (AE) such as death, life-threatening, permanent or severe disability or loss of function, the need for hospitalization or prolonged hospitalization, and congenital abnormalities or birth defects that occurs after the subject receives the investigational drug.

[0102] Dose-limiting toxicity (DLT) was defined as grade ≥3 non-hematologic toxicity or grade ≥4 hematologic toxicity occurring within 21 days (1 cycle) after the first dose.

[0103] The maximum tolerated dose (MTD) was defined as the highest dose level of the drug at which no more than one of six subjects experienced DLT during the 21-day treatment period (one cycle) after the first dose.

[0104] Experimental Example 1 ELISA method

[0105] After diluting the IL-2 ELISA kit with PSB, coat the plate with 4 μg / mL capture antibody at 50 μL / well. Seal the plate and incubate overnight. Aspirate the coating solution and add 100 μL of SmartBlock (#NC0104327, Fisher Scientific) to each well. Incubate the plate for 1 hour. Aspirate the blocking solution and add the supernatant sample diluted 15-fold in ELISA buffer to the plate along with the IL-2 standard provided in the ELISA kit. Incubate for 2 hours. Then wash the plate using a plate washer programmed for three cycles of wash and aspiration. Add 50 μL of the IL-2 detection antibody from the ELISA kit to a concentration of 0.100 μg / mL in ELISA buffer to each well and incubate the plate for 1 hour. Wash the plate again on the plate washer. Add 50 μL of streptavidin-conjugated HRP (#21140, Pierce) diluted 1:5000 in ELISA buffer to each well and incubate for 30 minutes. Wash the plate using a plate washer. Add 50 μL of TMB substrate (#tmbw-1000-01, SurModics) to each well and develop at room temperature for 4 minutes. Add 50 μL of 1N H2SO4 to each well to terminate the reaction. The plate is then read at 450-570 nm. The raw data is transferred to a graphing software (GraphPad Prism), where the data is converted to a sigmoid curve fit. Subsequent analysis (linear regression calculation) is performed in GraphPad to generate the EC 50 value.

[0106] Experimental Example 2 Protein content determination

[0107] Protein content was determined using a Lunatic microspectrophotometer. Purified water was used for blank subtraction, and the UV absorption at 280 nm was corrected by light scattering at 330 nm. Protein concentration was calculated according to Formula 1, where the extinction coefficient ε is 1.64 (L / g·cm -1 ).

[0108] Experimental Example 3 Size Exclusion Chromatography (SEC-HPLC)

[0109] Size exclusion chromatography was used to quantify aggregates, monomers, and fragments. This method utilized a Waters Xbridge BEH SEC The HPLC system was run on a 7.8 × 300 mm column using a Waters E2695-2489 HPLC system. The mobile phase consisted of 100 mM sodium phosphate, 150 mM sodium chloride, pH 6.8. The sample was diluted to 1 mg / mL with the mobile phase, and the injection volume was 25 μL. Isocratic elution was performed at a flow rate of 0.5 mL / min for 30 min, with detection at 215 nm. Integration was performed using Empower 3 software, and the percentage of each component was calculated by area normalization.

[0110] Experimental Example 4: Imaging Capillary Isoelectric Focusing (iCIEF)

[0111] In imaging capillary isoelectric focusing (IEF), an ampholyte carrier creates a uniform pH gradient from the anode to the cathode within the capillary under the influence of an electric field. Protein variants with different charges are focused and separated within the ampholyte pH gradient according to their isoelectric points. The focusing process was monitored in real time at 280 nm. A 100 μm inner diameter FC-coated fused silica capillary was used, with an effective separation length of 5 cm. Samples were treated with a final concentration of 1% Cytiva Pharmalyte 3-10, 0.5% Cytiva Pharmalyte 8-10.5, 0.35% HPMC, and 1 M urea, for a final sample concentration of 0.5 mg / ml. Focusing voltage and time were 1.5 kV for 1 min and 3 kV for 6 min, respectively. Chromatograms were integrated using Empower 3 software. The contents of the acidic, main, and basic regions were calculated using peak area percentages, and the pI values ​​of target peaks were calculated using marker pI values.

[0112] Experimental Example 5 Hyaluronidase Activity Detection

[0113] Hyaluronidase activity was assayed using a turbidimetric assay. Hyaluronidase can enzymatically hydrolyze the hyaluronic acid substrate. Excess hyaluronic acid solution can form a stable colloidal solution with acidified serum. A standard curve was constructed by measuring the absorbance of different concentrations of standard samples and their corresponding values, thereby determining the activity of hyaluronidase. A low-protein-adsorbed 96-well sample plate containing the test sample and standard sample and a 96-well substrate plate containing hyaluronic acid working solution were incubated at 37°C. The test sample and standard sample were transferred from the sample plate to the substrate plate for reaction. The serum working solution was then added to the substrate plate to terminate the reaction. The absorbance of the plate was measured at 640 nm, and the activity of the test sample was calculated based on the standard curve.

[0114] Example

[0115] The following specific embodiments are listed to illustrate the present disclosure. It should be understood that these examples are only for illustrating the present disclosure, rather than limiting the scope of the present disclosure.

[0116] Example 1 Obtaining the hybrid antibody ZPML265

[0117] A hybrid antibody produced by a single host cell line was prepared (see PCT / US2017 / 030676). The hybrid antibody contains two active ingredients, namely a recombinant humanized IgG1 monoclonal antibody targeting human CTLA4 and a recombinant humanized IgG4 monoclonal antibody targeting human PD1. The hybrid antibody can specifically bind to human CTLA4 and PD1 at the same time. After the two antibodies are simultaneously expressed by a single host cell line, they are collected, purified, and combined with a pharmaceutically acceptable carrier to form a single hybrid antibody ZPML265. The amino acid sequences of the anti-CTLA4 antibody and the anti-PD1 antibody are shown in Table 1 below. In addition, the molar ratio of the anti-PD-1 antibody to the anti-CTLA-4 antibody in ZPML265 is 2:1.

[0118] Table 1 Amino acid sequences of the components of the mixed antibody ZPML265

[0119] Example 2 Buffer Types for Low-Concentration Formulations

[0120] To optimize the buffering agent in the ZPML265 formulation, 10 mM acetic acid-sodium hydroxide, 10 mM histidine-hydrochloric acid, and 10 mM histidine-acetic acid buffers were selected to investigate the stability of the formulations. All formulations were sterile filtered through a 0.22 μm low protein-binding filter and aseptically filled into sterile 2 ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulation solutions were stored at the following conditions: 1 M at 40°C, 1 M at 2-8°C, and 1 M at 25°C to investigate sample stability. Table 2 provides information on the different formulations (R1, R2, and R3), and Table 3 provides the stability results for the different formulations.

[0121] Table 2 Prescription information of the preparation

[0122] Table 3 Stability data

[0123] The above results show that after one month of stability study, there is no significant difference between formulations R1 and R3, and both acetic acid-sodium hydroxide and histidine-acetic acid can be used as buffers.

[0124] Example 3 Buffer Concentration of Low-Concentration Formulation Prescription

[0125] To optimize the buffer concentration in the ZPML265 formulation, acetic acid-sodium hydroxide buffers were selected at concentrations of 10 mM, 20 mM, and 50 mM, respectively, to investigate the stability of the formulations. All formulations were sterile-filtered through a 0.22 μm low-protein-binding filter and aseptically filled into sterile 2 ml vials, sealed with film-coated rubber stoppers and aluminum-plastic combination caps. These formulations were stored at 40°C for 3 weeks to investigate sample stability. Table 4 provides information on the different formulations, and Table 5 presents the stability results for the different formulations.

[0126] Table 4 Prescription information of the preparation

[0127] Table 5 Stability data

[0128] The above results show that after 3 weeks at 40°C, the aggregate content in the formulation containing 50 mM buffer is higher than that in the formulations containing 10 mM and 20 mM buffer. Therefore, the formulations with buffer concentrations of 10 mM and 20 mM are preferred.

[0129] Example 4 Surfactant of low concentration formulation

[0130] To optimize the type and concentration of surfactants in the ZPML265 formulation, polysorbate 20 and polysorbate 80 were selected as surfactants at concentrations of 0.2 mg / ml and 0.5 mg / ml, respectively, to investigate the stability of the formulations. All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 2 ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations were placed under the following conditions: 40°C 3W and shaken (150 rpm, 25°C) for 24 hours to investigate sample stability. Table 6 shows information on different formulations, and Table 7 shows the stability results for different formulations.

[0131] Table 6 Prescription information of preparations

[0132] Table 7 Stability data

[0133] The above results show that the number of microparticles in the formulation without polysorbate is significantly greater than that in the formulation containing polysorbate; there is no significant difference between the use of polysorbate 20 and polysorbate 80. After stability testing, the main peak purity of the formulation containing 0.2 mg / ml polysorbate 20 is slightly higher, so 0.2 mg / ml polysorbate 20 is preferred as a surfactant.

[0134] Example 5 DOE test of low concentration preparation prescription

[0135] Nine formulations were tested under design-of-effect (DOE) conditions, with protein concentration, polysorbate 20 content, sucrose content, and pH as variables. The buffer used was 10 mM acetic acid-sodium hydroxide. The effects of the interactions between polysorbate 20, pH, protein concentration, and sucrose content on formulation stability were investigated. All formulations were sterile filtered through a 0.22 μm low-protein-binding filter and aseptically filled into sterile 2 ml vials, sealed with film-coated rubber stoppers and aluminum-plastic combination caps. These formulations were stored at the following conditions: 40°C for 1 minute and 2-8°C for 3 minutes to investigate sample stability. Table 8 shows information for the different formulations, and Table 9 shows the stability results for the different formulations.

[0136] Table 8 Prescription information of preparations

[0137] Table 9 Stability data

[0138] Example 6 In vivo pharmacodynamic study of anti-PD-1 component, anti-CTLA-4 component and ZPML265 in humanized NCG mouse HCC827 tumor model

[0139] NCG immunodeficient mice were used as experimental mice. Human tumor cells were first inoculated, and then human PBMCs were injected intravenously to establish a tumor-bearing mouse model with a human immune system. ZPML265, anti-human PD-1 antibody (the anti-PD-1 component of ZPML265), and anti-human CTLA-4 antibody (the anti-CTLA-4 component of ZPML265) were then administered. The pharmacodynamic effects of anti-PD-1 antibody, anti-CTLA-4 antibody, and ZPML265 were investigated in the humanized NCG mouse HCC827 tumor model, and whether ZPML265 had a synergistic effect was explored.

[0140] Peripheral blood from healthy donors was centrifuged by density gradient to separate PBMCs, which were then resuspended in PBS to a volume of 1×10 8 After adjusting the density of HCC827 tumor cells with PBS, the cells were plated at 5×10 6 / 100μL / mouse was inoculated subcutaneously on the right flank of NCG mice, and the day of tumor cell inoculation was recorded as day 0. When the average tumor volume reached 60-80mm 3 (approximately 5 days after tumor cell inoculation), 1×10 7 PBMC / 100μL / animal, intraperitoneal administration started 1 hour after PBMC inoculation. Before administration, use StudyDirector according to the tumor volume of the selected animal. TMThe software randomly divided the mice into 4 groups (5 mice per group). NCG mice were intraperitoneally (ip) administered with a low-concentration formulation of ZPML265 (7.5 mg / kg), anti-PD-1 antibody (5 mg / kg), anti-CTLA-4 antibody (2.5 mg / kg), and human IgG1 (7.5 mg / kg). The drugs were administered twice a week for 3 consecutive weeks. The long and short diameters of the tumors were measured twice a week using a vernier caliper, and the body weight was weighed. The tumor volume was calculated using the following formula (mm) 3 Indicates that V = 0.5 × a × b 2 , where a and b are the long and short diameters of the tumor, respectively. In this study, tumor volume inhibition (TGI) and tumor volume were used as biological indicators of pharmacodynamics (PD).

[0141] During the experiment, the animals showed no abnormal clinical signs. As shown in Figure 1, a low-concentration ZPML265 formulation significantly inhibited tumor growth (TGI = 93%, 7.5 mg / kg), while anti-PD-1 or anti-CTLA-4 antibodies administered alone had no significant effect.

[0142] In summary, in the humanized NCG mouse HCC827 tumor model experiment, the inhibitory effect of low-concentration ZPML265 preparation on HCC827 tumors was better than that of anti-PD-1 antibody or anti-CTLA-4 antibody alone, and ZPML265 had a synergistic effect.

[0143] Example 7 High Concentration Preparation Prescription

[0144] To prepare high-concentration formulation solutions of ZPML265, ZPML265 was buffer-exchanged with the intended excipient solution and concentrated by ultrafiltration to the desired antibody concentration. After ultrafiltration and concentration, the desired excipients were added to the antibody solution as a stock solution. Finally, the protein concentration was adjusted to the desired concentrations, such as approximately 100 mg / ml, 120 mg / ml, 150 mg / ml, and 200 mg / ml, using buffer. All formulations were sterile-filtered through a 0.22 μm low-protein-binding filter and aseptically filled into sterile 2 ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations were placed under the following conditions: 25°C for 2 minutes and 40°C for 1 minute to investigate sample stability. Table 10 shows information for different formulations, and Table 11 shows the stability results for different formulations.

[0145] Table 10 Prescription information of the preparation

[0146] Table 11 Stability data

[0147] The above results show that: after stability investigation, after the protein concentration exceeds 120 mg / ml, the aggregate content increases with the increase of protein concentration, so the preferred protein concentration is 120 mg / ml; there is no significant difference between prescriptions with different sucrose concentrations compared with prescription 1 and prescription 4, and prescription 2 and prescription 5; after high temperature 40℃1M stability investigation, the aggregate content in the methionine-containing prescription is lower than that of prescription 5, so 10mM methionine is preferred.

[0148] Example 8 Surfactant of high concentration formulation

[0149] In the high-concentration formulation, the surfactant and arginine hydrochloride content were further optimized, and polysorbate 20 at concentrations of 0.5 mg / ml and 1 mg / ml, poloxamer 188 at concentrations of 0.2 mg / ml and 0.5 mg / ml, and arginine hydrochloride at concentrations of 0 mg / ml and 100 mg / ml were selected, respectively, to investigate the stability of the formulation. All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 2 ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations were placed under the following conditions: freeze-thaw three times, shaken for 5 days (25°C), 40°C for 1 M, 2-8°C for 6 M, and 25°C for 3 M to investigate sample stability. Table 12 shows the information for different formulations, Table 13 shows the stability results for different formulations, and Table 14 shows the particle results for different formulations.

[0150] Table 12 Prescription information of preparations

[0151] Table 13 Stability data

[0152] Table 14 Particle data

[0153] The above results show that: after stability investigation, compared with prescription 1 and prescription 2, the presence or absence of arginine hydrochloride has no obvious effect on the purity of the main peak of the prescription; compared with prescriptions 2, 3, 4, and 5, prescriptions 4 and 5 have slightly more protein aggregates in the ≥5μm particles; at the same time, compared with prescriptions 2 and 3, there is no significant difference between the prescriptions with different concentrations of polysorbate 20, and polysorbate 20 is preferred as a surfactant.

[0154] Example 9 pH of high concentration formulation

[0155] In order to improve the subcutaneous absorption of high-concentration formulations, hyaluronidase was introduced into the formulation, and the pH value of the formulation was further optimized. The stability of the formulation was investigated at pH 5.0, 5.2, 5.4, 5.6, and 5.8, with an enzyme activity of 2000 IU / ml. All prepared formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 2 ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations were placed under the following conditions: 40°C 1M, 2-8°C 3M, and 25°C 1M to investigate sample stability. Table 15 shows the information of different formulations, and Table 16 shows the stability results of different formulations.

[0156] Table 15 Prescription information of preparations

[0157] Table 16 Stability data

[0158] The above results show that: after stability investigation, there is no significant difference in the purity of the SEC main peak at 2-8℃3M, 25℃1M, and 40℃1M, and the optimal pH value is 5.4.

[0159] Example 10 Buffer for high concentration formulation

[0160] The buffer was further optimized in the high-concentration formulation, and 10mM and 20mM acetic acid-sodium hydroxide and 10mM and 20mM histidine-hydrochloric acid were selected as buffers, respectively, to investigate the stability of the formulation. All formulations were sterile filtered through a 0.22μm low protein binding filter and aseptically filled into sterile 2ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations were placed under the following conditions: 40℃ 1M, 2-8℃ 3M, 25℃ 3M and 30℃ 3M to investigate the stability of the samples. Table 17 shows the information of different formulations, and Table 18 shows the stability results of different formulations.

[0161] Table 17 Prescription information of preparations

[0162] Table 18 Stability data

[0163] The above results show that: after the stability test at 30℃3M, the enzyme activities of formulations 3 and 4 are high, and the aggregates of formulation 4 are low, so 20mM histidine-hydrochloric acid is preferred as the buffer.

[0164] Example 11 Study on the enzyme activity stability of high concentration preparations

[0165] Enzyme activity stability was further investigated in high-concentration formulations. All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 2 ml vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulations were placed under the following conditions: 40°C for 7 days, 2-8°C for 3 M, 6 M, and 9 M, and 25°C for 1 M, 3 M, and 6 M to investigate sample stability. Table 19 shows information on different formulations, Table 20 shows the stability results for different formulations, and Table 21 shows the enzyme activity results for formulation R1.

[0166] Table 19 Prescription information of preparations

[0167] Table 20 Stability data

[0168] Table 21 Enzyme activity data (IU / ml)

[0169] The above results show that: after stability investigation, there is no significant difference in purity between prescription 1 and prescription 2, indicating that hyaluronidase has no significant effect on stability; and there is no significant change in the enzyme activity of prescription 1, and the enzyme activity stability is good.

[0170] Example 12 Evaluation of the Antitumor Effects of ZPML265H Injection and ZPML265 Injection in a B6-hPD1hCTLA4 Mouse Subcutaneous Transplantation MC38-hPDL1 Model

[0171] MC38-hPDL1 tumor cells were cultured at a density of 1×10 6 The cells / 100 μl / mouse were inoculated subcutaneously on the right flank of B6-hPD1 / hCTLA4 humanized mice (Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd.). When the average tumor volume reached 86.72 mm 3At the time of the study, 56 mice were randomly divided into seven groups of eight based on body weight and tumor volume. Group G1 mice were given NaCl plus the excipient of ZPML265H injection (with hyaluronidase rHuPH20) as a control. Groups G2-G4 mice were given different doses of ZPML265 injection (0.1 mg / kg for G2, 0.3 mg / kg for G3, and 1 mg / kg for G4). Groups G5-G7 mice were given different doses of ZPML265H injection (0.1 mg / kg for G5, 0.3 mg / kg for G6, and 1 mg / kg for G7). Dosing was twice weekly for a total of eight doses. In group G1, NaCl was administered via the tail vein, and the excipient of ZPML265H injection (with hyaluronidase rHuPH20) was administered subcutaneously. Groups G2-G4 mice were administered via the tail vein, and groups G5-G7 mice were administered subcutaneously. The day of grouping was defined as D0, the dosing period was from D0 to D28, the dosing dates were D0, D3, D7, D10, D14, D17, D21, D24, and the experiment ended on D28. After the start of dosing, the tumor size was measured and the mice were weighed on D0, D3, D7, D10, D14, D17, D21, D24, and D28. The tumor volume was calculated as follows: tumor volume (mm 3 ) = 0.5 × tumor long diameter × tumor short diameter 2 At the end of the experiment, the animals were euthanized and the changes in tumor volume (TGITV), tumor weight (TGITW), and body weight were analyzed. The experimental results are expressed as mean ± standard error (Mean ± SEM). Data were analyzed using SPSS24, and the independent sample T-test (T-Test) was used to compare the two groups of samples. P < 0.05 was considered to be significantly different. The graphics software was Graphpad Prism 9.

[0172] As shown in Figure 2, at the experimental endpoint D28, the tumor volume inhibition rates (TGITV) in the ZPML265-treated groups at doses of 0.1, 0.3, and 1 mg / kg were 56.42%, 66.36%, and 89.44%, respectively, and in the ZPML265H-treated groups at doses of 0.1, 0.3, and 1 mg / kg, were 66.85%, 80.35%, and 99.78%, respectively. Compared to the control group (NaCl+ZPML265H injection excipient (hyaluronidase rHuPH20 added), the tumor volumes in the ZPML265 and ZPML265H-treated groups showed significant differences, indicating that different doses of ZPML265 and ZPML265H had a significant inhibitory effect on tumor volume in a dose-dependent manner.

[0173] As shown in Figure 3, the tumor weight results showed a consistent trend with the tumor volume results. The tumor weight inhibition rates (TGITW) in the ZPML265-treated groups at doses of 0.1, 0.3, and 1 mg / kg were 52.46%, 68.02%, and 85.54%, respectively. The tumor weight inhibition rates in the ZPML265H-treated groups at doses of 0.1, 0.3, and 1 mg / kg were 71.14%, 79.83%, and 99.66%, respectively. Compared to the control group, which included the excipient of the NaCl+ZPML265H injection (hyaluronidase rHuPH20), both the ZPML265 and ZPML265H-treated groups significantly reduced tumor weight in a dose-dependent manner.

[0174] Comparison of the efficacy of ZPML265H and ZPML265 injections at the same dose showed no significant difference in tumor volume or weight between the ZPML265H and ZPML265 groups. These results indicate that, at the same dose, ZPML265H injection has a comparable tumor inhibitory effect as ZPML265 injection.

[0175] As shown in Figure 4, compared with the excipients of the control group NaCl+ZPML265H injection (hyaluronidase rHuPH20 added), except for the significant decrease in animal body weight in ZPML265 (1 mg / kg dose), there was no significant difference in the body weight of mice in the other dose-administered groups of ZPML265 and ZPML265H, indicating that the safety of ZPML265H injection tends to be better than that of ZPML265 injection at the same dosage.

[0176] These research results show that different doses of ZPML265 and ZPML265H injections can significantly inhibit tumor growth in a dose-dependent manner. The efficacy of ZPML265H injection at the same dose is comparable to that of ZPML265 injection, and the safety profile of ZPML265H injection tends to be superior to that of ZPML265 injection.

[0177] Example 13 A Phase II Clinical Study to Evaluate the Safety and Efficacy of ZPML265 Injection Combined with Paclitaxel-Cisplatin / Carboplatin in Patients with Recurrent or Metastatic Cervical Cancer

[0178] Dosage:

[0179] A Phase IⅠ clinical study conducted in China plans to enroll 30 patients with recurrent or stage IVB cervical cancer. The patients are over 18 years old, have an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1, and are expected to survive ≥ 3 months. The functional level of vital organs before the first use of the investigational drug is:

[0180] 1) Absolute neutrophil count ≥1.5×109 / L;

[0181] 2) Platelets ≥100×10 9 / L;

[0182] 3) Hemoglobin ≥90 g / L;

[0183] 4) serum albumin ≥30 g / L;

[0184] 5) Alanine aminotransferase (AST) and aspartate aminotransferase (ALT) ≤ 2.5 × upper limit of normal reference value (ULN) (for patients with liver cancer or liver metastasis, ≤ 5 × ULN is allowed);

[0185] 6) Total bilirubin ≤1.5×ULN (≤3×ULN is allowed for patients with Gilbert syndrome);

[0186] 7) serum creatinine ≤1.5×ULN. If the patient's creatinine level is >1.5×ULN, the creatinine clearance (CLcr) calculated by the Cockcroft-Gault equation is ≥50 mL / min;

[0187] 8) Left ventricular ejection fraction (LVEF)>50%;

[0188] 9) Proteinuria <2+ (when urine protein ≥2+, 24-hour urine protein quantification should be performed, and patients can be selected when ≤1g).

[0189] 10) International normalized ratio (INR) ≤ 1.5; activated partial thromboplastin time (APTT) ≤ 1.5 × ULN.

[0190] Dosage regimen: The dose of ZPML265 injection is 5 mg / kg, once every three weeks, administered by intravenous infusion.

[0191] The medications used in this study are as follows: The following drugs are administered once every 3 weeks, with each cycle consisting of 21 days (3 weeks). Paclitaxel and cisplatin / carboplatin are administered for a maximum of 6 cycles. After 6 cycles, subjects receive ZPML265 injection for maintenance treatment. ZPML265 injection is administered until disease progression, intolerable toxicity, initiation of other anti-cancer treatments, loss to follow-up, death, or study withdrawal (whichever occurs first). The dosing window is the planned dosing date (calculated from the date of the first dose) ± 3 days. Exceeding the dosing window is considered early or delayed dosing, and subsequent dosing dates must be recalculated based on the actual date of the last dose.

[0192] ZPML265 injection: 5 mg / kg, intravenous infusion (IV), D1, Q3W; the intravenous infusion time is more than 30 minutes. After the infusion is completed, 20 ml of the same solvent is used to flush the tube. The entire infusion time including the flushing is 60 minutes (± 5 minutes); ZPML265 injection must not be injected by intravenous push.

[0193] Chemotherapy drug regimen:

[0194] If the subject has previously received a cumulative dose of cisplatin ≤ 300 mg, the following regimen will be used:

[0195] For subjects who have not received definitive radiation / adjuvant pelvic external beam radiation therapy, paclitaxel 175 mg / m 2 D1, cisplatin 70mg / m 2 D2, dosing frequency was Q3W;

[0196] For subjects who have received radical radiotherapy, paclitaxel was administered at 135 mg / m 2 D1, cisplatin 50 mg / m 2 D2, dosing frequency was Q3W;

[0197] For subjects who have received adjuvant pelvic external beam radiation (≥50 Gy), paclitaxel was administered at a dose of 135 mg / m 2 D1, cisplatin 60 mg / m 2 D2, dosing frequency was Q3W;

[0198] If the cumulative dose of cisplatin received by the subject before enrollment is greater than 300 mg, the following regimen will be used:

[0199] For subjects who have not received definitive radiation / adjuvant pelvic external beam radiation therapy, paclitaxel 175 mg / m 2 D1, carboplatin used AUC 6D1, and the dosing frequency was Q3W;

[0200] For subjects who have received radical radiotherapy, paclitaxel was administered at 135 mg / m 2 D1, carboplatin using AUC 5D1, dosing frequency was Q3W;

[0201] For subjects who have received adjuvant pelvic external beam radiation (≥50 Gy), paclitaxel was administered at a dose of 135 mg / m 2 For D1, carboplatin was administered at AUC 5-6D1, and the dosing frequency was Q3W.

[0202] Effectiveness Results:

[0203] As of April 24, 2023, efficacy data for evaluable cases showed an objective response rate (ORR) of 78.6% (22 / 28), a disease control rate (DCR) of 100.0% (28 / 28), and a median progression-free survival (mPFS) of 12.5 months. These efficacy rates are superior to those of the currently marketed, similarly approved drug, pembrolizumab (a PD-1 inhibitor).

[0204] Safety results:

[0205] As of April 24, 2023, safety data for 30 subjects were collected. All subjects (100%) experienced treatment-emergent adverse events (TEAEs), 73.3% (22 patients) experienced grade ≥3 treatment-related adverse events (TRAEs), most of which were chemotherapy-related adverse events. No treatment-related deaths occurred, and the safety and tolerability of the drug were good.

[0206] Preliminary conclusions:

[0207] ZPML265 injection combined with paclitaxel-cisplatin / carboplatin showed a significant efficacy trend in patients with recurrent or metastatic cervical cancer, with controllable overall safety and good tolerability.

[0208] Example 14: Phase II clinical study evaluating the safety and efficacy of ZPML265 injection combined with paclitaxel-cisplatin / carboplatin and bevacizumab for the first-line treatment of patients with recurrent or metastatic cervical cancer

[0209] Dosage:

[0210] A Phase IⅠ clinical study conducted in China plans to enroll 30 patients with recurrent or stage IVB cervical cancer. The patients are over 18 years old, have an Eastern Cooperative Oncology Group (ECOG) performance status score of 0 or 1, and are expected to survive ≥ 3 months. The functional level of vital organs before the first use of the investigational drug is:

[0211] 1) Absolute neutrophil count ≥1.5×10 9 / L;

[0212] 2) Platelets ≥100×10 9 / L;

[0213] 3) Hemoglobin ≥90 g / L;

[0214] 4) serum albumin ≥30 g / L;

[0215] 5) Alanine aminotransferase (AST) and aspartate aminotransferase (ALT) ≤ 2.5 × upper limit of normal reference value (ULN) (for patients with liver cancer or liver metastasis, ≤ 5 × ULN is allowed);

[0216] 6) Total bilirubin ≤1.5×ULN (≤3×ULN is allowed for patients with Gilbert syndrome);

[0217] 7) serum creatinine ≤1.5×ULN. If the patient's creatinine level is >1.5×ULN, the creatinine clearance (CLcr) calculated by the Cockcroft-Gault equation is ≥50 mL / min;

[0218] 8) Left ventricular ejection fraction (LVEF)>50%;

[0219] 9) Proteinuria <2+ (when urine protein ≥2+, 24-hour urine protein quantification should be performed, and patients can be selected when ≤1g).

[0220] 10) International normalized ratio (INR) ≤ 1.5; activated partial thromboplastin time (APTT) ≤ 1.5 × ULN.

[0221] Dosage regimen:

[0222] (1) ZPML265, 5 mg / kg, intravenous infusion, D1, Q3W;

[0223] (2) Paclitaxel, 175 mg / m 2 or 135 mg / m 2 , intravenous infusion, D1, Q3W;

[0224] (3) Cisplatin, 50 mg / m 2 , intravenous infusion, D1 or D2, Q3W;

[0225] (4) Carboplatin, AUC 5, intravenous infusion, D1, Q3W;

[0226] (5) Bevacizumab, 15 mg / kg, intravenous infusion, D1, Q3W.

[0227] The choice of cisplatin or carboplatin in the dosing regimen was determined by the investigator based on the subject's condition.

[0228] ZPML265 injection combined with paclitaxel-cisplatin / carboplatin and bevacizumab was used until disease progression, intolerable toxicity, initiation of new anti-tumor treatment, loss to follow-up, death, withdrawal from the study, or other withdrawal criteria (whichever occurred first).

[0229] Based on the investigator's judgment, the subject should receive 6 cycles of chemotherapy if tolerated. Subsequently, chemotherapy can be stopped and treatment with ZPML265 and bevacizumab can be continued according to the investigator's decision and / or the subject's wishes until disease progression or other exit criteria are met.

[0230] When a subject experiences disease progression and does not meet any of the study treatment exit criteria, if the investigator believes that the subject can still benefit from continued treatment with ZPML265, the subject will be allowed to continue receiving ZPML265 monotherapy until the investigator determines that there is no longer clinical benefit, or the toxicity is intolerable, or the subject starts a new anti-tumor treatment, is lost to follow-up, dies, withdraws from the study, or other study treatment exit criteria (whichever occurs first).

[0231] Participants in the study received study treatment for a maximum of 2 years (24 months).

[0232] Evaluation indicators: The endpoints of this study are safety and efficacy indicators and other indicators.

[0233] include:

[0234] 1) Safety indicators;

[0235] 2) objective response rate (ORR) (according to RECIST v1.1);

[0236] 3) duration of response (DOR) (according to RECIST v1.1);

[0237] 4) disease control rate (DCR) (according to RECIST v1.1);

[0238] 5) progression-free survival (PFS) (according to RECIST v1.1);

[0239] 6) Overall survival (OS);

[0240] 7) Pharmacokinetic parameters of ZPML265;

[0241] 8) Immunogenicity of ZPML265.

[0242] Effectiveness Results:

[0243] As of April 24, 2023, efficacy data for evaluable cases showed an objective response rate (ORR) of 83.3% (25 / 30), a disease control rate (DCR) of 96.7% (29 / 30), and a median progression-free survival (mPFS) of 16.4 months. These efficacy rates are superior to those of the currently marketed, similarly approved drug, pembrolizumab (a PD-1 inhibitor).

[0244] Safety results:

[0245] As of April 24, 2023, safety data for 30 subjects were collected. All subjects (100%) experienced treatment-emergent adverse events (TEAEs), 70.0% (21 patients) experienced grade ≥3 treatment-related adverse events (TRAEs), most of which were chemotherapy-related adverse events. No treatment-related deaths occurred, and the safety and tolerability of the drug were good.

[0246] Preliminary conclusions:

[0247] ZPML265 injection combined with paclitaxel-cisplatin / carboplatin and bevacizumab showed a significant efficacy trend in patients with recurrent or metastatic cervical cancer, with controllable overall safety and good tolerability.

[0248] The above research results demonstrate that the formulation of the ZPML265 injection disclosed herein has excellent effects in terms of stability, safety, and efficacy.

[0249] The embodiments of the present disclosure described above are merely exemplary, and any person skilled in the art will recognize or be able to determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All such equivalents are within the scope of the present disclosure and are encompassed by the claims.

Claims

1. An aqueous pharmaceutical composition of a mixed antibody of anti-CTLA-4 and anti-PD-1, comprising: 1) a mixed antibody of an anti-CTLA-4 antibody and an anti-PD-1 antibody, wherein the amino acid sequences of the heavy chain and the light chain of the anti-CTLA-4 antibody are shown in SEQ ID NOs: 1 and 2, respectively, and the amino acid sequences of the heavy chain and the light chain of the anti-PD-1 antibody are shown in SEQ ID NOs: 3 and 4, respectively; 2) a stabilizer, wherein the stabilizer is selected from sucrose or trehalose; 3) a surfactant, wherein the surfactant is selected from polysorbate 80, polysorbate 20 or poloxamer 188; 4) a buffer selected from acetic acid-sodium hydroxide, histidine-hydrochloric acid or histidine-acetic acid; in, The pH of the aqueous pharmaceutical composition is about 4.0-6.0, and the molar ratio of the anti-CTLA-4 antibody to the anti-PD-1 antibody in the mixed antibody is about 1:

2.

2. The aqueous pharmaceutical composition of claim 1, wherein the concentration of the mixed antibody is about 20-200 mg / ml.

3. The aqueous pharmaceutical composition of claim 1, wherein the stabilizer is present in an amount of about 50-100 mg / ml.

4. The aqueous pharmaceutical composition of claim 1, wherein the surfactant is present in an amount of about 0.1-2 mg / ml.

5. The aqueous pharmaceutical composition of claim 1, wherein the buffer is present in an amount of about 5-50 mM.

6. The aqueous pharmaceutical composition of claims 1-5, comprising about 20-30 mg / ml of the mixed antibody; about 50-100 mg / ml of sucrose or about 50-100 mg / ml of trehalose; about 0.1-2 mg / ml of polysorbate 80 or about 0.1-2 mg / ml of polysorbate 20 or about 0.1-2 mg / ml of poloxamer 188; about 5-50 mM of acetic acid-sodium hydroxide or about 5-50 mM of histidine-hydrochloric acid or about 5-50 mM of histidine-acetic acid; wherein the pH of the aqueous pharmaceutical composition is about 4.0-6.

0.

7. The aqueous pharmaceutical composition of claim 6, comprising about 20-30 mg / ml of the mixed antibody; about 80-100 mg / ml of sucrose or about 80-100 mg / ml of trehalose; about 0.1-0.5 mg / ml of polysorbate 80 or about 0.1-0.5 mg / ml of polysorbate 20 or about 0.1-0.5 mg / ml of poloxamer 188; about 10-50 mM of acetic acid-sodium hydroxide or about 10-50 mM of histidine-hydrochloric acid or about 10-50 mM of histidine-acetic acid; wherein the pH of the aqueous pharmaceutical composition is about 4.7-5.

3.

8. The aqueous pharmaceutical composition according to claim 1 or 2, further comprising an antioxidant and / or a dispersing agent.

9. The aqueous pharmaceutical composition of claim 8, wherein the antioxidant is methionine, and its content is about 0-50 mM; and / or, the diffusing agent is hyaluronidase, and its content is about 0-4000 IU / ml.

10. The aqueous pharmaceutical composition of claim 9, further comprising arginine hydrochloride in an amount of about 0-150 mM.

11. The aqueous pharmaceutical composition of claim 10, comprising about 100-200 mg / ml of the mixed antibody; about 50-100 mg / ml of sucrose or about 50-100 mg / ml of trehalose; about 0.1-2 mg / ml of polysorbate 80 or about 0.1-2 mg / ml of polysorbate 20 or about 0.1-2 mg / ml of poloxamer 188; about 5-50 mM of acetic acid-sodium hydroxide or about 5-50 mM of histidine-hydrochloric acid or about 5-50 mM of histidine-acetic acid; wherein the pH of the aqueous pharmaceutical composition is about 4.0-6.0; optionally, further comprising about 0-50 mM of methionine, and / or, about 0-4000 IU / ml of hyaluronidase, and / or, about 0-150 mM of arginine hydrochloride.

12. The aqueous pharmaceutical composition of claim 11, comprising about 100-200 mg / ml of the mixed antibody; about 50-100 mg / ml of sucrose or about 50-100 mg / ml of trehalose; about 0.1-2 mg / ml of polysorbate 80 or about 0.1-2 mg / ml of polysorbate 20 or about 0.1-2 mg / ml of poloxamer 188; about 5-50 mM of acetic acid-sodium hydroxide or about 5-50 mM of histidine-hydrochloric acid or about 5-50 mM of histidine-acetic acid; about 5-50 mM of methionine; about 500-4000 IU / ml of hyaluronidase; wherein the pH of the aqueous pharmaceutical composition is about 4.0-6.0; optionally, further comprising about 20-150 mM of arginine hydrochloride.

13. The aqueous pharmaceutical composition of claim 12, comprising about 100-200 mg / ml of the mixed antibody; about 60-90 mg / ml of sucrose or about 60-90 mg / ml of trehalose; about 0.2-1 mg / ml of polysorbate 80 or about 0.2-1 mg / ml of polysorbate 20 or about 0.2-1 mg / ml of poloxamer 188; about 10-20 mM of acetic acid-sodium hydroxide or about 10-20 mM of histidine-hydrochloric acid or about 10-20 mM of histidine-acetic acid; about 10 mM of methionine; about 2000 IU / ml of hyaluronidase; wherein the pH of the aqueous pharmaceutical composition is about 5.0-5.8; optionally, it also comprises about 100 mM of arginine hydrochloride.

14. A lyophilized preparation, which is obtained by lyophilizing the aqueous pharmaceutical composition according to any one of the preceding items, or the aqueous pharmaceutical composition according to any one of the preceding items is obtained by reconstituting the lyophilized preparation.

15. Use of the pharmaceutical composition or lyophilized preparation according to any of the preceding claims in the preparation of a drug for treating tumors.

16. The use according to claim 15, wherein the tumor is selected from esophageal squamous cell carcinoma, melanoma, cervical cancer, small cell lung cancer, non-small cell lung cancer, colorectal cancer, liver cancer, and kidney cancer.

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